METHODS OF TREATING UROTHELIAL CARCINOMA WITH A PD-1 AXIS BINDING ANTAGONIST AND AN RNA VACCINE
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 63/621,511, filed January 16, 2024, which is hereby incorporated by reference in its entirety.
FIELD
[0002] The present disclosure relates to methods for treating an individual with urothelial carcinoma (UC), e.g., muscle-invasive urothelial carcinoma (MIUC), with an individualized cancer vaccine and a PD-1 axis antagonist.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] The content of the electronic sequence listing (146392068140seqlist.xml; Size: 62,149 bytes; and Date of Creation: December 18, 2024) is herein incorporated by reference in its entirety.
BACKGROUND
[0004] Urothelial carcinoma (UC) is the most common cancer of the urinary system worldwide, with the majority of cases originating in the bladder (Leow et al. (2017) Upper tract urothelial carcinoma: a different disease entity in terms of management. ESMO Open 2017;l :e000126). Approximately 1 in 3 new cases of urothelial carcinoma are diagnosed as muscle invasive disease (cT2-T4aNxM0) (Kaufman et al. (2009) Bladder cancer. Lancet 2009;374:239-49). The term “muscle invasive urothelial carcinoma” (MIUC) collectively refers to muscle invasive bladder cancer (MIBC; sometimes referred to as “bladder cancer”) and muscle invasive upper tract urothelial cancer (UTUC, which can include, for instance, cancer of the kidney and/or ureter).
[0005] Most urothelial tumors arise in the bladder, with transitional cell carcinoma (TCC; also called UC) being the most common histologic subtype. Patients with MIBC often undergo bladder resection (cystectomy). Patients with UTUC often undergo kidney and/or ureter resection (nephroureterectomy). Transitional cell carcinoma accounts for 90% of all MIBC cases in the industrialized world (Chalasani et al. (2009) Histologic variants of urothelial bladder cancer and nonurothelial histology in bladder cancer. Can Urol Assoc J 2009;3(Suppl 4): S 193-8). For patients undergoing radical cystectomy alone, recurrence-free survival and OS in male and female patients is reported as 66%-68% and 58%-66% at 5 years, and 60%-73% and 43%-49% at 10 years, respectively (Stein et al. (2001) Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. J Clin Oncol 2001;19:666-75; Hautmann et al. (2012) Radical cystectomy for urothelial carcinoma of the bladder without neoadjuvant or adjuvant therapy: long-term results in 1100 patients. Eur Urol 2012;61 : 1039-47; Madersbacher et al. (2003) Radical cystectomy for bladder cancer today — a homogeneous series without neoadjuvant therapy. J Clin Oncol 2003;21 :690-6). In node-positive patients, 10-year DSS and OS rates decrease to 27.7% and 20.9%, respectively (Gschwend et al. (2002) Disease specific survival as endpoint of outcome for bladder cancer patients following radical cystectomy. Eur Urol 2002;41 :440-8).
Radical Cystectomy
[0006] For MIBC, radical cystectomy with bilateral pelvic lymphadenectomy is the backbone of management. This surgery primarily involves resection of the bladder, adjacent organs, and regional lymph nodes, although there are also sex -based differences in the surgical approach. The perioperative mortality rate is approximately 2%-3% when cystectomy is performed at centers of excellence (Stein et al. 2001; Madersbacher et al. 2003).
[0007] Despite radical cystectomy, MIBC recurs in many patients, who subsequently present with pain or constitutional symptoms such as fatigue, weight loss, anorexia, and failure to thrive. Approximately half of the patients with MIBC will develop a local and/or metastatic recurrence of their disease within 2 years of cystectomy and will eventually die from their disease (Raghavan et al. (1990) Biology and management of bladder cancer. N Engl J Med 1990;322: 1129-38; Stein et al. 2001; Stenzl et al. (2009) The updated EAU guidelines on muscle-invasive and metastatic bladder cancer. Eur Urol 2009;55:815-25). For those with pathological high-risk features (pT3 T4a or pN+) who have not received neoadjuvant chemotherapy (NAC), the overall 5 year survival rates range from 10%-40% (Stein et al. 2001). Despite numerous clinical trials, no adjuvant therapies have shown improved survival in MIBC (Sternberg et al. (2014) Thoughts on a systematic review and meta-analysis of adjuvant chemotherapy in muscle-invasive bladder cancer. Eur Urol 2014;66:55-6).
Neoadjuvant and Adjuvant Treatments [0008] Because of the high risk of relapse with surgery alone, neoadjuvant and adjuvant treatments have been utilized in conjunction with radical cystectomy for MIBC. The rationale for perioperative chemotherapy is based on the response in patients with metastatic UC, in which cisplatin-based chemotherapy has demonstrated efficacy with a median survival of approximately 15 months with responses in 40%-60% of patients (von der Maase et al. (2005) Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer. J Clin Oncol 2005;23:4602-8). However, there is an urgent need for effective regimens for patients who decline or are ineligible for NAC or adjuvant chemotherapy. Many of the latter patients have poor performance status, comorbidities, or renal insufficiency, for whom carboplatin based regimens appear to generate suboptimal outcomes in both perioperative and advanced settings (Abol-Enein et al. (1997) Neo-adjuvant chemotherapy in the treatment of invasive transitional bladder cancer: a controlled prospective randomised study. Br J Urol 1997;79(Suppl 4): 174; De Santis et al. (2009) Randomized phase II/III trial assessing gemcitabine/carboplatin and methotrexate/carboplatin/vinblastine in patients with advanced urothelial cancer "unfit" for cisplatin-based chemotherapy: phase II results of EORTC study 30986. J Clin Oncol 2009;27:5634-9).
[0009] Neoadjuvant chemotherapy (NAC) has provided a modest survival benefit of 5% in MIBC (Advanced Bladder Cancer Meta-Analysis Collaboration 2003). The disadvantage of NAC is that it potentially delays definitive surgical management with radical cystectomy. Despite treatment with NAC, 60% of patients still have muscle-invasive disease at time of cystectomy (Rosenblatt et al. (2012) Pathologic downstaging is a surrogate marker for efficacy and increased survival following neoadjuvant chemotherapy and radical cystectomy for muscle-invasive urothelial bladder cancer. Eur Urol 2012;61 : 1229-38). Patients with MIBC who achieve a pathologic complete response (pCR; pTONOMO stage) or who are down-staged to non muscle-invasive disease after NAC demonstrate longer overall survival (OS) than patients who fail to achieve pCR or are not down-staged (Grossman et al. (2003) Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med 2003;349:859-66; Petrelli et al. (2014) Correlation of pathologic complete response with survival after neoadjuvant chemotherapy in bladder cancer treated with cystectomy: a meta-analysis. Eur Urol 2014;65:350-7). For patients with residual muscle-invasive disease (pT2-4a) or lymph node-positive disease (pN+) at cystectomy, the median survival is only 3.7 and 2.4 years, respectively (Sonpavde et al. (2010) Second-line systemic therapy and emerging drugs for metastatic transitional-cell carcinoma of the urothelium. Lancet Oncol 2010; 11 :861-70).
[0010] Many patients with MIBC choose to undergo definitive surgery (i.e., radical cystectomy) first or remain high-risk pathologically post-NAC. Patients at high risk for recurrence based on pathologic staging are recommended to consider adjuvant chemotherapy given its potential to eradicate micrometastatic disease. For example, both the European Association of Urology (EAU; 2023) and National Comprehensive Cancer Network (NCCN; 2023) guidelines suggest adjuvant cisplatin-based chemotherapy be considered for patients with pT3 T4 or pN+ at surgery as a category 2A recommendation if no neoadjuvant therapy was received (Nadal et al. (2018) Overview of current and future adjuvant therapy for muscle-invasive urothelial carcinoma. Curr Treat Options Oncol 2018;28; 19:36). However, recent studies and meta-analyses (e.g., Vale (2005) Adjuvant chemotherapy in invasive bladder cancer: a systematic review and meta-analysis of individual patient data. Advanced Bladder Cancer (ABC) Meta-Analysis Collaboration. Eur Urol 2005;48: 189-201; Meeks et al. (2012) A systematic review of neoadjuvant and adjuvant chemotherapy for muscle- invasive bladder cancer. Eur Urol 2012;62:523-33; Paz Ares et al. (2010) Randomized phase III trial comparing adjuvant paclitaxel/gemcitabine/cisplatin (PGC) to observation in patients with resected invasive bladder cancer: results of the Spanish Oncology Genitourinary Group (SOGUG) 99/01study [abstract LB A4518], J Clin Oncol 2010;28(Suppl 18):LBA4518; Stadler et al. (2011) Phase III study of molecularly targeted adjuvant therapy in locally advanced urothelial cancer of the bladder based on p53 status. J Clin Oncol 2011;29:3443-9; Cognetti et al. (2012) Adjuvant chemotherapy with cisplatin and gemcitabine versus chemotherapy at relapse in patients with muscle-invasive bladder cancer submitted to radical cystectomy: an Italian, multicenter, randomized phase III trial. Ann Oncol 2012;23:695-700; Sternberg et al. (2015) Immediate versus deferred chemotherapy after radical cystectomy in patients with pT3-pT4 or N+ M0 urothelial carcinoma of the bladder (EORTC 30994): an intergroup, open-label, randomised phase 3 trial. Lancet Oncol 2015;16:76-86) failed to demonstrate that adjuvant chemotherapy improved survival outcomes, even in the highest-risk patients with pathologic extravesical and/or node-positive disease. Thus, the benefit and utility of cisplatin-based adjuvant chemotherapy remains highly controversial.
[0011] Nivolumab has been approved as an adjuvant treatment for patients with MIUC who are at high risk of recurrence after undergoing radical resection (see, e.g., Opdivo® U.S. Package Insert) and for patients with MIUC with tumor cell PD-L1 expression ≥ 1% (see, e.g., Opdivo E.U. Summary of Product Characteristics). However, in the CheckMate 274 trial, approximately half of all patients treated with nivolumab had a DFS event (e.g., recurrence and or metastasis of the UC or death from any cause) within 2 years, which is indicative of still a large unmet need in this population.
[0012] To date, no adjuvant therapies have shown improved survival in MIUC. Once MIUC becomes metastatic, 5-year OS is a dismal 5% (American Cancer Society 2020); therefore, adjuvant treatment of muscle invasive disease is a potentially important opportunity to avoid metastasis and achieve cure.
[0013] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.
SUMMARY
[0014] Provided herein is a method for treating a urothelial carcinoma (UC) in a human patient in need thereof, comprising administering to the patient: (a) an individualized RNA vaccine comprising one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC specimen obtained from the patient, and (b) a PD-1 axis binding antagonist; wherein the RNA vaccine and the PD-1 axis binding antagonist are administered to the patient at least during a priming phase and a booster phase after the priming phase, wherein: (i) the priming phase comprises administering to the patient at least six doses of the RNA vaccine and at least one dose of the PD-1 axis binding antagonist, and (ii) the booster phase comprises administering to the patient at least two doses of the RNA vaccine and at least six doses of the PD-1 axis binding antagonist.
[0015] In some embodiments, the priming phase comprises administering a first dose and a second dose of the at least six doses of the RNA vaccine before administering the at least one dose of the PD-1 axis binding antagonist. In some embodiments, the priming phase comprises administering one dose of the PD-1 axis binding antagonist before administering a third dose of the at least six doses of the RNA vaccine.
[0016] In some embodiments, the UC is a muscle invasive urothelial carcinoma (MIUC). In some embodiments, the MIUC is a muscle invasive bladder cancer (MIBC). In some embodiments, the MIUC is a urinary tract urothelial cancer (UTUC). In some embodiments, the UC is resectable.
[0017] In some embodiments, the priming phase begins at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, or at least about 17 weeks after resection of the UC from the patient. In some embodiments, the priming phase begins at least about 28 days after resection of the UC from the patient. In some embodiments, the priming phase begins less than about 5 weeks, less than about 6 weeks, less than about 7 weeks, less than about 8 weeks, less than about 9 weeks, less than about 10 weeks, less than about 11 weeks, less than about 12 weeks, less than about 13 weeks, less than about 14 weeks, less than about 15 weeks, less than about 16 weeks, less than about 17 weeks, or less than about 18 weeks after resection of the UC from the patient. In some embodiments, the priming phase begins less than about 124 days after resection of the UC from the patient. In some embodiments, the priming phase begins between about 4 weeks and about 18 weeks after resection of the UC from the patient.
[0018] In some embodiments, the at least one dose of the PD-1 axis binding antagonist in the priming phase is administered at least 24 hours after administration of the second dose of the RNA vaccine. In some embodiments, the priming phase comprises administering at least two doses of the PD-1 axis binding antagonist. In some embodiments, the priming phase comprises administering at two doses of the PD-1 axis binding antagonist.
[0019] In some embodiments, the priming phase comprises administering a dose of the PD- 1 axis binding antagonist on day 9 of a first 28-day Cycle of the priming phase. In some embodiments, the priming phase comprises administering a dose of the PD-1 axis binding antagonist on day 8 of a second 28-day Cycle of the priming phase, ±3 days. In some embodiments, the method further comprises administering a dose of the PD-1 axis binding antagonist on day 8 of a third 28-day Cycle, ±3 days, wherein at least day 1 of the third 28- day cycle is within the priming phase. In some embodiments, the method further comprises administering the PD-1 axis binding antagonist once every four weeks beginning after administration of two doses of the RNA vaccine.
[0020] In some embodiments, the second dose of the PD-1 axis binding antagonist is administered after administration of the third dose of the RNA vaccine. In some embodiments, the second dose of the PD-1 axis binding antagonist is administered after administration of the fifth dose of the RNA vaccine. In some embodiments, the second dose of the PD-1 axis binding antagonist is administered on the same day as administration of the sixth dose of the RNA vaccine. In some embodiments, the second dose of the PD-1 axis binding antagonist is administered approximately 30 minutes after administration of the sixth dose of the RNA vaccine.
[0021] In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist during weeks 2 and week 6 of the priming phase, ±3 days, and wherein the method further comprises administering the PD-1 axis binding antagonist during week 10, ±3 days, timing starting with week 1 of the priming phase. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist on day 9 of a first 28-day Cycle of the priming phase, on day 8 of a second 28-day Cycle of the priming phase, and wherein the method further comprises administering the PD-1 axis binding antagonist on day 8 of a third 28-day Cycle of the priming phase. In some embodiments, the RNA vaccine doses are administered at least 48 hours apart from each other.
[0022] In some embodiments, the priming phase comprises administering 6, 7, or 8 doses of the RNA vaccine. In some embodiments, the priming phase comprises administering 8 doses of the RNA vaccine. In some embodiments, the method comprises administering one dose of the PD-1 axis binding antagonist one day after administration of the second dose of the RNA vaccine. In some embodiments, the method comprises administering the third dose of the RNA vaccine six days, ±3 days, after administration of one dose of the PD-1 axis binding antagonist.
[0023] In some embodiments, neither the RNA vaccine nor the PD-1 axis binding antagonist are administered during the eighth week, ±3 days, of the priming phase. In some embodiments, neither the RNA vaccine nor the PD-1 axis binding antagonist are administered on day 22±3 of the second 28-day Cycle of the priming phase.
[0024] In some embodiments, the eighth dose of the RNA vaccine is administered one week before administration of a third dose of the PD-1 axis binding antagonist. In some embodiments, the sixth dose of the RNA vaccine is administered on the same day as a second dose of the PD-1 axis binding antagonist. In some embodiments, second dose of the PD-1 axis binding antagonist is administered after the sixth dose of the RNA vaccine.
[0025] In some embodiments, neither the RNA vaccine nor the PD-1 axis binding antagonist is administered in weeks 11, 12, or 13, each ±3 days, timing starting with week 1 of the priming phase. In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, 6, 7, and 9, each ±3 days, of the priming phase. In some embodiments, the first, second, third, fourth, fifth, seventh, and eighth doses of the RNA vaccine administered to the patient during the priming phase are not administered on the same day as administration of a dose of the PD-1 axis binding antagonist.
[0026] In some embodiments, the priming phase comprises nine weeks. In some embodiments, the RNA vaccine is administered on day 1 of weeks 1, 2, 3, 4, 5, 6, 7, and 9 of the priming phase, and the PD-1 axis binding antagonist is administered on day 2 of week 2 of the priming phase, on day 1 of week 6 of the priming phase, and on day 1 of week 10, timing starting with week 1 of the priming phase.
[0027] In some embodiments, the method further comprises at least one tumor assessment. In some embodiments, the tumor assessment comprises monitoring for tumor recurrence before, during, and/or after treatment. In some embodiments, the tumor assessment comprises evaluating data. In some embodiments, the tumor assessment comprises evaluating data collected from physical examination of the chest, abdomen, upper urinary tracts, and/or pelvis. In some embodiments, the tumor assessment comprises evaluating imaging data. In some embodiments, the imaging data comprise at least one imaging assessment of the chest, abdomen, upper urinary tracts, and/or pelvis. In some embodiments, the imaging assessment comprises imaging of the upper urinary tracts collected by one or more methods selected from the group consisting of: IVP, CT urography, renal ultrasound with retrograde pyelogram, ureteroscopy, and MRI urogram. In some embodiments, at least a portion of the data is collected on at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 41, 15, 61, 17, 18, 19, 20, or 21 different days. In some embodiments, at least a portion of the data is collected on at least 1 day within ± 1 week of Weeks 12, 24, 36, and/or 48, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected on at least 1 day within ± 2 weeks of Weeks 60, 72, 84, and/or 96, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected on at least 1 day within ± 2 weeks of Weeks 112, 128, and/or 144, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected on at least 1 day within ± 2 weeks of Weeks 168, 192, 216, and/or 240, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected on at least 1 day within ± 2 weeks of Week 288, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected within about 18 weeks prior to administration of the first priming dose of the RNA vaccine. In some embodiments, at least a portion of the data is collected within about 4 weeks prior to administration of the first priming dose of the RNA vaccine. In some embodiments, at least a portion of the data is collected within 1 week prior to administration of the first priming dose of the RNA vaccine. In some embodiments, at least a portion of the data is collected every 12 weeks ± 1 week in the first year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 12 weeks ± 2 weeks in the second year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 16 weeks ± 2 weeks in the third year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 24 weeks ± 2 weeks in the fourth and fifth years after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected in approximately week 288, timing starting with day 1 of the priming phase. In some embodiments, at least a portion of the data is collected within 1 week prior to the administering in the priming phase and every 3 months thereafter for at least 81 weeks, timing starting with day 1 of the priming phase. In some embodiments, at least a portion of the data is collected in week 60, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected in week 84, timing starting with week 1 of the priming phase.
[0028] In some embodiments, the booster phase begins no later than about week 14, timing starting with week 1 of the priming phase. In some embodiments, the booster phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after the last priming phase dose of the RNA vaccine. In some embodiments, the booster phase begins about 6 weeks after administration of the last priming dose of the RNA vaccine. In some embodiments, the booster phase begins about 6 weeks after administration of the 8th priming dose of the RNA vaccine. In some embodiments, the first booster dose of the RNA vaccine is administered 6 weeks after administration of the 8th priming dose of the RNA vaccine.
[0029] In some embodiments, the method comprises administering one dose of the PD-1 axis binding antagonist between the last priming dose of the RNA vaccine and the first booster dose of the RNA vaccine. In some embodiments, there are six weeks between administration of the priming phase and the booster phase. In some embodiments, the weeks between the priming phase and the booster phase are weeks 10-13, timing starting with week 1 of the priming phase. In some embodiments, priming phase comprises 9 weeks, and the booster phase begins no later than week 14, timing starting with week 1 of the priming phase. [0030] In some embodiments, the PD-1 axis binding antagonist is administered every four weeks starting in week 2 and every four weeks thereafter, timing starting with week 1 of the priming phase. In some embodiments, the PD-1 axis binding antagonist is administered every four weeks starting in week 2 and every four weeks thereafter for up to one year, timing starting with week 1 of the priming phase. In some embodiments, the method comprises administration of 13 doses of the PD-1 axis binding antagonist, wherein one dose of the PD-1 axis binding antagonist is administered every 28 days over approximately 1 year. In some embodiments, the PD-1 axis binding antagonist is administered on day 2 of week 2 and on day 1 of weeks 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, and 50, timing starting with week 1 of the priming phase. In some embodiments, the PD-1 axis binding antagonist is not administered during a week selected from the group consisting of the following weeks, timing starting with week 1 of the priming phase: 1, 3, 4, 5, 7, 8, 9, 11, 12, 13, 15, 16, 17, 19, 20, 21, 23, 24, 25, 27, 28, 29, 31, 32, 33, 35, 36, 37, 39, 40, 41, 43, 44, 45, 47, 48, 49, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, and 81. In some embodiments, the PD-1 axis binding antagonist is not administered during any of weeks 1, 3, 4, 5, 7, 8, 9, 11, 12, 13, 15, 16, 17, 19, 20, 21, 23, 24,
25, 27, 28, 29, 31, 32, 33, 35, 36, 37, 39, 40, 41, 43, 44, 45, 47, 48, 49, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or
81, timing starting with week 1 of the priming phase. In some embodiments, the PD-1 axis binding antagonist is not administered during any of weeks 1, 3, 4, 5, 7, 8, 9, 11, 12, 13, 15, 16, 17, 19, 20, 21, 23, 24, 25, 27, 28, 29, 31, 32, 33, 35, 36, 37, 39, 40, 41, 43, 44, 45, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or later, timing starting with week 1 of the priming phase. In some embodiments, the one dose of the PD-1 axis binding antagonist that is administered between the priming phase and the booster phase is administered one week after the last booster dose of the RNA vaccine.
[0031] In some embodiments, the RNA vaccine is not administered between the priming phase and the booster phase. In some embodiments, one dose of the PD-1 axis binding antagonist is administered between the priming phase and the booster phase, and no doses of the RNA vaccine are administered between the priming phase and the booster phase.
[0032] In some embodiments, the booster phase begins in week 14, timing starting with week 1 of the priming phase. In some embodiments, the booster phase begins on day 1 of week 14, timing starting with day 1 week 1 of the priming phase.
[0033] In some embodiments, the booster phase comprises administering a first booster dose of the RNA vaccine on day 1 of week 14. In some embodiments, the booster phase comprises administering 2, 3, or 4 booster doses of the RNA vaccine. In some embodiments, the booster phase comprises administering a second booster dose of the RNA vaccine on day 1 of week 38. In some embodiments, the booster phase comprises administering 3 or 4 booster doses of the RNA vaccine. In some embodiments, the method comprises conducting a tumor assessment comprising evaluating data collected within ± 2 weeks of week 60, and administering the third booster dose of the RNA vaccine after the tumor assessment comprising evaluating data collected within ± 2 weeks of week 60, timing starting with week 1 of the priming phase. In some embodiments, the method comprises administering 4 booster doses of the RNA vaccine. In some embodiments, the method comprises conducting a tumor assessment comprising evaluating data collected within ± 2 weeks of week 84, and administering the fourth booster dose of the RNA vaccine after the tumor assessment comprising evaluating data collected within ± 2 weeks of week 84, timing starting with week 1 of the priming phase.
[0034] In some embodiments, the booster phase comprises administering 6, 7, 8, 9, or 10 doses of the PD-1 axis binding antagonist. In some embodiments, the booster phase comprises administering 10 doses of the PD-1 axis binding antagonist. In some embodiments, the booster phase comprises administering 10 doses of the PD-1 axis binding antagonist and 4 doses of the RNA vaccine. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist once every four weeks for up to 10 administrations. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and every four weeks thereafter. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and every four weeks thereafter for up to one year after the first administration of the PD-1 axis binding antagonist.
[0035] In some embodiments, the booster phase begins in week 14 on day 8 of Cycle 4 and comprises days 8-28 of Cycle 4 and at least the nine 28-day Cycles thereafter, and the booster phase comprises administering the RNA vaccine on day 8 of Cycles 4 and 10 and administering the PD-1 axis binding antagonist on day 8 of Cycles 4-13, timing starting with Cycle 1 beginning on week 1 day 1 of the priming phase. In some embodiments, the booster phase begins in week 14 on day 8 of Cycle 4 and comprises days 8-28 of Cycle 4 and the seventeen 28-day Cycles thereafter, the booster phase comprises administering the RNA vaccine on day 8 of Cycles 4, 10, 16, and 21, and the booster phase comprises administering the PD-1 axis binding antagonist on day 8 of Cycles 4-13, timing starting with Cycle 1 beginning on week 1 day 1 of the priming phase. In some embodiments, the PD-1 axis binding antagonist is not administered in Cycles 14-21. [0036] In some embodiments, administrations of the RNA vaccine and the PD-1 axis binding antagonist during Cycles 4 and 10 of the booster phase occur on the same day. In some embodiments, the PD-1 axis binding antagonist is administered approximately 30 minutes after administration of the RNA vaccine during Cycles 4 and 10, timing starting with Cycle 1 of the priming phase.
[0037] In some embodiments, the booster phase comprises 68 weeks. In some embodiments, the RNA vaccine and the PD-1 axis binding antagonist are administered in weeks 1 and 25 of the booster phase, timing starting with administration of the first booster dose of the RNA vaccine. In some embodiments, the administrations of the RNA vaccine and the PD-1 axis binding antagonist in weeks 1 and 25 of the booster phase occur on the same day, timing starting with administration of the first booster dose of the RNA vaccine. In some embodiments, the RNA vaccine and the PD-1 axis binding antagonist are administered on day 1 of weeks 1 and 25 of the booster phase, timing starting with administration of the first booster dose of the RNA vaccine. In some embodiments, the PD-1 axis binding antagonist is administered approximately 30 minutes after administration of the RNA vaccine in weeks 1 and 25 of the booster phase, timing starting with administration of the first booster dose of the RNA vaccine.
[0038] In some embodiments, the RNA vaccine is administered in weeks 1, 25, 48, and 68 of the booster phase, and the PD-1 axis binding antagonist is administered in weeks 1 and 25 of the booster phase, timing starting with administration of the first booster dose of the RNA vaccine. In some embodiments, the RNA vaccine is administered on day 1 of weeks 1, 25, 48, and 68 of the booster phase, and the PD-1 axis binding antagonist is administered on day 1 of weeks 1 and 25 of the booster phase, timing starting with administration of the first booster dose of the RNA vaccine. In some embodiments, the RNA vaccine is not administered in weeks 2-24 of the booster phase, timing starting with administration of the first booster dose of the RNA vaccine. In some embodiments, the PD-1 axis binding antagonist is not administered in weeks 2-3 or after week 25 of the booster phase, timing starting with administration of the first booster dose of the RNA vaccine.
[0039] In some embodiments, the booster phase comprises administering a dose of the RNA vaccine approximately 15 months after administration of the first dose of the PD-1 axis binding antagonist in the priming phase. In some embodiments, the booster phase comprises administering the third booster dose of the RNA vaccine approximately 15 months after administration of the first dose of the PD-1 axis binding antagonist in the priming phase. In some embodiments, the booster phase comprises administering a dose of the RNA vaccine approximately 21 months after administration of the first dose of the PD-1 axis binding antagonist in the priming phase. In some embodiments, the booster phase comprises administering the fourth dose of the RNA vaccine approximately 21 months after administration of the first dose of the PD-1 axis binding antagonist in the priming phase. [0040] In some embodiments, one or more doses of the RNA vaccine in the booster phase and/or the priming phase are missed, and/or one or more doses of the PD-1 axis binding antagonist in the booster phase and/or the priming phase are missed, due to treatment delay due to toxicity. In some embodiments, one or more make-up doses of the RNA vaccine are administered to make up for a missed booster dose or missed priming dose of the RNA vaccine. In some embodiments, one or more make-up priming dose of the RNA vaccine are administered. In some embodiments, one or more make-up priming dose of the RNA vaccine are administered no more frequently than weekly, ±2 days.
[0041] In some embodiments, (a) the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, 6, 7, and 9 of the priming phase, and administering the PD-1 axis binding antagonist on day 2 of week 2 and on day 1 of week 6 of the priming phase; (b) a third dose of the PD-1 axis binding antagonist is administered on day 1 of week ten; and (c) the booster phase comprises administering (i) a first booster dose of the RNA vaccine on day 1 of week 14, a second booster dose of the RNA vaccine on day 1 of week 38, a third booster dose of the RNA vaccine approximately 15 months after the first priming phase administration of the PD-1 axis binding antagonist, and a fourth booster dose of the RNA vaccine in approximately 21 months after the first priming phase administration of the PD-1 axis binding antagonist; and (ii) the PD-1 axis binding antagonist on day 1 of weeks 14, 18, 22, 26, 30, 34, 38, 42, 46, and 50; wherein timing starts with week 1 day 1 of the priming phase.
[0042] In some embodiments, (a) the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, 6, 7, and 9 of the priming phase, and administering the PD-1 axis binding antagonist on day 2 of week 2 and on day 1 of week 6 of the priming phase; (b) a third dose of the PD-1 axis binding antagonist is administered on day 1 of week ten; (c) the booster phase comprises administering (i) a first booster dose of the RNA vaccine on day 1 of week 14, a second booster dose of the RNA vaccine on day 1 of week 38, a third booster dose of the RNA vaccine within approximately weeks 58-66, and a fourth booster dose of the RNA vaccine within approximately weeks 82-92; and (ii) the PD-1 axis binding antagonist on day 1 of weeks 14, 18, 22, 26, 30, 34, 38, 42, 46, and 50; and (d) tumor assessments comprising evaluating physical examination and/or imaging data collected approximately every 3 months for at least about 21 months, starting with day 1 of the priming phase; wherein timing starts with week 1 day 1 of the priming phase.
[0043] In some embodiments, the priming phase begins between about 4 weeks and about 18 weeks after resection of the UC from the patient.
[0044] In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD-1 antibody is nivolumab.
[0045] In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody is avelumab or durvalumab. In some embodiments, the anti-PD-Ll antibody comprises: (a) a heavy chain variable region (VH) that comprises an HVR-H1 comprising an amino acid sequence GFTFSDSWIH (SEQ ID NO: 1), an HVR-H2 comprising an amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO:2), and HVR-H3 comprising an amino acid sequence RHWPGGFDY (SEQ ID NO:3), and (b) a light chain variable region (VL) that comprises an HVR-L1 comprising an amino acid sequence RASQDVSTAVA (SEQ ID NO:4), an HVR-L2 comprising an amino acid sequence SASFLYS (SEQ ID NO:5), and an HVR-L3 comprising an amino acid sequence QQYLYHPAT (SEQ ID NO:6). In some embodiments, the anti-PD-Ll antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO:7 and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO:8. In some embodiments, the anti-PD-Ll antibody is atezolizumab.
[0046] In some embodiments, the anti-PD-1 antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 11, and a light chain comprising an amino acid sequence of SEQ ID NO: 12.
[0047] In some embodiments, the PD-1 axis binding antagonist is administered intravenously to the patient. In some embodiments, the anti-PD-1 antibody is administered to the patient at a dose of about 480 mg. In some embodiments, the anti-PD-1 antibody is nivolumab, and the nivolumab is administered intravenously to the patient at a dose of about 480 mg.
[0048] In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding at least 5 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids. In some embodiments, the one or more polynucleotides of the RNA vaccine and the one or more lipids form a lipid nanoparticle. In some embodiments, the one or more polynucleotides of the RNA vaccine and the one or more lipids form a lipoplex. In some embodiments, the lipoplex comprises one or more lipids that form a multilamellar structure that encapsulates the one or more polynucleotides of the RNA vaccine. In some embodiments, the one or more lipids comprise at least one cationic lipid and at least one helper lipid. In some embodiments, the one or more lipids comprise (R) N,N,N- trimethyl-2,3-dioleyloxy-l-propanaminium chloride (DOTMA) and 1,2-di oleoyl -sn-glycero- 3 -phosphoethanolamine (DOPE). In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipid nanoparticle or lipoplex is 1.3:2 (0.65). In some embodiments, the one or more polynucleotides of the RNA vaccine are RNA molecules, optionally messenger RNA molecules.
[0049] In some embodiments, the RNA vaccine is administered to the patient at a dose of about 15 pg, about 21 pg, about 21.3 pg, about 25 pg, about 38 pg, or about 50 pg. In some embodiments, the RNA vaccine is administered to the patient at a dose of about 25 pg. In some embodiments, the RNA vaccine is administered intravenously to the patient.
[0050] In some embodiments, the RNA vaccine comprises an RNA molecule comprising, in the 5’->3’ direction: (1) a 5’ cap; (2) a 5’ untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding the one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; (5) a polynucleotide sequence encoding at least a portion of a transmembrane and cytoplasmic domain of a major histocompatibility complex (MHC) molecule; (6) a 3’ UTR comprising: (a) a 3’ untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (7) a poly(A) sequence. In some embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker; wherein the polynucleotide sequences encoding the amino acid linker and a first of the one or more neoepitopes form a first linker-neoepitope module; and wherein the polynucleotide sequences forming the first linker-neoepitope module are between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule in the 5’->3’ direction. In some embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 39). In some embodiments, the polynucleotide sequence encoding the amino acid linker comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO:37). In some embodiments, the RNA molecule further comprises, in the 5’->3’ direction: at least a second linker-neoepitope module, wherein the at least second linker-neoepitope module comprises a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope; wherein the polynucleotide sequences forming the second linker- neoepitope module are between the polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule in the 5’->3’ direction; and wherein the neoepitope of the first linker-neoepitope module is different from the neoepitope of the second linker-neoepitope module.
[0051] In some embodiments, the RNA molecule comprises 5 linker-neoepitope modules, and the 5 linker-neoepitope modules each encode a different neoepitope. In some embodiments, the RNA molecule comprises 10 linker-neoepitope modules, and the 10 linker- neoepitope modules each encode a different neoepitope. In some embodiments, the RNA molecule comprises 20 linker-neoepitope modules, and the 20 linker-neoepitope modules each encode a different neoepitope.
[0052] In some embodiments, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is between the polynucleotide sequence encoding the neoepitope that is most distal in the 3’ direction and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule.
[0053] In some embodiments, the 5’ cap of the RNA molecule comprises a DI diastereoisomer of the structure:
[0054] In some embodiments, the 5’ UTR of the RNA molecule in the RNA vaccine comprises the sequence UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO:23). In some embodiments, the 5’ UTR comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID N0:21). In some embodiments, the RNA vaccine comprises an RNA molecule that comprises a secretory signal peptide comprising the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO:27). In some embodiments, the RNA vaccine comprises an RNA molecule that comprises a polynucleotide sequence encoding a secretory signal peptide that comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGC CCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO:25). In some embodiments, the at least one portion of the transmembrane and cytoplasmic domain of the MHC molecule comprises the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO:30). In some embodiments, the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule comprises the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU GACAGCC (SEQ ID NO:28). In some embodiments, the 3’ untranslated region of the AES mRNA comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCC GAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACU CACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO:33). In some embodiments, the method comprises the non-coding RNA of the mitochondrially encoded 12S RNA comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGG AAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO:35). In some embodiments, the 3’ UTR comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGG UACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGC CCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCA GCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACC UUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCA AUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID N0:31). In some embodiments, the poly(A) sequence comprises 120 adenine nucleotides.
[0055] In some embodiments, the RNA vaccine comprises an RNA molecule comprising, in the 5’->3’ direction: the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAU GAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCC UGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19); a polynucleotide sequence encoding the one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU GACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCU UUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUC CCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGC ACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACA GCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACC CCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCU AGCCGCGUCGCU (SEQ ID NO:20).
[0056] In some embodiments, the RNA vaccine comprises autogene cevumeran.
[0057] In some embodiments, the RNA vaccine is administered in a liquid composition that is formulated for direct administration to the patient without dilution. In some embodiments, the composition further comprises sodium chloride at a concentration of about 10 mM or less, a stabilizer at a concentration of more than about 10% weight by volume percent (% w/v) and less than about 15% weight by volume percent (% w/v), and a buffer. In some embodiments, the concentration of salt and/or stabilizer in the composition is at about the value required for physiological osmolality. In some embodiments, the stabilizer is a carbohydrate selected from a monosaccharide, a disaccharide, a tri saccharide, a sugar alcohol, an oligosaccharide or its corresponding sugar alcohol, and a straight chain polyalcohol. In some embodiments, the the stabilizer is sucrose or trehalose, optionally wherein the sucrose at a concentration from about 12 to about 14% (w/v). In some embodiments, the buffer is selected from the group consisting of 2-[4-(2 -hydroxy ethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), histidine, acetic acid/sodium acetate, and MES (2-(N-morpholino)ethanesulfonic acid). [0058] In some embodiments, the UC is MIBC with a tumor stage of (y)pT3-T4a or (y)pN+ and MO upon cystectomy prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the UC is UTUC with an upper tract tumor stage of (y)pT3-T4 or (y)pN+ and MO upon nephroureterectomy prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the UC is a resectable MIUC identified based on a CT scan as cT3-T4 or N+ prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the UC is a resectable MIUC identified at surgical resection prior to administration of the RNA vaccine and the PD-1 axis binding antagonist as tumor stage of (y)pT3-4a or (y)pN+ and MO. In some embodiments, the UC is a resectable MIBC with pathological staging of (y)pT3-4a or (y)pN+ at cystectomy with or without administration of platinum-based neoadjuvant chemotherapy (NAC) treating the UC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist; and/or the UC is a resectable UTUC with pathological staging of (y)pT3-4 or (y)pN+ at nephroureterectomy with or without platinum-based NAC treating the UC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0059] In some embodiments, the patient received neither platinum-based NAC and nor prior cisplatin-based adjuvant chemotherapy for treating the UC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0060] In some embodiments, the cystectomy comprises radical cystectomy. In some embodiments, the radical cystectomy comprises bilateral pelvic lymphadenectomy.
[0061] In some embodiments, the UC is a resectable UTUC with pathological staging of (y)pT3-4 or (y)pN+ and M0 prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, the UC is a resectable MIUC comprising one or more characteristics selected from the group consisting of: having been histologically confirmed as muscle-invasive UC of the bladder or upper urinary tract, wherein patients with mixed or variant histologies have a dominant urothelial pattern; a TNM classification at pathological examination of surgical resection specimen as tumor stage of (y)pT3-4a or (y)pN+ and M0; a TNM classification at pathological examination of surgical UTUC resection specimen as tumor stage of (y)pT3-4 or (y)pN+ and M0; PD-L1 expression per PD-L1 IHC 28-8 pharmDx absence of metastatic disease; and absence of residual disease. In some embodiments, the UC is a resectable MIBC or resectable UTUC upper tract, wherein, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, the patient comprises one or more characteristics selected from the group consisting of: having recovered from a surgical resection of muscle-invasive UC of the bladder; having recovered from a radical cystectomy or radical nephroureterectomy performed by the open, laparoscopic, or robotic approach, wherein the radical cystectomy included bilateral lymph node dissection, and optionally wherein the radical cystectomy extended at a minimum from the mid common iliac artery proximally to Cooper's ligament distally, laterally to the genitofemoral nerve, and/or inferiorly to the obturator nerve; a negative surgical margin at the distal ureteral or urethral margin; and carcinoma in situ (CIS) at the distal ureteral or urethral margin. In some embodiments, the UC is a resectable UTUC, wherein, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, the patient has a radical nephroureterectomy (RNU) with excision of the bladder cuff, wherein the RNU was performed by the open or laparoscopic approach, wherein the RNU must included lymph node dissection (LND), optionally wherein the LND included para-aortic, paracaval and/or interaortocaval nodes from the renal hilum to the inferior mesenteric artery in renal pelvis and proximal ureteral tumors, or nodes from the renal hilum to the bifurcation of the common iliac artery and ipsilateral pelvic nodes in mid and lower ureteral tumors, respectively. In some embodiments, the UC has a negative surgical margin prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0062] In some embodiments, the patient has not received (a) platinum-based neoadjuvant chemotherapy comprising at least three cycles of a platinum-containing regimen; or (b) cisplatin-based adjuvant chemotherapy, for the UC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, the patient comprises one or more characteristics selected from the group consisting of: impaired renal function; hearing loss; Grade 2 or greater peripheral neuropathy; recovery from cystectomy or nephroureterectomy within 120 days following surgery; Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; ECOG performance status of 2, wherein the patient has not received cisplatin based neoadjuvant chemotherapy and is ineligible for cisplatin adjuvant chemotherapy agreement to remain abstinent or use contraception, wherein the patient is female; agreement to remain abstinent or use a condom, and agreement to refrain from donating sperm, wherein the patient is female; and age 18 years or older. In some embodiments, within 14 days prior to administration of the RNA vaccine, the patient comprises one or more hematologic and/or end-organ function characteristics selected from the group consisting of: ANC ≥ 1000 cells/pL; WBC > 2000/pL; platelet count ≥ 100,000/pL; hemoglobin ≥ 9.0 g/dL, optionally wherein the patient is transfused or receiving erythropoietic treatment; AST, ALT < 3.0 x the upper limit of normal (ULN); serum bilirubin < 1.5 x ULN; serum bilirubin < 3 x ULN, wherein the patient has been diagnosed with Gilbert disease; PTT/PT < 1.5 x ULN or INR < 1.7 x ULN, wherein the patient is not receiving therapeutic anticoagulation; serum creatinine < 1.5 x ULN; creatinine clearance (CrCl) ≥ 30 mL/min; negative HIV test at screening; positive HIV test at screening, wherein the patient is stable on anti-retroviral therapy, has a CD4 count of ≥ 200/ pL, and has an undetectable viral load; no evidence of active hepatitis B; a negative HBsAg test and a positive total hepatitis B core antibody (HBcAb) test, wherein a hepatitis B virus (HBV) DNA test demonstrate absence of active infection; negative hepatitis C virus (HCV) antibody test; and positive HCV antibody test followed by a negative HCV RNA test.
[0063] In some embodiments, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, a tumor specimen is prepared from a pretreatment tumor biopsy or surgical resection, wherein the tumor specimen comprises one or more characteristics selected from the group consisting of: being a formalin-fixed paraffin-embedded (FFPE); being in paraffin blocks; being an intact FFPE tumor block; comprising at least about 10 slides comprising unstained, freshly cut serial sections derived from an FFPE tumor block; having an associated pathology report; evaluable for tumor PD-L1 expression; at least 5 identified cancer-specific neoepitopes; good quality based on total and viable tumor content; and comprising a muscle invasive component. In some embodiments, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, a tumor specimen is prepared from a pretreatment tumor biopsy or surgical resection, wherein the tumor specimen comprises at least 5 identified cancer-specific neoepitopes. In some embodiments, the pretreatment tumor biopsy is a TURBT or wherein the surgical resection is a cystectomy or a nephroureterectomy. In some embodiments, the surgical resection is a radical cystectomy or a radical nephroureterectomy. In some embodiments, the method further comprises preparing one or more additional tissue samples taken at one or more additional times or anatomical sites.
[0064] In some embodiments, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, a post- TURBT or post-surgery matched blood sample is prepared from the patient. In some embodiments, within about four weeks prior to administration of the RNA vaccine, the patient comprises absence of residual disease and absence of metastasis, as confirmed by a negative baseline computed tomography (CT) or magnetic resonance imaging (MRI) scan of the pelvis, abdomen, and chest. In some embodiments, the UC is MIBC, and wherein imaging of the upper urinary tracts is completed no more than about four weeks prior to administration of the RNA vaccine and includes intravenous pyelogram (IVP), CT urography, renal ultrasound with retrograde pyelogram, ureteroscopy, and/or MRI urogram. In some embodiments, the UC is UTUC, wherein cystoscopy and urine cytology are completed no more than about four weeks prior to administration of the RNA vaccine and include upper tract imaging, wherein absence of contralateral disease is confirmed. In some embodiments, the UC is both primary MIBC and primary UTUC, wherein upper tract imaging and urine cytology are completed no more than about four weeks prior to administration of the RNA vaccine and include upper tract imaging, wherein absence of contralateral disease is confirmed.
[0065] In some embodiments, at least five neoepitopes resulting from cancer-specific somatic mutations are present in the tumor specimen obtained from the patient prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0066] In some embodiments, the patient is not pregnant, breastfeeding, or intending to become pregnant during the administration or within 28 days after the final dose of the RNA vaccine or within 5 months after the final dose of the PD-1 axis binding antagonist. In some embodiments, the patient is female and has a negative serum pregnancy test result within 14 days prior to administration of the RNA vaccine.
[0067] In some embodiments, the patient does not have a partial cystectomy in the setting of a bladder cancer primary tumor or a partial nephrectomy in the setting of a renal pelvis primary tumor. In some embodiments, the patient does not have any approved anti -cancer therapy, including chemotherapy, or hormonal therapy, excluding hormone-replacement therapy and oral contraceptives, within 3 weeks prior to administration of the RNA vaccine. In some embodiments, the patient does not have any neoadjuvant immunotherapy prior to administration of the RNA vaccine. In some embodiments, the patient does not have adjuvant chemotherapy or radiation therapy for UC following surgical resection prior to administration of the RNA vaccine. In some embodiments, the patient received primary chemoradiation for bladder preservation before cystectomy or before nephroureterectomy. In some embodiments, the patient has UTUC and does not have antegrade or retrograde instillation of chemotherapy or BCG prior to administration of the RNA vaccine. In some embodiments, the patient has a single dose of intravesical chemotherapy post nephroureterectomy prior to administration of the RNA vaccine. In some embodiments, the patient is not treated with an investigational agent that is not an individualized RNA vaccine and/or a PD-1 axis binding antagonist within about one month or five half-lives of the investigational agent, whichever is longer, prior to administration of the RNA vaccine. In some embodiments, the patient is not diagnosed with a malignancy other than UC within 5 years prior to administration of the RNA vaccine. In some embodiments, the patient is not diagnosed with UTUC with tumor stage (y)pT3-4 or ypN+ within 5 years prior to administration of the RNA vaccine.
[0068] In some embodiments, the patient is diagnosed with localized prostate cancer with tumor stage <T2b, Gleason score <7 and treated with curative intent and without prostatespecific antigen (PSA) recurrence within 5 years prior to administration of the RNA vaccine. In some embodiments, the localized prostate cancer has a PSA <20 ng/mL at prostate cancer diagnosis. In some embodiments, the patient is diagnosed with prostate cancer with tumor stage Tl/T2a, Gleason score <7 and PSA <10 ng/mL, and is treatment-naive and undergoing active surveillance, within 5 years prior to administration of the RNA vaccine.
[0069] In some embodiments, the patient is diagnosed with one or more malignancies of a negligible risk of metastasis or death, wherein the malignancy is treated with expected curative intent, and wherein there is no evidence of recurrence or metastasis by follow-up imaging and any disease-specific tumor markers, within 5 years prior to administration of the RNA vaccine. In some embodiments, the negligible risk of metastasis or death comprises risk of metastasis or death <5% at 5 years. In some embodiments, the malignancy comprises carcinoma in situ of the cervix, basal or squamous cell skin cancer, and/or ductal carcinoma, and wherein treatment with expected curative intent comprises surgical treatment.
[0070] In some embodiments, the patient does not have a major surgical procedure, other than for diagnosis or for resection of UC, within < 6 weeks prior to prior to administration of the RNA vaccine. In some embodiments, the patient does not have an anticipated need for a major surgical procedure for about 6 years following initiation of the priming phase. In some embodiments, the patient has a central venous access catheter placed within about 5 years prior to administration of the RNA vaccine. In some embodiments, the patient does not have significant cardiovascular disease within 3 months prior to administration of the RNA vaccine. In some embodiments, the significant cardiovascular disease is a New York Heart Association Class II or greater cardiac disease, a myocardial infarction, or a cerebrovascular accident. In some embodiments, the patient does not have unstable arrhythmia. In some embodiments, the patient does not have unstable angina. In some embodiments, the patient does not have clinically significant liver disease. In some embodiments, the clinically significant liver disease comprises an active viral disease, alcoholic or other hepatitis, cirrhosis, and/or inherited liver disease. In some embodiments, the patient does not exhibit alcohol abuse. In some embodiments, the patient does not have an autoimmune disease or immune deficiency or a history thereof. In some embodiments, the autoimmune disease or immune deficiency comprises myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, antiphospholipid antibody syndrome, granulomatosis with polyangiitis, Sjogren syndrome, Guillain-Barre syndrome, and/or multiple sclerosis. In some embodiments, the patient has a history of autoimmune-related hypothyroidism and is on thyroid replacement hormone. In some embodiments, the patient has controlled Type 1 diabetes mellitus and is on an insulin regimen. In some embodiments, the patient does not have psoriatic arthritis. In some embodiments, the patient has been diagnosed with a disease selected from the group consisting of eczema, psoriasis, lichen simplex chronicus, and vitiligo; wherein the disease has only dermatologic manifestations; wherein the patient does not have a rash covering >10% of body surface area; wherein the disease is well-controlled upon initiation of the priming phase and requires only low-potency topical corticosteroids; and wherein the disease is not treated with psoralen plus ultraviolet A radiation, methotrexate, retinoids, biologic agents, oral calcineurin inhibitors, and/or high potency or oral corticosteroids within 12 months prior to administration of the RNA vaccine.
[0071] In some embodiments, the patient does not have one or more of a known cellular primary immunodeficiency or a known combined T- and/or B-cell immunodeficiency. In some embodiments, the cellular primary immunodeficiency comprises DiGeorge syndrome and/or T-negative severe combined immunodeficiency (SCID). In some embodiments, the combined T- and/or B-cell immunodeficiency comprises T- and B-negative SCID, Wiskott- Aldrich syndrome, ataxia telangiectasia, and/or common variable immunodeficiency.
[0072] In some embodiments, the patient does not have ongoing treatment with monoamine oxidase inhibitors (MAOIs). In some embodiments, the patient is not treated with monoamine oxidase inhibitors (MAOIs) within 3 weeks prior to administration of the RNA vaccine. In some embodiments, the patient is not treated with a systemic immunostimulatory agent within 4 weeks or 5 drug-elimination half-lives, whichever is longer, prior to administration of the RNA vaccine. In some embodiments, the systemic immunostimulatory agent comprises interferon and/or IL-2. In some embodiments, the patient is not treated with a systemic immunosuppressive medication within 2 weeks prior to administration of the first priming dose of the RNA vaccine, or wherein the patient does not have anticipated need for systemic immunosuppressive medication for about 6 years following initiation of the priming phase. In some embodiments, the systemic immunosuppressive medication comprises a corticosteroid, a cyclophosphamide, an azathioprine, a methotrexate, a thalidomide, and/or an anti-TNF agent. [0073] In some embodiments, the patient receives acute, low-dose systemic immunosuppressant medication and/or a one-time pulse dose of systemic immunosuppressant medication within 2 weeks prior to administration of the first priming dose of the RNA vaccine. In some embodiments, the one-time pulse dose of systemic immunosuppressant medication comprises 48 hours of corticosteroids for a contrast allergy. In some embodiments, the patient receives one or more of a mineralocorticoid, an inhaled or low dose corticosteroid for chronic obstructive pulmonary disease or asthma, and/or a low-dose corticosteroids for orthostatic hypotension or adrenal insufficiency, within 2 weeks prior to administration of the first priming dose of the RNA vaccine. In some embodiments, the mineralocorticoid comprises fludrocortisone, and/or wherein the inhaled or low dose corticosteroid comprises <10 mg oral prednisone per day or daily equivalent.
[0074] In some embodiments, the patient does not have one or more of a characteristic selected from the group consisting of: a history of idiopathic pulmonary fibrosis; a history of organizing pneumonia; a history of drug-induced pneumonitis; a history of idiopathic pneumonitis; a history of severe allergic anaphylactic reactions to chimeric or humanized antibodies or fusion proteins; a known hypersensitivity to Chinese hamster ovary cell products; a known hypersensitivity or allergy to a component of a product comprising the RNA vaccine; a known hypersensitivity or allergy to a component of a product comprising the PD-1 axis binding antagonist; evidence of active pneumonitis by chest CT scan within about 1 month prior to administration of the first priming dose of the RNA vaccine; known active or latent tuberculosis; severe infection within 4 weeks prior to administration of the first priming dose of the RNA vaccine; prior allogeneic stem cell or solid organ transplantation; any other disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding that contraindicates the use of the RNA vaccine and/or the PD-1 axis binding antagonist, may affect interpretation results of treatment, and/or may render the patient at high risk from treatment complications. In some embodiments, the organizing pneumonia comprises bronchiolitis obliterans. In some embodiments, the severe infection comprises hospitalization for complications of infection, hospitalization for complications of bacteremia, hospitalization for complications of severe pneumonia, and/or any active infection that could impact patient safety.
[0075] In some embodiments, the patient is not treated with a live, attenuated vaccine within 4 weeks prior to administration of the first priming dose of the RNA vaccine, and/or wherein the patient does not have anticipated need for treatment with a live, attenuated vaccine between initiation of the priming phase and up to about 5 months after the end of the booster phase. In some embodiments, the patient receives an influenza vaccination during influenza season. In some embodiments, the patient does not receive an influenza vaccination not during influenza season between initiation of the priming phase and up to about 5 months after the end of the booster phase. In some embodiments, the patient does not receive an mRNA vaccine within about 7 days prior to administration of the first priming dose of the RNA vaccine. In some embodiments, the mRNA vaccine is a CO VID-19 vaccine.
[0076] In some embodiments, the patient has a known increased risk for infection with Mycobacterium tuberculosis within about 20 weeks prior to administration of the first priming dose of the RNA vaccine, and wherein latent tuberculosis diagnostic procedures are followed prior to administration of the first priming dose of the RNA vaccine. In some embodiments, the patient has a spleen prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has not had loss of spleen due to splenectomy, splenic injury/infarction, or functional asplenia prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0077] In some embodiments, the method further comprises assessing disease-free survival (DFS) of the patient after treatment with the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, administration of the RNA vaccine and the PD-1 axis binding antagonist results in an improvement in DFS of the patient as compared to DFS of a corresponding patient not administered the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the method further comprises assessing overall survival (OS) of the patient after treatment with the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, administration of the RNA vaccine and the PD-1 axis binding antagonist results in an improvement in OS of the patient as compared to OS of a corresponding patient not administered the RNA vaccine and the PD-1 axis binding antagonist.
[0078] In some embodiments, the method further comprises performing one or more clinical assessments of the patient before, during and/or after treatment with the RNA vaccine and the PD-1 axis binding antagonist, wherein the one or more clinical assessments are selected from the group consisting of European Organisation for Research and Treatment of Cancer QLQ-C30 Questionnaire (EORTC QLQ-C30), European Organisation for Research and Treatment of Cancer QLQ-PAN26 Questionnaire (EORTC QLQ PAN26), National Cancer Institute’s Patient-Reported Outcomes Common Terminology Criteria for Adverse Events (PRO CTCAE), and European Organisation for Research and Treatment of Cancer Item Library 46 Questionnaire (EORTC IL46). In some embodiments, administration of the RNA vaccine and the PD-1 axis binding antagonist results in an improvement in the one or more clinical assessments as compared to the one or more clinical assessments in the patient prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, and/or as compared to the one or more clinical assessments in a corresponding patient not administered the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the method further comprises assessing one or more of the following in the patient before, during, and/or after treatment with the RNA vaccine and the PD-1 axis binding antagonist: a relationship between biomarkers; a level of biomarkers of blood and/or tumor tissue; a change and/or clearance of circulating tumor DNA (ctDNA); a mean and/or mean changes in one or more symptoms other than patient-reported Pain, Physical and Role Functioning, and GHS/QoL scales, as assessed through use of the EORTC QLQ-C30; a health utility and visual analogue scale (VAS) score of an EQ-5D-5L questionnaire; a plasma concentration of DOTMA; a serum concentration of the PD-1 axis binding antagonist; a prevalence of antidrug antibodies (AD As) to the PD-1 axis binding antagonist; and antigen- and/or tumorspecific T-cell responses. In some embodiments, administration of the RNA vaccine and the PD-1 axis binding antagonist treatment results in an improved and/or altered relationship between biomarkers, level of biomarkers of blood and/or tumor tissue, level of ctDNA, symptom assessed by EORTC QLQ-C30, VAS score of an EQ-5D-5L questionnaire, plasma concentration of DOTMA, serum concentration of the PD-1 axis binding antagonist, prevalence of AD As to the PD-1 axis binding antagonist, and/or antigen- and/or tumorspecific T-cell responses in the patient as compared to prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, and/or as compared to a corresponding patient not administered the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the corresponding patient is a patient with a corresponding UC, optionally wherein the UC is a MIUC and the corresponding patient has MIUC, and optionally wherein the UC is a UTUC and the corresponding patient has UTUC. In some embodiments, the corresponding patient was treated with a standard of care treatment for UC, MIUC, UTUC, or resectable or resected UC, MIUC, UTUC. In some embodiments, the standard of care treatment comprises a cystectomy, a nephroureterectomy and/or adjuvant nivolumab. In some embodiments, the UC is MIUC, and wherein cystectomy comprises bilateral pelvic ly mphadenectomy .
[0079] In some embodiments, the RNA vaccine dose is administered to the patient in two equal half-doses. In some embodiments, the two equal half-doses are administered sequentially, optionally with an observation period between the administered equal half- doses. In some embodiments, the dose of about 25 pg is split into two equal half-doses of about 12.5 pg, each administered over 1 minute, optionally with a 5-minute observation period between the administered equal half-doses.
[0080] In one aspect is provided an individualized RNA vaccine for use in a method for treating a urothelial carcinoma (UC) in a human patient in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method of any one of the preceding embodiments, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancerspecific somatic mutations present in a UC specimen obtained from the patient.
[0081] In one aspect is provided a PD-1 axis binding antagonist for use in a method for treating a UC in a human patient in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method of any one of the preceding embodiments, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC specimen obtained from the patient.
[0082] In one aspect is provided a use of an individualized RNA vaccine in the manufacture of a medicament for treating a UC in a human patient in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method of any one of the preceding embodiments, and wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC specimen obtained from the patient. [0083] In one aspect is provided a use of a PD-1 axis binding antagonist in the manufacture of a medicament for treating a UC in a human patient in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method of any one of the preceding embodiments, and wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC specimen obtained from the patient.
[0084] In one aspect is provided a kit comprising an individualized RNA vaccine, for use in a method for treating a UC in a human patient in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method of any one of the preceding embodiments, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC specimen obtained from the patient. [0085] In one aspect is provided a kit comprising a PD-1 axis binding antagonist for use in a method for treating a UC in a human patient in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method of any one of the preceding embodiments, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC specimen obtained from the patient.
[0086] In some embodiments, prior to the administering step, the patient is selected by a method comprising: obtaining a tumor specimen from the patient and administering a radical surgical resection of the UC, and (a) administering a CT scan prior to the radical surgical resection and, from the CT scan, identifying the UC as having tumor stage cT3-T4 or N+; and/or (b) from the radical surgical resection, identifying the UC as having tumor stage of (y)pT3-4a or (y)pN+ and MO, wherein the UC is MIBC and wherein the radical surgical resection is a radical cystectomy; and/or (c) from the radical surgical resection, identifying the UC as having tumor stage of (y)pT3-4 or (y)pN+ and MO, wherein the UC is UTUC and wherein the radical surgical resection is an RNU; wherein the radical surgical resection is administered no more than about 120 days prior to administration of the RNA vaccine; and wherein the patient has no residual disease or metastases within about 30 days prior to administration of the RNA vaccine. In some embodiments, the tumor specimen is a transurethral resection of the bladder tumor (TURBT) specimen. In some embodiments, the tumor specimen is a surgical resection specimen from cystectomy or from nephroureterectomy obtained no more than about 120 days prior to administration of the RNA vaccine. In some embodiments, the tumor specimen comprises a representative formalin-fixed paraffin-embedded (FFPE) tumor specimen from a pretreatment tumor biopsy prior to the administering step. In some embodiments, the pretreatment tumor biopsy comprises transurethral resection of the bladder tumor (TURBT). In some embodiments, the tumor specimen comprises a representative formalin-fixed paraffin-embedded (FFPE) surgical resection specimen prior to the administering step. In some embodiments, the method further comprises obtaining a post- TURBT or post-surgery matched blood sample from the patient prior to the administering step. In some embodiments, the method comprises identifying at least 5 neoepitopes resulting from cancer-specific somatic mutations in the tumor specimen obtained from the patient.
[0087] In some embodiments, the UC exhibits a nodal stage of N+ within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the UC exhibits a nodal stage of NO within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the UC exhibits a PD-L1 IHC score of <1% within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the UC exhibits a PD-L1 IHC score of ≥1% within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the UC exhibits an indeterminate PD-L1 IHC score within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the patient has received neoadjuvant therapy for treatment of the UC prior to administration of the RNA vaccine. In some embodiments, the patient has not received neoadjuvant therapy for treatment of the UC prior to administration of the RNA vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] FIG. 1 provides a non-limiting schematic of the design of the two-part screening period (Part A and Part B; shown in the top and bottom panels, respectively) and an overview of the treatment scheme for the Phase II study described in Example 1 (bottom right). The top panel shows the treatments, sample collection and analysis, and upstream and downstream manufacturing of the individualized cancer vaccines that occur during Screening Part A. As shown at top, patients with MIUC (either MIBC or UTUC) undergo transurethral resection of the bladder tumor (TURBT), from which blood and tissue samples may be submitted for sequencing and vaccine design (middle left). Following TURBT and before cystectomy (if MIBC) or before nephroureterectomy (if UTUC) (top center), neoadjuvant chemotherapy (NAC) may (top) or may not (top middle) be administered to the patient. Screening Part B (bottom panel) begins after cystectomy/nephroureterectomy. In parallel, the individualized cancer vaccine is designed and manufactured to encode at least 5 cancer-specific neoantigen epitopes (also called neoepitopes or NEs) based on results from whole exome sequencing (WES) and/or RNA sequencing of each patient’s blood and tissue samples. Upstream manufacturing includes WES and RNA sequencing of the blood and tissue samples as part of individualized cancer vaccine design to identify cancer-specific NEs for each patient. The superscriptsa alongside the TURBT and cystectomy/nephroureterectomy labels, and the dashed line under cystectomy/nephroureterectomy, denote that tumor specimens from TURBT are strongly encouraged to be submitted for upstream manufacturing of individualized cancer vaccine, but that patients identified after surgical resection (i.e., after cystectomy/nephroureterectomy) may have either TURBT or surgical resection specimens submitted for individualized cancer vaccine manufacturing. WES results obtained for vaccine design may also be used for subsequent circulating tumor DNA (ctDNA) testing for patients enrolled in Part B. The superscriptb indicates that PD-L1 (programmed death ligand 1) testing will be performed on tissue samples from surgical resection, and that PD-L1 testing may be performed on TURBT samples if there is tissue remaining after sequencing and vaccine design (i.e., TURBT tissue is prioritized for autogene cevumeran upstream manufacturing). The superscriptc indicates that at least 5 cancer-specific NEs are required per patient for eligibility. Following identification of NEs, downstream manufacturing takes place, and patients proceed to Screening Part B after pathological confirmation of disease status from radical cystectomy or radical nephroureterectomy. The bottom panel shows a schematic of Screening Part B and an overview of the treatment scheme. Screening Part B takes place after Screening Part A and before treatment, and includes eligibility criteria screening and stratification and randomization in parallel with individualized cancer vaccine downstream manufacturing in preparation for treatment administration. Part B begins within 120 days post-cystectomy (in the case of patients with MIBC) or within 120 days post- nephroureterectomy (in the case of patients with UTUC) and includes patients with 5 or more NEs and sufficient tumor material and absence of residual disease or metastases within about 28 days before randomization (R). Patients who, at cystectomy, exhibited MIUC of bladder (MIBC) can be included in Screening Part B and beyond if they were found to have pathological staging of (y)pT3-T4a or (y)pN+ and MO at cystectomy. Patients who, at nephroureterectomy, exhibited MIUC of upper tract (UTUC) can be included in Part B if they were found to have pathological staging of (y)pT3-T4 or (y)pN+ and MO at nephroureterectomy, though the number of patients with UTUC will be capped at no more than approximately 10% of the study population. Initially, up to 12 patients are enrolled in a safety run-in phase (bottom middle) in which they receive an individualized cancer vaccine (such as, for instance, autogene cevumeran) and a PD-1 axis binding antagonist (such as, for example, nivolumab). The superscriptd indicates that an Internal Monitoring Committee (IMC) reviews safety data after the first 6 patients have completed Cycle 1, Day 28. After 12 patients have been enrolled in the safety run-in phase or the IMC has determined it is safe to proceed, approximately 350 patients globally are randomized in a 1 : 1 ratio (R 1 : 1) to one of the following arms: arm A (experimental arm; labeled “Autogene cevumeran + nivolumab” in FIG. 1, in which patients will receive an individualized cancer vaccine (such as, for instance, autogene cevumeran) and a PD-1 axis binding antagonist (such as, for example, nivolumab) for 1 year and then booster doses of the individualized cancer vaccine every 6 months (Q6M) until approximately 21 months have elapsed since randomization); or arm B (control arm; labeled “Placebo + nivolumab” in FIG. 1, in which patients will receive a placebo and a PD-1 axis binding antagonist (such as, for example, nivolumab) for 1 year and then booster doses of the placebo every 6 months until approximately 21 months have elapsed since randomization. Randomization will be stratified by the following factors: Pathologic nodal stage (N+ vs. NO), PD-L1 immunohistochemistry (IHC) status (greater than or equal to 1% vs. <1% or indeterminate), and prior neoadjuvant chemotherapy (yes vs. no). Randomization must occur within 120 days after radical surgical resection of the primary tumor (e.g., within 120 days after radical cystectomy, in the case of patients with MIBC; or within 120 days after radical nephroureterectomy (RNU), in the case of patients with UTUC), and administration of the individualized cancer vaccine or placebo in the experimental or control arm, respectively, begins within 3 calendar days after randomization.
[0089] FIGS. 2A-2B provide non-limiting diagrams of the design of the study priming phase and at least a portion of the booster phase for arms 1 and 2 of the study described in Example 1 and shown in the bottom right portion of FIG. 1. FIG. 2A shows that, over the course of, for example, 21 28-day Cycles (e.g., over the course of about 21 months postrandomization), patients in the experimental arm receive, for example, 25 pg of individualized cancer vaccine (such as, for example, autogene cevumeran; annotated as “cevu”) in each of, for example, 8 priming doses during the priming phase, and in each of, for example, four booster doses during the booster phase. Patients in the experimental arm also receive 480 mg of PD-1 axis binding antagonist (such as, for example, nivolumab; annotated as “nivo”) every 4 weeks (Q4W) beginning after administration of the first two priming doses of the individualized cancer vaccine (i.e., on day (D) 9 of Cycle 1 and then on D8 of every cycle thereafter) for up to 1 year of treatment. In parallel, patients in the control arm receive, for example, 25 pg of placebo in each of, for example, 8 “priming” doses during the priming phase, and in each of, for example, four “booster” doses during the booster phase. Patients in the control arm also receive 480 mg of PD-1 axis binding antagonist (such as, for example, nivolumab) Q4W beginning after administration of the first two “priming” doses of the placebo (i.e., on D9 of Cycle 1 and then on D8 of every cycle thereafter) for up to 1 year of treatment. The priming doses of individualized cancer vaccine or placebo occur, for example, at a rate of about one dose per week, except that no priming dose is given the week before the last priming dose. In the scheme shown in FIG. 2A, neither individualized cancer vaccine/placebo nor the PD-1 axis binding antagonist are administered during week 8 (e.g., D22 of Cycle 2) in either arm. The vertical dashed line in week 2 (between days 8-9 of Cycle 1) indicates that that the individualized cancer vaccine (in the experimental arm) or the placebo (in the control arm) in cycle (C) 1 week (W) 2 is administered on a different day (D8) than the PD-1 axis binding antagonist in C1W2, which is administered on D9. Thus, the priming phase includes, for example, 8 doses of individualized cancer vaccine or placebo and at least the first two of 13 planned doses of PD-1 axis binding antagonist over the first 9 weeks of drug administration following randomization. The booster phase and the priming phase are named based on the administration schedule of the individualized cancer vaccine. Thus, there may further be, for example, at least one dose of the PD-1 axis binding antagonist administered between the priming phase and the booster phase, such as, for example, a third dose of the PD-1 axis binding antagonist administered one week after the last priming dose of the individualized cancer vaccine, shown on W10D8 and W9D1, respectively. The booster phase includes, for example, four booster doses of individualized cancer vaccine or placebo, of which the first two are administered on Day 8 of Cycles 4 and 10, respectively. Superscripta indicates that, in the pictured scheme, the booster dose of individualized cancer vaccine or placebo in Cycle 10 / Week 38 is administered on the same day as a dose of the PD-1 axis binding antagonist. The row labeled “Imagingb” indicates that imaging is performed at screening/baseline and every 12 weeks starting from randomization for the first 2 years and then according to the schedule as outlined herein, including at least at about week 60 and about week 84 (asterisks), prior to administration of the third and fourth booster doses, respectively, of individualized cancer vaccine/placebo. FIG. 2B shows an extended timeline of the same events shown in FIG. 2A but without Day or Week labels, and in which the timing of a discontinuation visit (DV) and follow-up are marked. Four booster doses of individualized cancer vaccine or placebo are labeled Bl, B2, B3, and B4, respectively.
DETAILED DESCRIPTION
I. Definitions
[0090] Before describing the invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0091] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.
[0092] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
[0093] The term “adverse event” as used herein refers to any untoward medical occurrence in a patient or clinical study participant temporally associated with the use of a study treatment, whether or not considered related to the study treatment.
[0094] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. [0095] The term “PD-1 axis binding antagonist” refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis - with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.
[0096] The term “PD-1 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- 1 with one or more of its binding partners, such as PD-L1, PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD- 1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 binding antagonists are provided infra.
[0097] The term “PD-L1 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- L1 with either one or more of its binding partners, such as PD-1, B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonists include anti-PD-Ll antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B7-1. In one embodiment, a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L1 so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L1 binding antagonist is an anti-PD-Ll antibody. Specific examples of PD-L1 binding antagonists are provided infra.
[0098] The term “PD-L2 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- L2 with either one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L2 binding antagonist is an immunoadhesin.
[0099] “Sustained response” refers to the sustained effect on reducing tumor growth after cessation of a treatment. For example, the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration at least the same as the treatment duration, at least 1.5X, 2. OX, 2.5X, or 3. OX length of the treatment duration.
[0100] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
[0101] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with cancer are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals.
[0102] As used herein, “delaying progression of a disease” means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late-stage cancer, such as development of metastasis, may be delayed.
[0103] An “effective amount” is at least the minimum amount required to effect a measurable improvement or prevention of a particular disorder. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (z.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (z.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
[0104] As used herein, “in conjunction with” or “in combination with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” or “in combination with” refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.
[0105] A “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
[0106] The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.
[0107] “Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.
[0108] A “subject”, “patient” or an “individual” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
[0109] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
[0110] An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
[0111] “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
[0112] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHI, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
[0113] The “variable region” or “variable domain” of an antibody refers to the aminoterminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
[0114] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
[0115] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“X”), based on the amino acid sequences of their constant domains.
[0116] The term IgG “isotype” or “subclass” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
[0117] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, y, e, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides. [0118] The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
[0119] A “naked antibody” for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
[0120] “Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
[0121] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen -binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. [0122] “Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
[0123] The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. [0124] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.
[0125] The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
[0126] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
[0127] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467- 12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93
(1995).
[0128] The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
[0129] “Humanized” forms of non-human (e.g, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non- human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. [0130] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
[0131] A “species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody “binds specifically” to a human antigen (e.g., has a binding affinity (Kd) value of no more than about 1x1 O'7 M, preferably no more than about IxlO'8 M and preferably no more than about IxlO'9 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
[0132] The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446- 448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0133] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.
Loop Kabat AbM Chothia Contact LI L24-L34 L24-L34 L26-L32 L30-L36
L2 L50-L56 L50-L56 L50-L52 L46-L55
L3 L89-L97 L89-L97 L91-L96 L89-L96
Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering)
Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering)
H2 H50-H65 H50-H58 H53-H55 H47-H58
H3 H95-H102 H95-H102 H96-H101 H93-H101
[0134] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (Hl), 50-65 or 49-65 (H2) and 93- 102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.
[0135] HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (Hl), 50-65 or 49-65 (H2) and 93- 102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.
[0136] “Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.
[0137] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
[0138] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabaf ’ refers to the residue numbering of the human IgGl EU antibody.
[0139] The expression “linear antibodies” refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. [0140] As use herein, the term “binds”, “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < IpM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.
[0141] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and/or individual of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof. In some embodiments, the sample is a sample obtained from the cancer of an individual (e.g., a tumor sample) that comprises tumor cells and, optionally, tumor-infiltrating immune cells. For example, the sample can be a tumor specimen that is embedded in a paraffin block, or that includes freshly cut, serial unstained sections. In some embodiments, the sample is from a biopsy and includes 50 or more viable tumor cells (e.g., from a core-needle biopsy and optionally embedded in a paraffin block; excisional, incisional, punch, or forceps biopsy; or a tumor tissue resection).
[0142] By “tissue sample”, “tissue specimen” or “cell sample” is meant a collection of similar cells obtained from a tissue, for example a tumor, of a subject or individual. The source of the tissue or cell sample may be solid tissue (e.g., a tumor) as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease tissue/organ. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
[0143] A “reference sample”, “reference cell”, “reference tissue”, “control sample”, “control cell”, or “control tissue”, as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual. For example, healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual. In even another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the subject or individual.
[0144] A “reference level”, “reference number”, or “reference frequency”, as used herein, refers to a number, a frequency, an amount, etc. with a predetermined value. In this context, “level” encompasses the absolute number or amount, the relative number or amount, the frequency, the proportion, the percentage, etc. as well as any value or parameter which correlates thereto or can be derived therefrom. As the skilled artisan will appreciate, the reference level, reference number, or reference frequency is predetermined and set to meet routine requirements in terms of e.g., specificity and/or sensitivity. These requirements can vary, e.g., from regulatory body to regulatory body. For example, it may be that assay sensitivity and/or specificity has to be set to certain limits, e.g., 80%, 90%, 95%, 98%, 99%, or 100%. These requirements may also be defined in terms of positive or negative predictive values. Nonetheless, based on the teaching given in the present invention, it will be possible for a skilled artisan to arrive at the reference level, reference number, or reference frequency meeting those requirements. For example, the reference level, reference number, or reference frequency can be determined in reference samples obtained from patients prior to treatment administration or obtained from healthy individuals. The reference level, reference number, or reference frequency in one embodiment has been predetermined in reference samples from the disease entity to which the patient belongs. In certain embodiments, the reference level, reference number, or reference frequency can be statistically calculated or set as determined from the overall distribution of the values in reference samples from a disease entity investigated. In one embodiment the reference level, reference number, or reference frequency is set as a cutoff value as determined from the overall distribution of the values in a disease entity investigated, such that the cutoff value is indicative of the value at which, e.g.x the rate of predicting immunogenicity and/or immune response to the therapy described herein reaches a specificity of 100% and a sensitivity of 80%, The reference level, reference number, or reference frequency may vary from patient to patient or may vary depending on various physiological parameters of the patient such as age, gender, or subpopulation, as well as on the number of times treated or vaccinated and the methods used for the determination of (e.g.) the referred to herein. In one embodiment, the reference sample is from essentially the same type of cells, tissue, organ, or body fluid source as the sample from the individual or patient subjected to the method of the invention, e.g., if according to the invention, blood is used as a sample to determine the level of de novo SE TCR clones in the individual, the reference level, reference number, or reference frequency is also determined in blood or a part thereof.
[0145] An “effective response” of a patient or a patient's “responsiveness” to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer. [0146] A patient who “does not have an effective response” to treatment refers to a patient who does not have any one of extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.
[0147] A patient who is “likely to respond” to a therapy refers to a patient who has been identified based on one or more specific biological features or traits relative to the disease, disorder, or condition (such as cancer) that are correlated with treatment responsiveness (or effective response to treatment). This correlation can be identified statistically such that a patient identified as “likely to respond” can refer to a patient with a calculable statistical probability of likelihood to show an effective response to treatment.
[0148] The phrase “responsive to” in the context of the present invention indicates that a patient suffering from, being suspected to suffer or being prone to suffer from, or diagnosed with a disorder as described herein, shows a positive response to a treatment, for example an individualized RNA vaccine treatment described herein. Treatment response can be defined based on progression free survival (PFS), overall survival (OS), and overall response rate (ORR), including partial response or complete response to treatment.
[0149] The term “individualized” context of the present invention indicates that a therapy or treatment is unique to each patient, for example has been designed for, constructed, or otherwise manufactured based on the specifications, e.g., the genomic profile, immunological profile, metabolic profile, cancer type, cancer antigen profile, somatic mutation profile, age, sex, etc. or the treatment needs of an individual patient. For example, the RNA vaccine of the present disclosure is individualized to each patient such that the individualized RNA vaccine targets one or more neoepitopes resulting from cancer-specific somatic mutations present in, e.g., a UC specimen from each patient that may be unique to each patient.
[0150] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as disclosed, for example, in definitions herein.
[0151] A cancer or biological sample which “has human effector cells” is one which, in a diagnostic test, has human effector cells present in the sample (e.g., infiltrating human effector cells). [0152] A cancer or biological sample which “has FcR-expressing cells” is one which, in a diagnostic test, has FcR-expressing present in the sample (e.g., infiltrating FcR-expressing cells). In some embodiments, FcR is FcyR. In some embodiments, FcR is an activating FcyR. [0153] The phrase “selecting a patient” or “identifying a patient” as used herein refers to using the information or data generated relating to the number and/or frequency of TCR clones (such as significantly expanded (SE) TCR clones, e.g., de novo SE TCR clones) in a sample of a patient to identify or select the patient as likely to benefit from a therapy comprising an individualized RNA vaccine. The information or data used or generated may be in any form, e.g., written, oral, or electronic. In some embodiments, using the information or data generated includes communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, dispensing, or combinations thereof. In some embodiments, communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, dispensing, or combinations thereof are performed by a computing device, analyzer unit or combination thereof. In some further embodiments, communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, dispensing, or combinations thereof are performed by a laboratory or medical professional. In some embodiments, the information or data includes a comparison of the number and/or frequency of TCR clones (such as significantly expanded (SE) TCR clones, e.g., de novo SE TCR clones) to a reference level. In some embodiments, the information or data includes an indication that TCR clones (such as significantly expanded (SE) TCR clones, e.g., de novo SE TCR clones) are present in the sample. In some embodiments, the information or data includes an indication that the patient is more likely to respond to a therapy comprising an individualized RNA vaccine.
IL Methods of Treating Urothelial Carcinoma
[0154] Certain aspects of the present disclosure relate to methods for treating urothelial carcinoma, e.g., MIUC in a patient, such as a human patient in need thereof, by administering to the patient an effective amount of an individualized RNA vaccine and a PD-1 axis binding antagonist. In some embodiments, the individualized RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC specimen from the patient, e.g., as described in greater detail below.
[0155] Any of the individualized RNA vaccines and PD-1 axis binding antagonists described herein may be used in the methods of the disclosure. [0156] The RNA vaccine according to the present disclosure is designed to induce neoantigen- and/or tumor-specific immune responses in the patient, such as neoantigen- specific T cell responses, and may augment the magnitude and quality of pre-existing neoantigen-specific T cells. Blocking the PD-L1/PD-1 pathway in the patient, e.g., by administration of a PD-1 axis binding antagonist, in combination with the RNA vaccine may enhance priming and/or reactivation of T-cells, and/or improve the activity of dysfunctional T-cells after tumor antigen exposure, thereby enhancing RNA vaccine-induced immune responses. Hence, the combination of an RNA vaccine with a PD-1 axis binding antagonist may result in more robust anti-tumor immune responses leading to improved clinical efficacy, particularly for patient populations with high unmet medical needs, such as MIUC patients with residual muscle-invasive disease (pT2-4a) or lymph node-positive disease (pN+) following NAC, MIUC patients with high risk features (e.g., extravesical (pT3-T4a) or pN+) disease with a high risk of relapse) who have not received NAC, MIUC patients who are not eligible for cisplatin adjuvant therapy due to co-morbidities, and patients with high- risk UTUC with similar staging to MIBC (pT3-4 or pN+).
[0157] Thus, administering the RNA vaccine during a priming phase (including, e.g., at least 6 priming RNA vaccine doses, such as, for example, 8 priming RNA vaccine doses) alongside regular administration of a PD-1 axis binding antagonist (administered, for example, once every four weeks), in which, for example, the first two doses of the RNA vaccine are administered before administering the first dose of the PD-1 axis binding antagonist, but without administration of a chemotherapy, may result in enhanced neoantigen- and/or tumor-specific immune responses in such at-risk patients (see, e.g., Example 1, herein), which may be maximized and/or maintained when followed-up with a booster phase (including, e.g., at least two booster doses of the RNA vaccine, such as, for example, four booster doses of the RNA vaccine), leading to more robust anti-tumor immune responses and improved clinical efficacy for MIUC patients following surgical resection (e.g., cystectomy for MIBC patients and nephroureterectomy for UTUC patients), including, in particular, MIBC patients who have received prior neoadjuvant chemotherapy and have tumor staging of ypT3-4a or ypN+ at pathological examination of resected specimen and MO radiographically, UTUC patients who have received prior neoadjuvant chemotherapy and have tumor staging of ypT3-4 or ypN+ at pathological examination of resected specimen and MO radiographically, MIBC patients who have not received neoadjuvant chemotherapy and are ineligible for or declined treatment with cisplatin-based adjuvant chemotherapy and have tumor staging of pT34a or pN+ and MO, and/or UTUC patients who have not received neoadjuvant chemotherapy and are ineligible for or declined treatment with cisplatin-based adjuvant chemotherapy and have tumor staging of pT3-4 or pN+ and MO.
[0158] Accordingly, in some embodiments, the methods for treating urothelial carcinoma (UC) provided herein comprise administering an individualized RNA vaccine and a PD-1 axis binding antagonist at least during a priming phase and a subsequent booster phase, wherein the priming phase comprises administering at least six doses of the RNA vaccine (e.g., 8 priming RNA vaccine doses) and at least one dose of the PD-1 axis binding antagonist (e.g., 2-3 doses of the PD-1 axis binding antagonist), and the booster phase comprises administering at least two doses of the RNA vaccine (e.g., 4 booster RNA vaccine doses) and at least 6 doses of the PD-1 axis binding antagonist (e.g., 10 doses of the PD-1 axis binding antagonist). In some embodiments, the first two priming doses of the RNA vaccine are administered before initiation of PD-1 axis binding antagonist administration. In some embodiments, the PD-1 axis binding antagonist is administered once every four weeks (Q4W) until about one year has passed since initiation of the priming phase. In some embodiments, at lease one dose of the PD-1 axis binding antagonist is administered between administration of the last priming dose of the RNA vaccine and administration of the first booster dose of the RNA vaccine.
[0159] In some embodiments, a UC (e.g., an MIUC) to be treated according to the methods of the present disclosure is an MIBC. In some embodiments, the UC is a UTUC. In some embodiments, the MIUC to be treated according to the methods of the disclosure is a resectable MIBC or a resectable UTUC. In some embodiments, an MIBC patient treated according to the methods of the disclosure has a negative surgical margin (i.e., RO resection) or has carcinoma in situ (CIS) at the distal ureteral or urethral margin following cystectomy. In some embodiments, a UTUC patient treated according to the methods of the disclosure has a negative surgical margin (i.e., RO resection) following nephroureterectomy. In some embodiments, an absence of residual disease is confirmed by a negative baseline CT or MRI scan of the pelvis, abdomen, and chest in a patient treated according to the methods of the disclosure within about 28 days prior to administration of the first priming dose of the RNA vaccine. In some embodiments, an absence of metastasis is confirmed by a negative baseline CT or MRI scan of the pelvis, abdomen, and chest in a patient treated according to the methods of the disclosure within about 28 days prior to administration of the first priming dose of the RNA vaccine.
[0160] In some embodiments, the methods for treating MIUC provided herein comprise at least one tumor assessment. In some embodiments, the tumor assessment comprises monitoring for tumor recurrence before treatment, such as, e.g., during a baseline scan. In some embodiments, the tumor assessment comprises monitoring for tumor recurrence during treatment, such as, e.g., during the priming phase and/or the booster phase described herein and/or between the priming and booster phases. In some embodiments, the tumor assessment comprises monitoring for tumor recurrence following treatment, such as, e.g., after administration of at least one dose of the RNA vaccine; after administration of at least one dose of the PD-1 axis binding antagonist; after completion of the priming phase; and/or after completion of the booster phase described herein. In some embodiments, the tumor assessment comprises evaluating data, such as, e.g., imaging data, in order to assess recurrence of a MIUC tumor. In some embodiments, the tumor assessment comprises evaluating data collected from physical examination of the chest, abdomen, upper urinary tracts, and/or pelvis. In some embodiments, the tumor assessment comprises evaluating imaging data, such as, for example an imaging assessment of the chest, abdomen, upper urinary tracts, and/or pelvis, in which the data were collected by, for example, one or more methods selected from the group consisting of: IVP, CT urography, renal ultrasound with retrograde pyelogram, ureteroscopy, and MRI urogram. In some embodiments, at least a portion of the data is collected on at least 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 35-40, or 45- 50 different days. In some embodiments, at least a portion of the data is collected on the same day as administration of a dose of the RNA vaccine and/or a dose of the PD-1 axis binding antagonist. In some embodiments, at least a portion of the data is collected about 1-5, about 5-10, about 10-15, or about 15-30 days before administration of a dose of the RNA vaccine and/or a dose of the PD-1 axis binding antagonist. In some embodiments, at least a portion of the data is collected about 1-5 days before administration of a dose of the RNA vaccine and/or a dose of the PD-1 axis binding antagonist. In some embodiments, at least a portion of the data is collected about 1-5 days before administration of a booster dose of the RNA vaccine. In some embodiments, at least a portion of the data is collected about 1-5 days before administration of a third booster dose of the RNA vaccine. In some embodiments, at least a portion of the data is collected about 1-5 days before administration of a fourth booster dose of the RNA vaccine.
[0161] In some embodiments, at least a portion of the data is collected on within ± 1 week of weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,
208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,
244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,
280, 282, 284, 286, 288, 290, 292, 294, 296, 298, or 300, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during the first year following the first priming administration of the RNA vaccine. In some embodiments, at least a portion of the data is collected during or within ± 2 weeks of any of weeks 12, 24, 36, and/or 48, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during or within ± 1 week of any of weeks 12, 24, 36, and/or 48, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during the second year following the first priming administration of the RNA vaccine. In some embodiments, at least a portion of the data is collected during or within ± 2 weeks of any of weeks 60, 72, 84, and/or 96, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during or within ± 1 week of any of weeks 60, 72, 84, and/or 96, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during week 58, 59, 60, 61, and/or 62, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during week 60 timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during week 82, 83, 84, 85, and/or 86, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during week 84, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during the third year following the first priming administration of the RNA vaccine. In some embodiments, at least a portion of the data is collected during or within ± 2 weeks of any of weeks 112, 128, and/or 144, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during or within ± 1 week of any of weeks 112, 128, and/or 144, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during the fourth year following the first priming administration of the RNA vaccine. In some embodiments, at least a portion of the data is collected during the fifth year following the first priming administration of the RNA vaccine. In some embodiments, at least a portion of the data is collected during or within ± 2 weeks of any of weeks 168, 192, 216, and/or 240, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during or within ± 1 week of any of weeks 168, 192, 216, and/or 240, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected approximately 48 weeks after the last assessment conducted during the fifth year following the first priming administration of the RNA vaccine. In some embodiments, at least a portion of the data is collected during or within ± 2 weeks of week 288, timing starting with week 1 of the priming phase. In some embodiments, at least a portion of the data is collected during or within ± 1 week of week 288, timing starting with week 1 of the priming phase.
[0162] In some embodiments, the timing of at least one administration of the RNA vaccine and/or of at least one administration of the PD-1 axis binding antagonist is contingent on the timing of at least a portion of the data collection. In some embodiments, the timing of administration of the third RNA vaccine booster dose is contingent on the timing the imaging data collected during any of weeks 50-70, timing starting with week 1 of the priming phase. In some embodiments, the timing of administration of the third RNA vaccine booster dose is contingent on the timing the imaging data collected during week 60, timing starting with week 1 of the priming phase. In some embodiments, the timing of administration of the fourth RNA vaccine booster dose is contingent on the timing the imaging data collected during any of weeks 74-94, timing starting with week 1 of the priming phase. In some embodiments, the timing of administration of the third RNA vaccine booster dose is contingent on the timing the imaging data collected during week 84, timing starting with week 1 of the priming phase.
[0163] In some embodiments, at least a portion of the data is collected within about 30, about 25, about 20, about 18-20, about 16-18, about 14-16, about 12-14, about 10-12, about 8-10, about 6-8, about 4-6, about 2-4, about 1-2, or about 0-1 weeks prior to administration of the first priming dose of the RNA vaccine. In some embodiments, at least a portion of the data is collected within about 4 weeks prior to administration of the first priming dose of the RNA vaccine. In some embodiments, at least a portion of the data is collected within 1 week prior to administration of the first priming dose of the RNA vaccine.
[0164] In some embodiments, at least a portion of the data is collected every 6-8 weeks, every 8-10 weeks, every 10-12 weeks, every 12-14 weeks, every 14-16 weeks, every 16-18 weeks, every 18-20 weeks, every 20-22 weeks, or every 22-24 weeks in the first year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 12 weeks ± 2 weeks in the first year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 12 weeks ± 1 week in the first year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 6-8 weeks, every 8-10 weeks, every 10-12 weeks, every 12-14 weeks, every 14-16 weeks, every 16-18 weeks, every 18-20 weeks, every 20-22 weeks, or every 22-24 weeks in the second year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 12 weeks ± 2 weeks in the second year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 12 weeks ± 1 week in the second year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 6-8 weeks, every 8-10 weeks, every 10-12 weeks, every 12-14 weeks, every 14-16 weeks, every 16-18 weeks, every 18-20 weeks, every 20-22 weeks, or every 22-24 weeks in the third year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 16 weeks ± 2 weeks in the third year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 16 weeks ± 1 week in the third year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 10-12 weeks, every 12-14 weeks, every 14-16 weeks, every 16-18 weeks, every 18-20 weeks, every 20-22 weeks, every 22-24, every 24-26 weeks, every 26-28 weeks, every 28-30 weeks, every 30-32 weeks, or every 32-34 weeks in the third year after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 24 weeks ± 2 weeks in the fourth and fifth years after day 1 of the priming phase. In some embodiments, at least a portion of the data is collected every 24 weeks ± 1 week in the fourth and fifth years after day 1 of the priming phase.
(i) Priming Phase
[0165] In some embodiments, the methods for treating MIUC provided herein comprise administering to a patient, such as a human patient in need thereof, an individualized RNA vaccine and a PD-1 axis binding antagonist during a priming phase of treatment. The priming phase is named in reference to “priming” doses of the RNA vaccine, though as detailed below, at least one dose of a PD-1 axis binding antagonist may also be administered during the priming phase.
[0166] In some embodiments, the priming phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, or at least about 17 weeks after resection of a MIUC tumor from the patient, such as an MIUC tumor. In some embodiments, the priming phase begins at least about 28 days after resection of a MIUC tumor from the patient, such as an MIUC tumor.
[0167] In some embodiments, the priming phase begins less than about 1 week, less than about 2 weeks, less than about 3 weeks, less than about 4 weeks, less than about 5 weeks, less than about 6 weeks, less than about 7 weeks, less than about 8 weeks, less than about 9 weeks, less than about 10 weeks, less than about 11 weeks, less than about 12 weeks, less than about 13 weeks, less than about 14 weeks, less than about 15 weeks, less than about 16 weeks, less than about 17 weeks, less than about 18 weeks, less than about 19 weeks, or less than about 20 weeks after resection of a MIUC tumor from the patient, such as an MIUC tumor. In some embodiments, the priming phase begins less than about 18 weeks after resection of a MIUC tumor from the patient, such as an MIUC tumor. In some embodiments, the priming phase begins less than about 124 days after resection of a MIUC tumor from the patient, such as an MIUC tumor. In some embodiments, the priming phase begins about 28- 90 days after resection of a MIUC tumor from the patient, such as an MIUC tumor. In some embodiments, the priming phase begins about 90 days after resection of a MIUC tumor from the patient, such as an MIUC tumor. In some embodiments, the priming phase begins about 90-120 days after resection of a MIUC tumor from the patient, such as an MIUC tumor.
[0168] In some embodiments, the priming phase comprises any of between about 1 and about 12 weeks, between about 1 and about 10 weeks, or between about 1 and about 9 weeks. In some embodiments, the priming phase comprises 9 weeks.
[0169] In some embodiments, the priming phase comprises administering to the patient at least one dose of the RNA vaccine and at least one dose of the PD-1 axis binding antagonist. In some embodiments, the priming phase comprises administering to the patient any of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more, doses of the RNA vaccine. In some embodiments, between 1 and 8, between 6 and 8, or doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, 2 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, 3 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, 4 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, 5 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, 6 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, 7 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, 8 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, no more than 8 doses of the RNA vaccine are administered to the patient during the priming phase. In some embodiments, a dose of RNA vaccine may be administered as a single composition, or may be administered in more than one composition. For example, in some cases, an RNA vaccine dose is administered as two separate compositions administered sequentially.
[0170] In some embodiments, the priming phase comprises administering the RNA vaccine once per week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), or once every eight weeks (Q8W). In some embodiments, the priming phase comprises administering the RNA vaccine once per week (QW), e.g., once every 7 days. In some embodiments, the priming phase comprises at least one week in which the RNA vaccine is not administered. In some embodiments, the priming phase comprises one week in which the RNA vaccine is not administered. In some embodiments, the RNA vaccine is not administered during the second week. In some embodiments, the RNA vaccine is not administered during the third week. In some embodiments, the RNA vaccine is not administered during the fourth week. In some embodiments, the RNA vaccine is not administered during the fifth week. In some embodiments, the RNA vaccine is not administered during the sixth week. In some embodiments, the RNA vaccine is not administered during the seventh week. In some embodiments, the RNA vaccine is not administered during the eighth week. In some embodiments, the priming phase comprises administering the RNA vaccine once per week (QW), e.g., once every 7 days, except for during one week in which no RNA vaccine is administered. In some embodiments, administration of the RNA vaccine during priming phase begins on day 1 of week 1 of the priming phase. In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1 of the priming phase and once per week (QW), e.g., once every 7 days, thereafter, except for, in some embodiments, during one week in which no RNA vaccine is administered. In some embodiments, the one week in which no RNA vaccine is administered is the second, third, fourth, fifth, sixth, seventh, or eighth week. In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1 of the priming phase and once per week (QW), e.g., once every 7 days, thereafter, except for, in some embodiments, during one or more weeks selected from the group consisting of: the second, third, fourth, fifth, sixth, seventh, and eighth weeks. In some embodiments, the RNA vaccine is not administered during more than one week of the priming phase, and a make-up priming dose of the RNA vaccine is administered. In some embodiments, a priming phase comprising administration of a make-up priming dose of the RNA vaccine comprises administration of the RNA vaccine no more frequently than weekly (± 2 days). In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1 of the priming phase and once per week (QW), e.g., once every 7 days, thereafter, except for, in some embodiments, during the eighth week, in which no RNA vaccine is administered. [0171] In some embodiments, the priming phase comprises administering eight, nine, or ten doses of the RNA vaccine. For example, in some cases, the RNA vaccine is administered on eight of the following, nine of the following, or all of the following days of the priming phase, ±3 days: day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, day 1 of week 6, day 1 of week 7, day 1 of week 8, day 1 of week 9, and day 1 of week 10. In some embodiments, the RNA vaccine is administered on the following days of the priming phase, ±3 days: day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, day 1 of week 6, day 1 of week 7, and day 1 of week 9.
[0172] In some embodiments, 2 doses of the RNA vaccine are administered to the patient during the priming phase before administration of the PD-1 axis binding antagonist.
[0173] In some embodiments, the priming phase comprises administering to the patient at least one dose of the PD-1 axis binding antagonist. In some embodiments, the priming phase comprises administering to the patient any of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more doses of the PD-1 axis binding antagonist. In some embodiments, between 1 and 4, between 1 and 3, or either 2 or 3 doses of the PD-1 axis binding antagonist are administered to the patient during the priming phase. In some embodiments, 2 doses of the PD-1 axis binding antagonist are administered to the patient during the priming phase. In some embodiments, 3 doses of the PD-1 axis binding antagonist are administered to the patient during the priming phase.
[0174] In some embodiments, any dose of the PD-1 axis binding antagonist administered to the patient during the priming phase is administered on the same day as the administration of a dose of the RNA vaccine. In some such embodiments, a dose of the PD-1 axis binding antagonist is administered to the patient after completion of administration of that day’s dose of the RNA vaccine. In some such embodiments, the dose of the PD-1 axis binding antagonist is administered to the patient at least about 1-15, at least about 15-30, at least about 30-45, at least about 45-60, at least about 60-120, or at least about 120-180 minutes after completion of administration of that day’s dose of the RNA vaccine. In some such embodiments, the dose of the PD-1 axis binding antagonist is administered to the patient about 30 minutes after completion of administration of that day’s dose of the RNA vaccine. [0175] In some embodiments, the second dose of the PD-1 axis binding antagonist administered to the patient during the priming phase is administered on the same day as the administration of a dose of the RNA vaccine. In some embodiments, the second dose of the PD-1 axis binding antagonist administered to the patient during the priming phase is administered on the same day as the administration of the sixth dose of the RNA vaccine. In some embodiments, any dose of the PD-1 axis binding antagonist administered to the patient during or after the priming phase is administered the day after a day of administration of a dose of the RNA vaccine. In some embodiments, the first dose of the PD-1 axis binding antagonist administered to the patient during the priming phase is administered the day after a day of administration of a dose of the RNA vaccine. In some embodiments, the third dose of the PD-1 axis binding antagonist administered to the patient is administered the day after a day of administration of a dose of the RNA vaccine. In some embodiments, the first dose of the PD-1 axis binding antagonist administered to the patient during the priming phase is administered the day after administration of the second dose of the RNA vaccine. In some embodiments, the third dose of the PD-1 axis binding antagonist administered to the patient is administered the day after administration of the eighth dose of the RNA vaccine.
[0176] In some embodiments, the priming phase comprises administering to the patient two doses of the PD-1 axis binding antagonist. In some embodiments, the first dose of the PD-1 axis binding antagonist is administered to the patient no sooner than after administration of 2 doses of the RNA vaccine. In some embodiments, the first dose of the PD-1 axis binding antagonist is administered to the patient 1-4 days after administration of the second priming dose of the RNA vaccine. In some embodiments, the first dose of the PD-1 axis binding antagonist is administered to the patient about 1 day after administration of the second priming dose of the RNA vaccine.
[0177] In some embodiments, the first dose of the PD-1 axis binding antagonist is administered to the patient no sooner than after administration of more than 2 doses of the RNA vaccine, e.g., after administration of at least 3, at least 4, at least 5, at least 6, or at least 7 doses of the RNA vaccine. In some embodiments, the priming phase comprises administering to the patient one dose of the PD-1 axis binding antagonist. In some embodiments, the first dose of the PD-1 axis binding antagonist is administered to the patient 1-7 weeks after administration of the second priming dose of the RNA vaccine. In some embodiments, the first dose of the PD-1 axis binding antagonist is administered to the patient about 1 month after administration of the second priming dose of the RNA vaccine.
[0178] In some embodiments, the PD-1 axis binding antagonist is administered once per week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), or once every eight weeks (Q8W). In some embodiments, the PD-1 axis binding antagonist is administered once every four weeks (Q4W), e.g., once every 28 days, ±3 days. In some embodiments, administration of the PD-1 axis binding antagonist during priming phase begins the day after the day of administration of the second priming dose of the RNA vaccine. In some embodiments, administration of the PD-1 axis binding antagonist during priming phase begins on day 2 of week 2 of the priming phase. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist on day 2 of week 2 of the priming phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±3 days. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist on the day after the day of administration of the second priming dose of the RNA vaccine and once every four weeks (Q4W), e.g., once every 28 days, thereafter. In some embodiments, Q4W administration of the PD-1 axis binding antagonist continues through the end of the priming phase. In some embodiments, Q4W administration of the PD-1 axis binding antagonist continues between the end of the priming phase and the beginning of the booster phase. In some embodiments, Q4W administration of the PD-1 axis binding antagonist continues through the beginning of the booster phase.
[0179] In some embodiments, administration of the PD-1 axis binding antagonist ends within 0-6 months, within 6-12 months, within 12-18 months, within 18-24 months, within 24-30 months, or within 30-36 months after day 1 of week 1 of the priming phase or after administration of the first dose of the PD-1 axis binding antagonist. In some embodiments, Q4W administration of the PD-1 axis binding antagonist ends up to one year after the first administration of the PD-1 axis binding antagonist. In some embodiments, the priming phase comprises administering the PD-1 axis binding antagonist on the day after the day of administration of the second priming dose of the RNA vaccine and once every four weeks (Q4W), e.g., once every 28 days, thereafter, for up to one year after the first administration of the PD-1 axis binding antagonist.
[0180] In some embodiments, the priming phase comprises administering two doses of the PD-1 axis binding antagonist. For example, in some cases, the PD-1 axis binding antagonist is administered on day 2 of week 2 of the priming phase and on day 1 of week 6 of the priming phase. In some embodiments, the a third dose of the PD-1 axis binding antagonist is administered during the priming phase (e.g., before or on the same day as administration of the last priming dose of the RNA vaccine). In some embodiments, the a third dose of the PD- 1 axis binding antagonist is administered on day 1 of week 10, ±3 days, and day 1 of week 10 ±3 days is within the priming phase (e.g., before or on the same day as administration of the last priming dose of the RNA vaccine), such that the third dose of the PD-1 axis binding antagonist is administered during the priming phase.
[0181] In some embodiments, the priming phase comprises administering the RNA vaccine on day 1 of week 1, day 1 of week 2, day 1 of week 3, day 1 of week 4, day 1 of week 5, day 1 of week 6, day 1 of week 7, and day 1 of week 9 of the priming phase, and the PD-1 axis binding antagonist on day 2 of week 2 and day 1 of week 6 of the priming phase. In some such embodiments, the PD-1 axis binding antagonist is further administered on day 1 of week 10 (i.e., after the last priming dose of the RNA vaccine).
[0182] An exemplary priming phase is provided in Table 1, below.
Table 1. Exemplary priming phase (and shortly thereafter).
[0183] In some embodiments, the RNA vaccine is administered to the patient during the priming phase at a dose of between about 15 pg to about 50 pg (e.g., any of about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 38 pg, about 40 pg, about 45 pg, or about 50 pg). In some embodiments, the RNA vaccine is administered to the patient at a dose of about 15 pg, about 21 pg, about 21.3 pg, about 25 pg, about 38 pg, or about 50 pg. In some embodiments, the RNA vaccine is administered to the patient at a dose of 25 pg. In some embodiments, the RNA vaccine is administered to the patient at a dose of about 21 pg. In some embodiments, the RNA vaccine is administered to the patient at a dose of about 21.3 pg. In certain embodiments, the RNA vaccine is administered intravenously to the patient. In some embodiments, a total dose of RNA vaccine may be administered as a single composition, or may be administered in more than one composition. For example, in some cases, the RNA vaccine is administered at a total dose of 25 pg, which is split into two compositions that are administered sequentially. In some embodiments, the RNA vaccine dose is administered to the patient in two equal half-doses. In some embodiments, the two equal half-doses are administered sequentially, optionally with an observation period between the administered equal half-doses. In some embodiments, the dose of about 25 pg is split into two equal half-doses of about 12.5 pg, each administered over 1 minute, optionally with a 5- minute observation period between the administered equal half-doses. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 or 10-20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen from the patient.
[0184] In some embodiments, the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids. In some embodiments, the RNA vaccine is formulated as a lipid nanoparticle, wherein the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipid nanoparticle. In some embodiments, the RNA vaccine is formulated as a lipoplex, wherein the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipoplex. In some embodiments, the lipoplex comprises one or more lipids that form a multilamellar structure that encapsulates the one or more polynucleotides of the RNA vaccine.
[0185] In some specific embodiments, the PD-1 axis binding antagonist is an anti-PD-Ll antibody, e.g., as described below. In some embodiments, the anti-PD-Ll antibody is nivolumab, avelumab, durvalumab, or atezolizumab. In one embodiment, the anti-PD-Ll antibody is nivolumab. In some embodiments, the anti-PD-Ll antibody is administered to the patient at a dose of about 240 mg, about 480 mg, about 1200 mg, or about 1680 mg. In some embodiments, the anti-PD-Ll antibody is administered to the patient at a dose of about 480 mg. In certain embodiments, the PD-1 axis binding antagonist is administered intravenously to the patient.
(ii) Booster Phase
[0186] In some embodiments, the methods for treating MIUC provided herein comprise administering to a patient, such as a human patient in need thereof, at least one dose of an RNA vaccine and at least one dose of a PD-1 axis binding antagonist during a booster phase after the end of a priming phase, e.g., as described above. The booster phase is named in reference to “booster” doses of the RNA vaccine (or placebo), though as detailed below, at least one dose of a PD-1 axis binding antagonist may also be administered during the booster phase, including, for example, after administration of the last booster dose of the RNA vaccine.
[0187] In some embodiments, the methods for treating MIUC provided herein further comprise administering to a patient at least one dose of a PD-1 axis binding antagonist before a boost phase after the end of a priming phase, e.g., as described above. In some embodiments, three doses of a PD-1 axis binding antagonist are administered before the booster phase. In some embodiments, one dose of a PD-1 axis binding antagonist is administered between the priming phase and the booster phase. In some embodiments, two doses of a PD-1 axis binding antagonist are administered between the priming phase and the booster phase. In some embodiments, no doses of a PD-1 axis binding antagonist are administered between the priming phase and the booster phase.
[0188] In some embodiments, the boost phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, or at least about 15 weeks after the end of the priming phase, e.g., after the last priming administration of the RNA vaccine. In some embodiments, the boost phase begins between about 3 weeks and about 12 weeks, or between about 4 weeks and about 6 weeks after the end of the priming phase, e.g., after the last priming administration of the RNA vaccine. In some embodiments, the boost phase begins 5 weeks after the end of the after the end of the priming phase, e.g., after the last priming administration of the RNA vaccine. In some embodiments, the boost phase begins 4 weeks after the end of the after the end of the priming phase, e.g., after the last priming administration of the RNA vaccine. In some embodiments, the boost phase begins no later than about 6 weeks after the end of the after the end of the priming phase, e.g., after the last priming administration of the RNA vaccine. In some embodiments, the booster phase begins on week 14 (e.g., day 1 of week 14), timing starting with week 1 of the priming phase.
[0189] In some embodiments, the booster phase comprises administering to the patient at least two doses of the PD-1 axis binding antagonist. In some embodiments, the booster phase comprises administering to the patient any of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or more than 15 doses of the PD-1 axis binding antagonist. In some embodiments, between 1 and 13, between 6 and 14, between 8 and 12, or either 9 or 10 doses of the PD-1 axis binding antagonist are administered to the patient during the booster phase. In some embodiments, 10 doses of the PD-1 axis binding antagonist are administered to the patient during the booster phase.
[0190] In some embodiments, any dose of the PD-1 axis binding antagonist administered to the patient during the booster phase is administered on the same day as the administration of a dose of the RNA vaccine. In some such embodiments, a dose of the PD-1 axis binding antagonist is administered to the patient after completion of administration of that day’s dose of the RNA vaccine. In some such embodiments, the dose of the PD-1 axis binding antagonist is administered to the patient at least about 1-15, at least about 15-30, at least about 30-45, at least about 45-60, at least about 60-120, or at least about 120-180 minutes after completion of administration of that day’s dose of the RNA vaccine. In some such embodiments, the dose of the PD-1 axis binding antagonist is administered to the patient about 30 minutes after completion of administration of that day’s dose of the RNA vaccine. In some embodiments, the first dose of the PD-1 axis binding antagonist administered to the patient during the booster phase is administered on the same day as the administration of a booster dose of the RNA vaccine. In some embodiments, the first dose of the PD-1 axis binding antagonist administered to the patient during the booster phase is administered on the same day as the administration of the first booster dose of the RNA vaccine. In some embodiments, a dose of the PD-1 axis binding antagonist administered to the patient during the booster phase is administered on the same day as the administration of the second booster dose of the RNA vaccine. In some embodiments, the seventh dose of the PD-1 axis binding antagonist administered to the patient during the booster phase is administered on the same day as the administration of the second booster dose of the RNA vaccine.
[0191] In some embodiments, the PD-1 axis binding antagonist is administered once per week (QW), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every five weeks (Q5W), once every six weeks (Q6W), once every seven weeks (Q7W), or once every eight weeks (Q8W) throughout at least a portion of the booster phase. In some embodiments, the PD-1 axis binding antagonist is administered once every four weeks (Q4W), e.g., once every 28 days, ±5 days throughout at least the first portion of the booster phase. In some embodiments, administration of the PD-1 axis binding antagonist during the booster phase begins the day of administration of the first booster dose of the RNA vaccine. In some embodiments, administration of the PD-1 axis binding antagonist during the booster phase begins on day 1 of week 1 of the booster phase. In some embodiments, administration of the PD-1 axis binding antagonist during the booster phase begins on day 1 of week 14, timing starting from week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until one year has passed since week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 doses of the PD-1 axis binding antagonist have been administered since week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of at least 10, at least 11, at least 12, or at least 13 doses of the PD-1 axis binding antagonist have been administered since week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of at least 12 doses of the PD-1 axis binding antagonist have been administered since week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of 13 doses of the PD-1 axis binding antagonist have been administered since week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 doses of the PD-1 axis binding antagonist have been administered during the booster phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of at least 9, at least 10, at least 11, or at least 12 doses of the PD-1 axis binding antagonist have been administered during the booster phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of at least 9 doses of the PD-1 axis binding antagonist have been administered during the booster phase. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist on day 1 of week 1 of the booster phase and once every four weeks (Q4W), e.g., once every 28 days, thereafter, ±5 days, until a total of 10 doses of the PD-1 axis binding antagonist have been administered during the booster phase. [0192] In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist 1-3 days after the day of administration of a booster dose of the RNA vaccine. In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist 1-3 days before the day of administration of a booster dose of the RNA vaccine.
[0193] In some embodiments, administration of the PD-1 axis binding antagonist continues through the end of the booster phase (e.g., until or after administration of the last booster dose of the RNA vaccine). In some embodiments, administration of the PD-1 axis binding antagonist ends before the end of the booster phase (e.g., before administration of the last booster dose of the RNA vaccine). In some embodiments, administration of the PD-1 axis binding antagonist stops after 1 year since week 1, day 1 of the priming phase. In some embodiments, administration of the PD-1 axis binding antagonist stops after 13 administrations of the PD-1 axis binding antagonist, including 10 administrations during the booster phase.
[0194] In some embodiments, the booster phase comprises administering the RNA vaccine to the patient approximately 2-4, approximately 8-10, approximately 13-15, and/or approximately 19-21 months after administration of the second priming dose of the RNA vaccine. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient approximately 3, approximately 9, approximately 14, and/or approximately 20 months after administration of the second priming dose of the RNA vaccine. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient approximately 3, approximately 9, approximately 14, and approximately 20 months after administration of the second priming dose of the RNA vaccine. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient on day 1 of week 14, ±5 days, and/or on day 1 of week 10, ±5 days, timing starting from week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient on day 1 of week 14, ±5 days, and/or on day 1 of week 10, ±5 days, timing starting from week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient approximately 15 months post-initiation of the PD-1 axis binding antagonist. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient after review of an imaging assessment conducted approximately 60 weeks after week 1, day 1 of the priming phase. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient approximately 21 months post-initiation of the PD-1 axis binding antagonist. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient after review of an imaging assessment conducted approximately 84 weeks after week 1, day 1 of the priming phase.
[0195] In some embodiments, administration of the RNA vaccine and/or the PD-1 axis binding antagonist during the booster phase begins on day 1 of week 1 of the booster phase. In some embodiments, the booster phase comprises administering the RNA vaccine and/or the PD-1 axis binding antagonist to the patient on day 1 of the boost phase, and administration of the PD-1 axis binding antagonist once every four weeks (Q4W), e.g., once every 28 days, thereafter, until 10 doses of the PD-1 axis binding antagonist have been administered during the booster phase. In some embodiments, the booster phase comprises administering the RNA vaccine and/or the PD-1 axis binding antagonist to the patient on day 1 of the boost phase, and administration of the RNA vaccine three more times thereafter, wherein administration of the second, third, and fourth booster doses are spaced approximately 6 months apart between week 1, day 1 of the booster phase and approximately 21 months thereafter.
[0196] In some embodiments, the booster phase comprises administering to the patient at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more, doses of the RNA vaccine. In some embodiments, between 1 and 8, between 6 and 8, between 2 and 6, or either 4 doses of the RNA vaccine are administered to the patient during the booster phase. In some embodiments, 4 doses of the RNA vaccine are administered to the patient during the booster phase. In some embodiments, 6 doses of the RNA vaccine are administered to the patient during the boost phase.
[0197] In some embodiments, a dose of RNA vaccine may be administered as a single composition, or may be administered in more than one composition. For example, in some cases, an RNA vaccine dose is administered as two separate compositions administered sequentially. In some embodiments, the two separate compositions administered sequentially each comprise a half-dose of the RNA vaccine. In some embodiments, a half-dose of the RNA vaccine comprises approximately 12.5 pg of the RNA vaccine.
[0198] In some embodiments, the booster phase comprises administering to the patient the RNA vaccine and/or the PD-1 axis binding antagonist in every sixth or seventh 4-week cycle (e.g., every sixth or seventh set of 28-day cycles), for example, starting on day 1 of week 1 of the boost phase. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient every sixth or seventh 4-week cycles at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or more, 4-week cycles (e.g., 28-day cycles). In some embodiments, the booster phase comprises administering the RNA vaccine to the patient every sixth 4-week cycle over at least 21 4-week cycles. In some embodiments, the booster phase comprises administering the RNA vaccine to the patient every seventh 4-week cycle over at least 21 4-week cycles. In some embodiments, the RNA vaccine is administered in 24-week cycles (e.g., 168-day cycles) for 1, 2, 3, 4, 5, 6, or more cycles during the booster base. In some embodiments, the RNA vaccine is administered in 24-week cycles (e.g., 168-day cycles) for 4 or more cycles during the booster phase. In some embodiments, the RNA vaccine is administered in 24-week cycles (e.g., 168-day cycles) for 4 cycles during the booster phase. In other embodiments, the RNA vaccine is administered in 24-week cycles (e.g., 168-day cycles) for at least 4 cycles during the booster phase. In other embodiments, the RNA vaccine is administered in 24-week cycles (e.g., 168-day cycles) for no more than 4 cycles during the booster phase.
[0199] In some embodiments, the booster phase comprises administering the PD-1 axis binding antagonist to the patient for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more, 4-week cycles (e.g, 28-day cycles). In some embodiments, the PD-1 axis binding antagonist is administered in 4-week cycles (e.g, 28-day cycles) for between 5 and 15, between 8 and 12, between 9 and 1, or 10 cycles during the booster base. In some embodiments, the PD-1 axis binding antagonist is administered in 4-week cycles (e.g., 28-day cycles) for 10 cycles during the booster phase. In other embodiments, the PD-1 axis binding antagonist is administered in 4-week cycles (e.g., 28-day cycles) for at least 10 cycles during the booster phase. In other embodiments, the PD-1 axis binding antagonist is administered in 4-week cycles (e.g., 28-day cycles) for no more than 10 cycles during the booster phase.
[0200] In some embodiments, the boost phase begins in week 14 (e.g., day 1 of week 14), timing starting with week 1 of the priming phase, e.g., as described above. In some embodiments, the boost phase comprises administering to the patient the RNA vaccine starting on day 1 of week 33, timing starting with week 1 of the priming phase, e.g., as described above, and approximately every 6 sets of 28-day cycles thereafter, for example, for a total of 4 administration of the RNA vaccine. For example, in some cases, the booster phase comprises administering to the patient the RNA vaccine on day 1 of week 14, day 1 of week 38, one day during week 60 or 61, and one day during week 84 or 85, timing starting with week 1 of the priming phase. In some embodiments, the booster phase comprises administering to the patient the PD-1 axis binding antagonist starting on day 1 of week 14, timing starting with week 1 of the priming phase, e.g., as described above, and every four weeks (Q4W; e.g., every 28 days) thereafter, for example, for a total of 10 administration of the PD-1 axis binding antagonist during the booster phase. For example, in some cases, the boost phase comprises administering to the patient the PD-1 axis binding antagonist on day 1 of week 14, day 1 of week 18, day 1 of week 22, day 1 of week 26, day 1 of week 30, day 1 of week 34, day 1 of week 38, day 1 of week 42, day 1 of week 46, and day 1 of week 50 of the booster phase, timing starting with week 1 of the priming phase. In some embodiments, the booster phase comprises administering to the patient the RNA vaccine on day 1 of week 14, day 1 of week 38, one day during week 60 or 61, and one day during week 84 or 85, timing starting with week 1 of the priming phase, and administering to the patient the PD-1 axis binding antagonist on day 1 of week 14, day 1 of week 18, day 1 of week 22, day 1 of week 26, day 1 of week 30, day 1 of week 34, day 1 of week 38, day 1 of week 42, day 1 of week 46, and day 1 of week 50 of the booster phase, timing starting with week 1 of the priming phase.
[0201] An exemplary booster phase is provided in Table 2, below.
Table 2. Exemplary booster phase.
[0202] In some embodiments, the RNA vaccine is administered to the patient during the booster phase at a dose of between about 15 pg to about 50 pg (e.g., any of about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 38 pg, about 40 pg, about 45 pg, or about 50 pg). In some embodiments, the RNA vaccine is administered to the patient at a dose of about 15 pg, about 21 pg, about 21.3 pg, about 25 pg, about 38 pg, or about 50 pg. In some embodiments, the RNA vaccine is administered to the patient at a dose of 25 pg. In some embodiments, the RNA vaccine is administered to the patient at a dose of about 21 pg. In some embodiments, the RNA vaccine is administered to the patient at a dose of about 21.3 pg. In certain embodiments, the RNA vaccine is administered intravenously to the patient. In some embodiments, a total dose of RNA vaccine may be administered as a single composition, or may be administered in more than one composition. For example, in some cases, the RNA vaccine is administered at a total dose of 25 pg, which is split into two compositions that are administered sequentially. In some embodiments, the RNA vaccine dose is administered to the patient in two equal half-doses. In some embodiments, the two equal half-doses are administered sequentially, optionally with an observation period between the administered equal half-doses. In some embodiments, the dose of about 25 pg is split into two equal half-doses of about 12.5 pg, each administered over 1 minute, optionally with a 5- minute observation period between the administered equal half-doses. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 or 10-20 neoepitopes resulting from cancer-specific somatic mutations present in a tumor specimen from the patient. [0203] In some embodiments, the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids. In some embodiments, the RNA vaccine is formulated as a lipid nanoparticle, wherein the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipid nanoparticle. In some embodiments, the RNA vaccine is formulated as a lipoplex, wherein the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipoplex. In some embodiments, the lipoplex comprises one or more lipids that form a multilamellar structure that encapsulates the one or more polynucleotides of the RNA vaccine.
[0204] In some specific embodiments, the PD-1 axis binding antagonist is an anti-PD-Ll antibody, e.g., as described below. In some embodiments, the anti-PD-Ll antibody is nivolumab, avelumab, durvalumab, or atezolizumab. In one embodiment, the anti-PD-Ll antibody is nivolumab . In some embodiments, the anti-PD-Ll antibody is administered to the patient at a dose of about 240 mg, about 480 mg, about 1200 mg or about 1680 mg. In some embodiments, the anti-PD-Ll antibody is administered to the patient at a dose of about 480 mg. In certain embodiments, the PD-1 axis binding antagonist is administered intravenously to the patient.
[0205] Any of the priming phases and booster phases described herein may be used in any combination in the methods for treating MIUC provided herein.
[0206] For example, in some embodiments, the methods of treating a MIUC provided herein comprise administering to the patient an individualized RNA vaccine and a PD-1 axis binding antagonist during a treatment period that comprises at least a priming phase and a booster phase after the priming phase, wherein: the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, 6, 7, and 9 of the priming phase, and the PD-1 axis binding antagonist on day 2 of week 2 of the priming phase and day 1 of week 6 of the priming phase; further administering the PD-1 axis binding antagonist on day 1 of week 10 (e.g., one week after the last priming dose of the RNA vaccine); and the booster phase comprises administering the RNA vaccine and the PD-1 axis binding antagonist on day 1 of weeks 14 and 38 and further administering the RNA vaccine during week 60 and week 84, timing starting with week 1 of the priming phase. In another example, the methods of treating a MIUC provided herein comprise administering to the patient an individualized RNA vaccine and a PD-1 axis binding antagonist during a treatment period that comprises at least a priming phase and a booster phase after the priming phase, wherein: the priming phase comprises administering the RNA vaccine on day 1 of weeks 1, 2, 3, 4, 5, 6, 7, and 9 of the priming phase, and the PD-1 axis binding antagonist on day 2 of week 2 of the priming phase and day 1 of week 6 of the priming phase; further administering the PD-1 axis binding antagonist on day 1 of week 10 (e.g., one week after the last priming dose of the RNA vaccine); and the booster phase comprises administering the RNA vaccine and the PD-1 axis binding antagonist on day 1 of weeks 14 and 38 and further administering the RNA vaccine during week 61 and week 85, timing starting with week 1 of the priming phase..
[0207] In some embodiments, the priming phase begins at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, or at least about 32 weeks after resection, e.g., after radical surgical resection, of a MIUC tumor from the patient, such as a MIBC and/or UTUC tumor. In some embodiments, the priming phase begins between about 4 and about 30 weeks after resection, e.g., after radical surgical resection, of a MIUC tumor from the patient, such as a MIBC and/or UTUC tumor. In some embodiments, the priming phase begins between about 4 and about 18 weeks after resection of a MIUC tumor from the patient, such as a MIBC and/or UTUC tumor. In some embodiments, the priming phase begins no more than about 120 days after resection of a MIUC tumor from the patient, such as a MIBC and/or UTUC tumor. In some embodiments, the priming phase begins no more than about 120 days after radical cystectomy of an MIBC tumor from the patient. In some embodiments, the priming phase begins no more than about 120 days after radical nephroureterectomy of a UTUC tumor from the patient.
Individuals with a Tumor
[0208] In some embodiments, the UC tumor is a resected MIUC tumor in patients who are at high-risk for recurrence and have no evidence of disease after surgery, which is assessed by imaging in the human patient with computed tomography (CT) scan with contrast or positron emission tomography-CT (PET-CT) prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, CT scan with contrast may be contraindicated. In some embodiments wherein CT scan with contrast is contraindicated, CT scan without contrast, magnetic resonance imaging (MRI), CT urography with reduced contrast, ureteroscopy, IVP x-ray, and/or renal ultrasound with retrograde pyelogram (X-ray) scan is used prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, MRI scan with contrast may be contraindicated. In some embodiments wherein MRI scan with contrast is contraindicated, non-contrast MRI urogram with static fluid T2-weighted sequences and/or ureteroscopy is used prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, other imaging techniques are performed if clinically indicated. Other imaging techniques may include, but are not limited to, positron emission tomography (PET), ultrasound, 3D ultrasound, radiography, etc. Contrast agents can include, for example, gadolinium, iron oxide, manganese(II), iodine (e.g., lohexol), barium-sulfate etc.
[0209] In some embodiments, the MIUC tumor is a resectable MIBC tumor comprising one or more characteristics selected from the group consisting of: having undergone and fully recovered from radical cystectomy with lymph node dissection within the previous 120 days, (y)pT3-T4a or (y)pN+ at time of radical cystectomy, MO, having received prior neoadjuvant chemotherapy, not having received neoadjuvant chemotherapy and being ineligible for or having declined treatment with cisplatin-based adjuvant chemotherapy, having provided a TURBT and/or cystectomy specimen, absence of residual disease within 31 days before administration of the RNA vaccine, and absence of metastatic disease within 31 days before administration of the RNA vaccine.
[0210] In some embodiments, the MIUC tumor is a resectable UTUC tumor comprising one or more characteristics selected from the group consisting of: having undergone and fully recovered radical nephroureterectomy (RNU) with excision of the bladder cuff within the previous 120 days, (y)pT3-T4 or (y)pN+ at time of radical cystectomy, M0, having received prior neoadjuvant chemotherapy, not having received neoadjuvant chemotherapy and being ineligible for or having declined treatment with cisplatin-based adjuvant chemotherapy, having provided a TURBT and/or nephroureterectomy specimen, absence of residual disease within 31 days before administration of the RNA vaccine, and absence of metastatic disease within 31 days before administration of the RNA vaccine.
[0211] In some embodiments, the human patient has a histologically confirmed diagnosis of MIBC and/or UTUC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received a histologically confirmed diagnosis of MIBC and has received radical cystectomy comprising bilateral lymph node dissection prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the radical cystectomy is performed by the open, laparoscopic, or robotic approach. In some embodiments, the radical cystectomy extends at a minimum from the mid common iliac artery proximally to Cooper's ligament distally, laterally to the genitofemoral nerve, and inferiorly to the obturator nerve. In some embodiments, the radical cystectomy has a negative surgical margin (i.e., RO resection). In some embodiments, the human patient has received a histologically confirmed diagnosis of MIBC and carcinoma in situ (CIS) at the distal ureteral or urethral margin. In some embodiments, the radical cystectomy does not have a positive R2 margin (i.e., a tumor identified at the inked perivesical fat margin surrounding the cystectomy specimen). In some embodiments, the radical cystectomy does not have an R1 margin (i.e., evidence of microscopic disease identified at the tumor margin). In some embodiments, the radical cystectomy has an R1 margin and the patient has carcinoma in situ (CIS) at the distal ureteral or urethral margin. [0212] In some embodiments, the human patient has a histologically confirmed diagnosis of UTUC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received a histologically confirmed diagnosis of UTUC and has received RNU comprising excision of the bladder cuff prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the RNU is performed by the open or laparoscopic approach. In some embodiments, the RNU includes lymph node dissection (LND). In some embodiments, the RNU includes the para-aortic, paracaval or interaortocaval nodes from the renal hilum to the inferior mesenteric artery in renal pelvis. In some embodiments, the RNU includes proximal ureteral tumors. In some embodiments, the RNU includes nodes from the renal hilum to the bifurcation of the common iliac artery in mid-ureteral tumors. In some embodiments, the RNU includes ipsilateral pelvic nodes in lower-ureteral tumors. In some embodiments, the RNU has a negative surgical margin (i.e., RO resection). In some embodiments, the RNU does not have a positive R1 margin. In some embodiments, the RNU does not have a positive R2 margin.
[0213] In some embodiments, the human patient has MIBC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has UTUC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has MIBC and UTUC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received platinum-based neoadjuvant chemotherapy (NAC) prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received at least three cycles of a platinum-containing neoadjuvant chemotherapy (NAC) prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0214] In some embodiments, the human patient has not received platinum -based neoadjuvant chemotherapy (NAC) prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has been ineligible to receive adjuvant cisplatin-based therapy either due to cisplatin ineligibility, patient refusal, or investigator decision prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has been ineligible to receive adjuvant cisplatin-based therapy due to cisplatin ineligibility prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has refused adjuvant cisplatin-based therapy prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0215] In some embodiments, the UC tumor has tumor, lymph node, metastasis (TNM) pathological staging values (y)pT3-4a or (y)pN+ for MIBC patients, or (y)pT3-4 or (y)pN+ for UTUC patients, and MO prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the staging values are assessed as per the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, 7th edition (Edge, S. et al., eds., American Joint Committee on Cancer (AJCC) Cancer Staging Manual; 8th ed. New York: Springer 2011).
[0216] In some embodiments, a surgical tumor specimen is provided from the patient. In some embodiments, the surgical tumor specimen is from TURBT. In some embodiments, the surgical tumor specimen is from cystectomy. In some embodiments, the surgical tumor specimen is from nephroureterectomy. In some embodiments, surgical tumor specimen is used for determining PD-L1 expression. In some embodiments, surgical tumor specimen is used for exploratory biomarker research. In some embodiments, nucleic acids from the surgical tumor specimen are sequenced. In some embodiments, the sequencing comprises whole exome sequencing. In some embodiments, the sequencing comprises RNA sequencing. In some embodiments, data (e.g., sequencing data) from the surgical tumor specimen are used in designing the RNA vaccine. In some embodiments, the surgical tumor specimen is provided with an associated pathology report. In some embodiments, the surgical tumor specimen comprises an FFPE tumor block. In some embodiments, the surgical tumor specimen comprises an intact FFPE tumor block. In some embodiments, the surgical tumor specimen comprises 1-10, 10-15, 15-20, 20-25, 25-30, 30-35, or 35-40 slides comprising sections derived from an FFPE tumor block. In some embodiments, the surgical tumor specimen comprises 10-30 slides comprising sections derived from an FFPE tumor block. In some embodiments, the surgical tumor specimen comprises about 20 slides comprising sections derived from an FFPE tumor block. In some embodiments, the sections comprise unstained, freshly cut serial sections derived from an FFPE tumor block. In some embodiments, the surgical tumor specimen comprises 20 slides comprising sections derived from an FFPE tumor block. In some embodiments, the surgical tumor specimen comprises 20 slides comprising unstained, freshly cut serial sections derived from an FFPE tumor block. [0217] In some embodiments, surgical tumor specimen is of good quality based on total and viable tumor content. In some embodiments, surgical tumor specimen comprises a muscle invasive component (i.e., T2). In some embodiments, a plurality of different surgical tumor specimens are provided. In some embodiments, the different surgical tumor specimens were taken from the same patient at different sites. In some embodiments, the different surgical tumor specimens were taken from the same patient at different times. In some embodiments, a specimen from the plurality of different surgical tumor specimens is from surgical resection of the patient’s primary tumor. In some embodiments, a specimen from the plurality of different surgical tumor specimens is from dissection of the patient’s lymph node. In some embodiments, a specimen from the surgical resection of the primary tumor is used for any of the specimen analyses described herein. In some embodiments, a specimen from lymph node dissection is used for any of the specimen analyses described herein. In some embodiments, a specimen from the most recently resected site of disease is used for any of the specimen analyses described herein. In some embodiments, a specimen from the most recently resected site of disease is used for PD-L1 IHC analysis. In some embodiments, additional archived tumor tissue from the most recent resection or TURBT that yielded the initial muscle invasive diagnosis is provided. In some embodiments, the additional archived tumor tissue is used in any of the specimen analyses described herein.
[0218] In some embodiments, the patient has fully recovered from cystectomy within about 20-30, about 30-50, about 50-70, about 70-90, about 90-100, about 100-110, about 110-115, about 115-120, about 120-125, about 125-130, about 130-150, about 150-170, about 170-190, about 190-210, or more than about 210 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has fully recovered from cystectomy within about 115-125 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has fully recovered from cystectomy within 120 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has fully recovered from cystectomy within 120 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0219] In some embodiments, the patient has fully recovered from nephroureterectomy within about 20-30, about 30-50, about 50-70, about 70-90, about 90-100, about 100-110, about 110-115, about 115-120, about 120-125, about 125-130, about 130-150, about 150-170, about 170-190, about 190-210, or more than about 210 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has fully recovered from nephroureterectomy within about 115-125 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has fully recovered from nephroureterectomy within 120 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has fully recovered from nephroureterectomy within 120 days following surgery prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0220] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 7, 18, 19, 20, or more than 20 neoepitopes resulting from cancer-specific somatic mutations are present in the tumor specimen obtained from the human patient prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, at least five neoepitopes resulting from cancer-specific somatic mutations are present in the tumor specimen obtained from the human patient prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0221] In some embodiments, the human patient has an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1 prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. ECOG Performance Status evaluates a patient’s ability to care for themself, their daily activity, and their physical ability (for example, walking, working, etc.). In some embodiments, the human patient has adequate hematologic and endorgan function prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. Adequate hematologic and end-organ function may comprise any of the following laboratory results obtained within 14 days prior to the administration of the RNA vaccine: ANC ≥ 1000 cells/pL; WBC >2000/pL; Platelet count ≥ 100,000/pL; Hemoglobin ≥ 9.0 g/dL; AST, ALT < 3.0 times the upper limit of normal (ULN); Serum bilirubin < 1.5 times ULN; PTT/PT < 1.5 x ULN or INR < 1.7 times ULN; Serum creatinine < 1.5 times ULN; creatinine clearance ≥ 30 mL/min (using the Cockcroft-Gault formula). In some embodiments, a patient is transfused to meet the hemoglobin ≥ 9.0 g/dL criteria. In some embodiments, a patient receives erythropoietic treatment to meet the hemoglobin ≥ 9.0 g/dL criteria. In some embodiments, a patient has known Gilbert disease and a serum bilirubin level < 3 times ULN. In some embodiments, a patient has PTT/PT < 1.5 x ULN or INR < 1.7 times ULN and is not receiving therapeutic anti coagulation. In some embodiments, a patient does not have PTT/PT < 1.5 x ULN or INR < 1.7 times ULN and is receiving a stable dose of therapeutic anti coagulation. In some embodiments, a patient has a negative HIV test prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, a patient has a positive HIV test prior to administration of the RNA vaccine and the PD-1 axis binding antagonist and is on anti retroviral therapy with a CD4 count ≥ 200/pL and an undetectable viral load. In some embodiments, a patient has no evidence of active hepatitis B prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. No evidence of active hepatitis B may comprise having a negative hepatitis B surface antigen (HbsAg) test prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, a patient has had a past or resolved hepatitis B infection prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. A past or resolved hepatitis B infection may comprise a negative HbsAg test, a positive total hepatitis B core antibody (HbcAb) test, and a hepatitis B virus (HBV) DNA test demonstrating absence of active infection prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, a patient has negative hepatitis C virus (HCV) antibody test prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, a patient has positive hepatitis C virus (HCV) antibody test followed by a negative HCV RNA test prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0222] In some embodiments, the human patient has not received a partial cystectomy in the setting of bladder cancer primary tumor prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received partial nephroureterectomy in the setting of renal pelvis primary tumor prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received an approved anti-cancer therapy, including chemotherapy, or hormonal therapy within 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, or more than 10 weeks prior to initiation of study treatment prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received an approved anti-cancer therapy, including chemotherapy, or hormonal therapy within 3 weeks prior to initiation of study treatment prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received adjuvant chemotherapy for MIUC following surgical resection prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received radiation therapy for MIUC following surgical resection prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received antegrade or retrograde instillation of chemotherapy or BCG for UTUC following surgical resection prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received a single dose of intravesical chemotherapy for UTUC following nephroureterectomy prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0223] In some embodiments, the human patient has not received a diagnosis of a malignancy other than a MIUC within 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 years prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received a diagnosis of a malignancy other than a MIUC within 5 years prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received a diagnosis of localized low risk prostate cancer (e.g, Stage < T2b, Gleason score < 7, and/or prostate-specific antigen (PSA) at prostate cancer diagnosis < 20 ng/mL), which was subsequently treated with curative intent and without PSA recurrence within 5 years prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received a diagnosis of low-risk prostate cancer (e.g., Stage Tl/T2a, Gleason score < 7, and/or PSA < 10 ng/mL) within 5 years prior to administration of the RNA vaccine and the PD-1 axis binding antagonist and is treatment- naive and undergoing active surveillance at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has received, within 5 years prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, a diagnosis of a malignancy with a negligible risk of metastasis or death. In some embodiments, the malignancy with a negligible risk of metastasis or death carries a risk of metastasis or death of less than 5% at 5 years. In some embodiments, the patient having received the diagnosis of a malignancy with a negligible risk of metastasis or death had the malignancy treated with expected curative intent within 5 years prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. Expected curative intent could include, for example, adequately treated carcinoma in situ of the cervix, basal or squamous cell skin cancer, and/or ductal carcinoma in situ treated surgically with curative intent. In some embodiments, the patient having received the diagnosis of a malignancy with a negligible risk of metastasis or death has no evidence of recurrence or metastasis by follow-up imaging and any disease-specific tumor markers by a time within 5 years prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0224] In some embodiments, the MIUC tumor exhibits a nodal stage of N+ prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the MIUC tumor exhibits a nodal stage of N+ within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the MIUC tumor exhibits a nodal stage of NO prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the MIUC tumor exhibits a nodal stage of NO within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the MIUC tumor exhibits a PD-L1 IHC score of <1% prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the MIUC tumor exhibits a PD-L1 IHC score of <1% within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the MIUC tumor exhibits a PD-L1 IHC score of ≥1% prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the MIUC tumor exhibits a PD-L1 IHC score of >1% within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the MIUC tumor exhibits an indeterminate PD-L1 IHC score prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the MIUC tumor exhibits an indeterminate PD-L1 IHC score within about a week, within about 5 days, within about 3 days, or less than 3 days before administration of the RNA vaccine. In some embodiments, the patient has received neoadjuvant therapy for treatment of the MIUC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has not received neoadjuvant therapy for treatment of the MIUC prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0225] In some embodiments, the human patient has not received a major surgical procedure, other than for diagnosis or for resection of the MIUC, within 0-1, 1-2, 2-3, 3-4, 4- 5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, or 19-10 weeks prior to prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received a major surgical procedure, other than for diagnosis or for resection of the MIUC, less than 6 weeks prior to prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has had a placement of a central venous access catheter (e.g., port or similar) within 6 weeks prior to prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient does not anticipate a major surgical procedure within about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, or about 7 years after the first administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0226] In some embodiments, the human patient has not received a diagnosis of a significant cardiovascular disease (such as, for example, New York Heart Association Class II or greater cardiac disease, myocardial infarction, cerebrovascular accident, unstable arrhythmia, and/or unstable angina), within 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, or 12-13 months prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not received a diagnosis of a significant cardiovascular disease (such as, for example, New York Heart Association Class II or greater cardiac disease, myocardial infarction, cerebrovascular accident, unstable arrhythmia, and/or unstable angina), within 3 months prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient does not have clinically significant liver disease at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist. Clinically significant liver disease may comprise active viral, alcoholic, or other hepatitis, cirrhosis, inherited liver disease, and/or current alcohol abuse.
[0227] In some embodiments, the patient does not have active autoimmune disease or immune deficiency at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient does not have a history of autoimmune disease or immune deficiency at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist. Exemplary autoimmune diseases include but are not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, antiphospholipid antibody syndrome, granulomatosis with polyangiitis, Sjogren syndrome, Guillain Barre syndrome, and multiple sclerosis. In some embodiments, the patient has a history of autoimmune-related hypothyroidism and is on thyroid replacement hormone at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has controlled Type 1 diabetes mellitus and is on an insulin regimen at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the patient has eczema, psoriasis, lichen simplex chronicus, or vitiligo with dermatologic manifestations only (e.g., not psoriatic arthritis) at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist, and meet at least one of the following criteria at the time of administration of the RNA vaccine and the PD-1 axis binding antagonist: the rash covers less than 10% of patient’s body surface area; the disease is well controlled at baseline and requires only low-potency topical corticosteroids; and there has been no occurrence of acute exacerbations of the underlying condition requiring psoralen plus ultraviolet A radiation, methotrexate, retinoids, biologic agents, oral calcineurin inhibitors, or high potency or oral corticosteroids within the previous 12 months.
[0228] In some embodiments, the human patient has a spleen prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not had loss of spleen due to splenectomy, splenic injury/infarction, or functional asplenia prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0229] In some embodiments, the human patient does not have a known primary cellular immunodeficiency (e.g., DiGeorge syndrome, T negative severe combined immunodeficiency (SCID)) prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient does not have a known primary combined T- and B-cell immunodeficiency (e.g., T- and B-negative SCID, Wiskott-Aldrich syndrome, ataxia telangiectasia, common variable immunodeficiency) prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0230] In some embodiments, the human patient has not been treated with: monoamine oxidase inhibitors (MAOIs) within 3 weeks, a systemic immunostimulatory agent within 4 weeks or 5 drug-elimination half-lives, whichever is longer, or a systemic immunosuppressive medication within 2 weeks, prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, the human patient has not had an allogeneic stem cell or solid organ transplantation prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
[0231] In some embodiments, the patient has not been treated with systemic immunosuppressive medication (including, but not limited to: corticosteroids, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents) within 2 weeks prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, or anticipation of need for systemic immunosuppressive medication during administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, a patient has received acute, low-dose systemic immunosuppressant medication or a one-time pulse dose of systemic immunosuppressant medication (e.g., 48 hours of corticosteroids for a contrast allergy). In some embodiments, a patient has received mineralocorticoids (e.g., fludrocortisone), inhaled or low dose corticosteroids (defined as less than or equal to 10 mg oral prednisone per day or daily equivalent) for chronic obstructive pulmonary disease or asthma, or low-dose corticosteroids for orthostatic hypotension or adrenal insufficiency. [0232] In some embodiments, a patient does not have a history of idiopathic pulmonary fibrosis, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced pneumonitis, or idiopathic pneumonitis, or evidence of active pneumonitis on screening chest CT scan prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, a patient does not have known active or latent tuberculosis prior to administration of the RNA vaccine and the PD-1 axis binding antagonist. In some embodiments, a patient does not have recent acute infection (e.g., severe infection within about 4 weeks prior to administration of the RNA vaccine and the PD-1 axis binding antagonist and including, but not limited to, for example, hospitalization for complications of infection, bacteremia, or severe pneumonia, or any active infection that could impact patient safety) prior to administration of the RNA vaccine and the PD-1 axis binding antagonist.
Response to Administration
[0233] In some embodiments, the method described herein further comprises assessing disease-free survival (DFS) of the human patient after treatment with the RNA vaccine and the PD-1 axis binding antagonist. DFS is measured as the time from patient randomization to the first of either the first recurrence of MIUC, as determined by the investigator, or death from any cause. In some embodiments, disease recurrence may comprise any of the following: local (pelvic) recurrence of MIUC (including soft tissue and regional lymph nodes); urinary tract recurrence of MIUC (including all pathological stages and grades); and/or distant metastasis of UC. In some embodiments, administration of the RNA vaccine and the PD-1 axis binding antagonist results in an improvement in DFS of the human patient as compared to DFS of a corresponding human patient who was not administered the RNA vaccine and the PD-1 axis binding antagonist.
[0234] In some embodiments, the method described herein further comprises assessing overall survival (OS) of the human patient after treatment with the RNA vaccine and the PD- 1 axis binding antagonist. OS is measured as the time from patient randomization to death from any cause. In some embodiments, administration of the RNA vaccine and the PD-1 axis binding antagonist results in an improvement in OS of the human patient as compared to OS of a corresponding human patient who was not administered the RNA vaccine and the PD-1 axis binding antagonist.
[0235] In some embodiments, the method described herein further comprises performing one or more clinical assessments of the human patient before, during and/or after treatment with the RNA vaccine and the PD-1 axis binding antagonist, wherein the one or more clinical assessments are selected from the group consisting of European Organisation for Research and Treatment of Cancer QLQ-C30 Questionnaire (EORTC QLQ C30), European Organisation for Research and Treatment of Cancer QLQ-PAN26 Questionnaire (EORTC QLQ PAN26), National Cancer Institute's Patient-Reported Outcomes Common Terminology Criteria for Adverse Events (PRO CTCAE), European Organisation for Research and Treatment of Cancer Item Library 46 Questionnaire (EORTC IL46), and EuroQol (EQ)-5D- 5L. In some embodiments, the clinical assessments are administered in the following order: EORTC QLQ-C30, PRO-CTCAE, EORTC IL46, and EQ-5D-5L. The QLQ-C30 consists of 30 questions that assess five aspects of patient functioning (physical, emotional, role, cognitive, and social), three symptom scales (fatigue, nausea and vomiting, pain), global health and quality of life, and six single items (dyspnea, insomnia, appetite loss, constipation, diarrhea, and financial difficulties) with a recall period of the previous week. Scale scores can be obtained for the multi-item scales. PRO CTCAE is used to characterize the presence, frequency of occurrence, severity, and/or degree of interference with daily function of 78 patient-reportable symptomatic treatment toxi cities (see, e.g., Basch et al., J Natl Cancer Inst;106:dju244 (2014); and Dueck et al., JAMA Oncol; 1 : 1051-1059 (2015)). The PRO- CTCAE contains 124 questions that are rated either dichotomously (for determination of presence vs. absence) or on a 5-point Likert scale (for determination of frequency of occurrence, severity, and interference with daily function). Treatment toxi cities can occur with observable signs (e.g., vomiting) or non-observable symptoms (e.g., nausea). The standard PRO-CTCAE recall period is the previous 7 days. IL46 is a validated single-item question used to assess the overall impact of side effects and is used along with the PRO CTCAE to assess treatment tolerability. Symptomatic adverse events from the PRO-CTCAE item bank include, but may not be limited to, mouth/throat sores, nausea, vomiting, diarrhea, shortness of breath, cough, rash, hair loss, hand-foot syndrome, neuropathy, dizziness, headache, arthralgia, fatigue, bruising, chills, nosebleeds, injection- or IV-site pain. The EQ- 5D-5L is a validated self-reported health status questionnaire that is used to calculate a health status utility score for use in health economic analyses (EuroQol Group 1990; Brooks 1996; Herdman et al. 2011; Janssen et al. 2013). There are two components to the EQ-5D-5L: a five-item health state profile that assesses mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, as well as a Visual Analog Scale (VAS) that measures health state. The EQ-5D-5L is designed to capture a patient’s current health status. Published weighting systems allow for creation of a single composite score of the patient’s health status. The EQ 5D-5L takes approximately 3 minutes to complete and will be used with the treatments described herein for informing pharmacoeconomic evaluations. In some embodiments, administration of the RNA vaccine and the PD-1 axis binding antagonist results in an improvement in the one or more clinical assessments as compared to the one or more clinical assessments in the human patient prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, and/or as compared to the one or more clinical assessments in a corresponding human patient who is not administered the RNA vaccine and the PD-1 axis binding antagonist.
[0236] In some embodiments, the method described herein further comprises assessing biomarkers, changes or clearance of ctDNA, plasma concentrations of DOTMA, blood concentration of mRNA, serum concentrations of nivolumab, and/or prevalence of anti-drug antibodies (AD As) to nivolumab prior to administration of the RNA vaccine and the PD-1 axis binding antagonist, during the priming phase described herein, after the priming phase described herein, during the booster phase described herein, and/or after the booster phase described herein.
[0237] In some embodiments, the corresponding human patient is a human patient with a corresponding MIUC. In some embodiments, the MIUC is a MIBC tumor, and the corresponding human patient has a MIUC tumor. In some embodiments, the MIUC is a UTUC tumor, and the corresponding human patient has a UTUC tumor. In some embodiments, the corresponding human patient was treated with a standard of care treatment for UC, MIUC, or resectable or resected MIUC. In some embodiments, the standard of care treatment comprises cystectomy, nephroureterectomy, neoadjuvant cisplatin-based therapy, adjuvant cisplatin therapy, and/or administration of nivolumab.
Adverse Events
[0238] Some embodiments involve assessment of adverse events. An adverse event in the context of the present disclosure may include any of the following: Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiological scans, vital sign measurements), including those that worsen from baseline, considered clinically significant in the medical and scientific judgment of the investigator (i.e., not related to progression of underlying disease); exacerbation of a chronic or intermittent preexisting condition, including either an increase in frequency and/or intensity of the condition; new condition detected or diagnosed after study treatment administration, even though it may have been present before the start of the study; signs, symptoms, or clinical sequelae of a suspected drug-drug interaction; signs, symptoms, or clinical sequelae of a suspected overdose of either study treatment or a concomitant medication; overdose per se is not reported as an adverse event or serious adverse event unless it is an intentional overdose taken with possible suicidal or self harming intent; such overdoses should be reported regardless of sequelae; “lack of efficacy” or “failure of expected pharmacological action” per se is not an adverse event or serious adverse event, but the signs, symptoms, and/or clinical sequelae resulting from lack of efficacy may be an adverse event or serious adverse event if they fulfill the definition of an adverse event or serious adverse event.
[0239] The following events are not considered adverse events as described herein: Any clinically significant abnormal laboratory findings or other abnormal safety assessments that are associated with the underlying disease, unless judged by the investigator to be more severe than expected for the patient’s condition; the disease or disorder being studied or expected progression, signs, or symptoms of the disease or disorder being studied, unless more severe than expected for the patient’s condition; medical or surgical procedure (e.g., endoscopy, appendectomy; the condition that leads to the procedure is the adverse event); situations in which an untoward medical occurrence did not occur (social and/or convenience admission to a hospital); and anticipated day-to-day fluctuations of a preexisting disease or condition present or detected at the start of the study that do not worsen. If an event is not an adverse event per the definition in Section A3-1, it cannot be a serious adverse event even if serious conditions are met (e.g., hospitalization for signs or symptoms of the disease under study, death due to progression of disease).
[0240] A serious adverse event is defined as any untoward medical occurrence that, at any dose: Results in death; is life threatening (i.e., an event in which the participant was at risk of death at the time of the event; not an event that hypothetically might have caused death if it were more severe); requires inpatient hospitalization or prolongation of existing hospitalization (complications that occur during hospitalization are adverse events; if a complication prolongs hospitalization or fulfills any other seriousness criteria, the event is serious; hospitalization for elective treatment of a preexisting condition that did not worsen from baseline is not considered an adverse event); results in persistent disability or incapacity (i.e., a substantial disruption of a person’s ability to conduct normal life functions; not intended to include experiences of relatively minor medical significance, such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental trauma (e.g., sprained ankle) that may interfere with or prevent everyday life functions but do not constitute a substantial disruption); is a congenital anomaly or birth defect; or other situations according to medical or scientific judgement.
[0241] In describing an adverse event herein, the terms “severe” and “serious” are not synonymous. Severity refers to the intensity of an adverse event (e.g., rated as mild, moderate, or severe, or according to National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE); see Section A3-3.2); the event itself may be of relatively minor medical significance (such as severe headache without any further findings)
Compositions for Use in Treating Muscle Invasive Urothelial Carcinoma
[0242] In one aspect, there is provided an individualized RNA vaccine for use in a method for treating a MIUC in a human patient in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC tumor specimen obtained from the human patient. In another aspect, there is provided a use of an individualized RNA vaccine in the manufacture of a medicament for treating a UC in a human patient in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC tumor specimen obtained from the human patient.
[0243] In one aspect, there is provided a PD-1 axis binding antagonist for use in a method for treating a UC in a human patient in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC tumor specimen obtained from the human patient. In another aspect, there is provided a use of a PD-1 axis binding antagonist in the manufacture of a medicament for treating a UC in a human patient in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC tumor specimen obtained from the human patient.
Methods of Administration and Additional Therapies
[0244] The PD-1 axis binding antagonist and the RNA vaccine may be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the RNA vaccine is administered (e.g., in a lipoplex or lipid nanoparticle) intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the RNA vaccine is administered (e.g., in a lipoplex) intravenously. In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are administered intravenously.
[0245] The PD-1 axis binding antagonist and the RNA vaccine may be administered in any order when administered on the same day, e.g., during the priming and/or boost phases of treatment. For example, the PD-1 axis binding antagonist and the RNA vaccine may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the RNA vaccine is administered before the PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are in separate compositions. In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are in the same composition.
[0246] More than one RNA vaccine may be administered to a patient, e.g., the patient may be administered one RNA vaccine with a combination of neoepitopes and also administered a separate RNA vaccine with a different combination of neoepitopes. In some embodiments, a first RNA vaccine with, for example 5-20 or 10-20 neoepitopes, is administered in combination with a second RNA vaccine with, for example 5-20 or 10-20 different or alternative neoepitopes.
[0247] In some embodiments, the methods for treating MIUC provided herein may further comprise administering to the patient an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., resection of a MIUC tumor), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti- metastatic agent. In some embodiments, the additional therapy is the administration of sideeffect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery, e.g., resection of a MIUC tumor. In some embodiments, the additional therapy is a combination of radiation therapy and surgery, e.g., resection of a MIUC tumor. In some embodiments, the additional therapy is gamma irradiation.
III. RNA Vaccines
[0248] Certain aspects of the present disclosure relate to individualized cancer vaccines (ICVs). In some embodiments, the individualized cancer vaccine is an RNA vaccine. Features of exemplary RNA vaccines are described infra. In some embodiments, the present disclosure provides an RNA polynucleotide comprising one or more of the features/sequences of the RNA vaccines described infra. In some embodiments, the RNA polynucleotide is a single-stranded mRNA polynucleotide. In other embodiments, the present disclosure provides a DNA polynucleotide encoding an RNA comprising one or more of the features/sequences of the RNA vaccines described infra.
[0249] Individualized cancer vaccines comprise individualized neoantigens (z.e., tumor- associated antigens (TAAs) that are specifically expressed in the patient's cancer) identified as having potential immunostimulatory activities. In the embodiments described herein, the individualized cancer vaccine is a nucleic acid, e.g., messenger RNA. Accordingly, without wishing to be bound by theory, it is believed that upon administration, the individualized cancer vaccine (e.g., an RNA vaccine of the disclosure) is taken up and translated by antigen presenting cells (APCs) and the expressed protein is presented via major histocompatibility complex (MHC) molecules on the surface of the APCs. This leads to an induction of both cytotoxic T-lymphocyte (CTL)-and memory T-cell -dependent immune responses against cancer cells expressing the TAA(s).
[0250] Individualized cancer vaccines (e.g., an RNA vaccine) typically include multiple neoantigen epitopes (“neoepitopes”), e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 neoepitopes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 neoepitopes, optionally with linker sequences between the individual neoepitopes. In some embodiments, a neoepitope as used herein refers to a novel epitope that is specific for a patient’s cancer but not found in normal cells of the patient. In some embodiments, the neoepitope is presented to T cells when bound to MHC. In some embodiments, the individualized cancer vaccine also includes a 5’ mRNA cap analogue, a 5’ UTR, a signal sequence, a domain to facilitate antigen expression, a 3’ UTR, and/or a polyA tail. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 or 10- 20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding at least 5 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-10 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen.
[0251] In some embodiments, the RNA vaccine comprises an individualized neoantigen specific immunotherapy (iNeST). In some embodiments, the RNA vaccine comprises autogene cevumeran. Autogene cevumeran is an individualized neoantigen-specific immunotherapy based on the immunotherapeutic targeting of unique mutations in a patient’s tumor. Autogene cevumeran is designed by using immunogenic neoantigen epitopes (neoepitopes) that are predicted from somatic mutations identified by paired next-generation sequencing (NGS) of a patient’s peripheral blood and tumor tissue and quantified by RNA sequencing. Without wishing to be bound by theory, it is believed that each patient’s cancer has its own unique repertoire of non-synonymous mutations that encode neoantigens; these neoantigens are believed to play an important role in anti-tumor immunity (Alexandrov et al. Signatures of mutational processes in human cancer. Nature 2013;500:415-421; van Rooij et al. Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab- responsive melanoma. J Clin Oncol 2013;31 :e439-42; Brown et al. Neoantigens predicted by tumor genome meta analysis correlate with increased patient survival. Genome Res 2014;24:743-50; Snyder et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 2014;371 :2189-99; Le et al. PD-1 blockade in tumors with mismatch -repair deficiency. N Engl J Med 2015;372:2509-20; Rizvi et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 2015;348: 124-8; Van Allen et al. Genomic correlates of response to CTLA 4 blockade in metastatic melanoma. Science 2015;350:207-11; Rosenberg et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum -based chemotherpay: a single-arm, multicentre, Phase 2 trial. Lancet 2016;387: 1909-20; Ramalingam et al. Abstract CT078: Tumor mutational burden (TMB) as a biomarker for clinical benefit from dual immune checkpoint blockade with nivolumab (nivo) + ipilimumab (ipi) in first-line (IL) non-small cell lung cancer (NSCLC): identification of TMB cutoff from CheckMate 568 [abstract]. Cancer Res 2018;78(Suppl 13):CT078). The neoantigen candidates are predicted from somatic mutations identified by paired next-generation sequencing (NGS) of a patient’s peripheral blood and tumor tissue and quantified by RNA sequencing. In some embodiments, an autogene cevumeran drug product comprises up to 2 messenger RNA (mRNA) molecules, each encoding up to 10 neoantigens (for a total of up to 20 neoantigens) specific to a patient’s cancer.
[0252] In some embodiments, the manufacture of an RNA vaccine of the present disclosure is a multi-step process, whereby somatic mutations in the patient's tumor are identified by next-generation sequencing (NGS) and immunogenic neoantigen epitopes (or "neoepitopes"; NEs) are predicted. The RNA cancer vaccine targeting the selected neoepitopes is manufactured on a per-patient basis. In some embodiments, the vaccine is an RNA-based cancer vaccine consisting of up to two messenger RNA molecules, each encoding up to 10 neoepitopes (for a total of up to 20 neoepitopes), which are specific to the patient's tumor. [0253] In some embodiments, the RNA vaccine is manufactured as described in Rojas et al. (Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature 618, 144-150 (2023). https://doi.org/10.1038/s41586-023-06063-y), which is hereby incorporated by reference in its entirely. For example, in some embodiments, individualized mRNA neoantigen vaccines are manufactured under good manufacturing practice conditions containing two uridine-based mRNA strands with noncoding sequences optimized for superior translational performance (see, e.g., Holtkamp et al., Modification of antigenencoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009-4017 (2006); Kreiter et al., Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692-696 (2015)). In some such embodiments, each mRNA strand encodes up to 10 MHCI and MHCII neoepitopes. In some embodiments, the mRNA strands of the mRNA vaccine are formulated in approximately 400 nm diameter lipoplex nanoparticles (e.g., as described in Kranz et al., Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534, 396-401 (2016)). In some such embodiments, the lipoplex nanoparticles comprise the synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleyloxy-l-propanaminium chloride (DOTMA) and the phospholipid l,2-dioleoyl-sn-glycero-3 -phosphatidylethanolamine (DOPE) to enable intravenous delivery.
[0254] In some embodiments, expressed non-synonymous mutations are identified by whole exome sequencing (WES) of tumor DNA and peripheral blood mononuclear cell (PBMC) DNA (as a source of healthy tissue from the patient) as well as tumor RNA sequencing (to assess expression). From the resulting list of mutant proteins, potential neoantigens are predicted using a bioinformatics workflow that ranks their likely immunogenicity on the basis of multiple factors, including the binding affinity of the predicted epitope to individual major histocompatibility complex (MHC) molecules, and the level of expression of the associated RNA. The mutation discovery, prioritization, and confirmation processes are complemented by a database that provides comprehensive information about expression levels of respective wild-type genes in healthy tissues. This information enables the development of a personalized risk mitigation strategy by removing target candidates with an unfavorable risk profile. Mutations occurring in proteins with a possible higher auto-immunity risk in critical organs are filtered out and not considered for vaccine production. In some embodiments, up to 20 MHCI and MHCII neoepitopes that are predicted to elicit CD8+ T-cell and/or CD4+ T-cell responses, respectively, for an individual patient are selected for inclusion into the vaccine. Vaccinating against multiple neoepitopes is expected to increase the breadth and magnitude of the overall immune response to individualized cancer vaccines and may help to mitigate the risk of immune escape, which can occur when tumors are exposed to the selective pressure of an effective immune response (Tran E, Robbins PF, Lu YC, et al. N Engl J Med 2016;375:2255-62; Verdegaal EM, de Miranda NF, Visser M, et al. Nature 2016;536:91-5).
[0255] In some embodiments, the RNA vaccine comprises one or more polynucleotide sequences encoding an amino acid linker. For example, amino acid linkers can be used between 2 tumor-specific neoepitope sequences, between a tumor-specific neoepitope sequence and a fusion protein tag (e.g., comprising sequence derived from an MHC complex polypeptide), or between a secretory signal peptide and a tumor-specific neoepitope sequence. In some embodiments, the RNA vaccine encodes multiple linkers. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-20 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen, and the polynucleotides encoding each epitope are separated by a polynucleotide encoding a linker sequence. In some embodiments, the RNA vaccine comprises one or more polynucleotides encoding 5-10 neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen, and the polynucleotides encoding each epitope are separated by a polynucleotide encoding a linker sequence. In some embodiments, polynucleotides encoding linker sequences are also present between the polynucleotides encoding an N- terminal fusion tag (e.g., a secretory signal peptide) and a polynucleotide encoding one of the neoepitopes and/or between a polynucleotide encoding one of the neoepitopes and the polynucleotides encoding a C-terminal fusion tag (e.g., comprising a portion of an MHC polypeptide). In some embodiments, two or more linkers encoded by the RNA vaccine comprise different sequences. In some embodiments, the RNA vaccine encodes multiple linkers, all of which share the same amino acid sequence.
[0256] In some embodiments, the RNA molecule further comprises a polynucleotide sequence encoding an amino acid linker; wherein the polynucleotide sequences encoding the amino acid linker and a first of the one or more neoepitopes form a first linker-neoepitope module; and wherein the polynucleotide sequences forming the first linker-neoepitope module are between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule in the 5’->3’ direction. In some embodiments, the RNA molecule further comprises, in the 5’->3’ direction: at least a second linker-neoepitope module, wherein the at least second linker-neoepitope module comprises a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope; wherein the polynucleotide sequences forming the second linker-neoepitope module are between the polynucleotide sequence encoding the neoepitope of the first linker- neoepitope module and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule in the 5 ’->3’ direction; and wherein the neoepitope of the first linker-neoepitope module is different from the neoepitope of the second linker-neoepitope module. In some embodiments, the RNA molecule comprises 5 linker-neoepitope modules, and wherein the 5 linker-neoepitope modules each encode a different neoepitope. In some embodiments, the RNA molecule comprises 10 linker- neoepitope modules, and wherein the 10 linker-neoepitope modules each encode a different neoepitope. In some embodiments, the RNA molecule comprises 20 linker-neoepitope modules, and wherein the 20 linker-neoepitope modules each encode a different neoepitope. In some embodiments, the RNA molecule further comprises a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is between the polynucleotide sequence encoding the neoepitope that is most distal in the 3’ direction and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule. [0257] A variety of linker sequences are known in the art. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises G, S, A, and/or T residues. In some embodiments, the linker consists of glycine and serine residues. In some embodiments, the linker is between about 5 and about 20 amino acids or between about 5 and about 12 amino acids in length, e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids in length. In some embodiments, the linker comprises the sequence GGSGGGGSGG (SEQ ID NO:39). In some embodiments, the linker of the RNA vaccine comprises the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO:37). In some embodiments, the linker of the RNA vaccine is encoded by DNA comprising the sequence GGCGGCTCTGGAGGAGGCGGCTCCGGAGGC (SEQ ID NO:38).
[0258] In some embodiments, the RNA vaccine comprises a 5’ cap. The basic mRNA cap structure is known to contain a 5 ’-5’ triphosphate linkage between 2 nucleosides (e.g., two guanines) and a 7-methyl group on the distal guanine, z.e., m7GpppG. Exemplary cap structures can be found, e.g., in U.S. Pat. Nos. 8,153,773 and 9,295,717 and Kuhn, A.N. et al. (2010) Gene Ther. 17:961-971. In some embodiments, the 5’ cap has the structure m27’2 ' °GppspG. In some embodiments, the 5’ cap is a beta-S-ARCA cap. The S-ARCA cap structure includes a 2’-0 methyl substitution (e.g., at the C2’ position of the m7G) and an S- substitution at one or more of the phosphate groups. In some embodiments, the 5’ cap comprises the structure:
[0259] In some embodiments, the 5’ cap is the DI diastereoisomer of beta-S-ARCA (see, e.g., U.S. Pat. No. 9,295,717). The * in the above structure indicates a stereogenic P center, which can exist in two diastereoisomers (designated DI and D2). The DI diastereomer of beta-S-ARCA or beta-S-ARCA(Dl) is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6x250 mm is used for separation, whereby a flow rate of 1.3 ml/min can be applied. In one embodiment, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH=5.9, within 15 min is used. UV- detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.
[0260] In some embodiments, the RNA vaccine comprises a 5’ UTR. Certain untranslated sequences found 5’ to protein-coding sequences in mRNAs have been shown to increase translational efficiency. See, e.g., Kozak, M. (1987) J. Mol. Biol. 196:947-950. In some embodiments, the 5’ UTR comprises sequence from the human alpha globin mRNA. In some embodiments, the RNA vaccine comprises a 5’ UTR sequence of UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO:23). In some embodiments, the 5’ UTR sequence of the RNA vaccine is encoded by DNA comprising the sequence TTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO:24). In some embodiments, the 5’ UTR sequence of RNA vaccine comprises the sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO:21). In some embodiments, the 5’ UTR sequence of RNA vaccine is encoded by DNA comprising the sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO:22).
[0261] In some embodiments of the methods provided herein, the constant region of an exemplary RNA vaccine comprises the ribonucleotide sequence (5'->3') of SEQ ID NO: 42. The linkage between the first two G residues is the unusual bond (5' - > 5')-ppsp-, e.g., as shown in Table 3. “N” refers to the position of polynucleotide sequence(s) encoding one or more (e.g., 1-20) neoepitopes (separated by optional linkers). The insertion site for tumorspecific sequences (C131-A132; marked in bold text) is depicted in bold text. See Table 3 for the modified bases and uncommon links in the exemplary RNA sequence.
Table 3
[0262] In some embodiments, the RNA vaccine comprises a polynucleotide sequence encoding a secretory signal peptide. As is known in the art, a secretory signal peptide is an amino acid sequence that directs a polypeptide to be trafficked from the endoplasmic reticulum and into the secretory pathway upon translation. In some embodiments, the signal peptide is derived from a human polypeptide, such as an MHC polypeptide. See, e.g., Kreiter, S. et al. (2008) J. Immunol. 180:309-318, which describes an exemplary secretory signal peptide that improves processing and presentation of MHC Class I and II epitopes in human dendritic cells. In some embodiments, upon translation, the signal peptide is N-terminal to one or more neoepitope sequence(s) encoded by the RNA vaccine. In some embodiments, the secretory signal peptide comprises the sequence MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO:27). In some embodiments, the secretory signal peptide of the RNA vaccine comprises the sequence AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGC CCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO:25). In some embodiments, the secretory signal peptide of the RNA vaccine is encoded by DNA comprising the sequence ATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCC TGACAGAGACATGGGCCGGAAGC (SEQ ID NO:26).
[0263] In some embodiments, the RNA vaccine comprises a polynucleotide sequence encoding at least a portion of a transmembrane and/or cytoplasmic domain. In some embodiments, the transmembrane and/or cytoplasmic domains are from the transmembrane/ cytoplasmic domains of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" relate to a complex of genes which occurs in all vertebrates. The function of MHC proteins or molecules in signaling between lymphocytes and antigen-presenting cells in normal immune responses involves them binding peptides and presenting them for possible recognition by T-cell receptors (TCR). MHC molecules bind peptides in an intracellular processing compartment and present these peptides on the surface of antigen-presenting cells to T cells. The human MHC region, also referred to as HLA, is located on chromosome 6 and comprises the class I region and the class II region. The class I alpha chains are glycoproteins having a molecular weight of about 44 kDa. The polypeptide chain has a length of somewhat more than 350 amino acid residues. It can be divided into three functional regions: an external, a transmembrane and a cytoplasmic region. The external region has a length of 283 amino acid residues and is divided into three domains, alphal, alpha2 and alpha3. The domains and regions are usually encoded by separate exons of the class I gene. The transmembrane region spans the lipid bilayer of the plasma membrane. It consists of 23 usually hydrophobic amino acid residues which are arranged in an alpha helix. The cytoplasmic region, z.e., the part which faces the cytoplasm and which is connected to the transmembrane region, typically has a length of 32 amino acid residues and is able to interact with the elements of the cytoskeleton. The alpha chain interacts with beta2 -microglobulin and thus forms alpha-beta2 dimers on the cell surface. The term "MHC class II" or "class II" relates to the major histocompatibility complex class II proteins or genes. Within the human MHC class II region there are the DP, DQ, and DR subregions for class II alpha chain genes and beta chain genes (z.e., DPalpha, DPbeta, DQalpha, DQbeta, DRalpha and DRbeta). Class II molecules are heterodimers each consisting of an alpha chain and a beta chain. Both chains are glycoproteins having a molecular weight of 31-34 kDa (a) or 26-29 kDA (beta). The total length of the alpha chains varies from 229 to 233 amino acid residues, and that of the beta chains from 225 to 238 residues. Both alpha and beta chains consist of an external region, a connecting peptide, a transmembrane region, and a cytoplasmic tail. The external region consists of two domains, alphal and alpha2 or betal and beta2. The connecting peptide is respectively beta and 9 residues long in alpha and beta chains. It connects the two domains to the transmembrane region which consists of 23 amino acid residues both in alpha chains and in beta chains. The length of the cytoplasmic region, z.e., the part which faces the cytoplasm and which is connected to the transmembrane region, varies from 3 to 16 residues in alpha chains and from 8 to 20 residues in beta chains. Exemplary transmembrane/cytoplasmic domain sequences are described in U.S. Pat. Nos. 8,178,653 and 8,637,006. In some embodiments, upon translation, the transmembrane and/or cytoplasmic domain is C-terminal to one or more neoepitope sequence(s) encoded by the RNA vaccine. In some embodiments, the transmembrane and/or cytoplasmic domain of the MHC molecule encoded by the RNA vaccine comprises the sequence
IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO:30). In some embodiments, the transmembrane and/or cytoplasmic domain of the MHC molecule comprises the sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU GACAGCC (SEQ ID NO:28). In some embodiments, the transmembrane and/or cytoplasmic domain of the MHC molecule is encoded by DNA comprising the sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCC GTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGC TACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACA GCC (SEQ ID NO:29).
[0264] In some embodiments, the RNA vaccine comprises both a polynucleotide sequence encoding a secretory signal peptide that is N-terminal to the one or more neoepitope sequence(s) and a polynucleotide sequence encoding a transmembrane and/or cytoplasmic domain that is C-terminal to the one or more neoepitope sequence(s). Combining such sequences has been shown to improve processing and presentation of MHC Class I and II epitopes in human dendritic cells. See, e.g., Kreiter, S. et al. (2008) J. Immunol. 180:309-318. [0265] In myeloid DCs, the RNA is released into the cytosol and translated into a poly-neoepitopic peptide. The polypeptide contains additional sequences to enhance antigen presentation. In some embodiments, a signal sequence (sec) from the MHCI heavy chain at the N-terminal of the polypeptide is used to target the nascent molecule to the endoplasmic reticulum, which has been shown to enhance MHCI presentation efficiency. Without wishing to be bound by theory, it is believed that the transmembrane and cytoplasmic domains of MHCI heavy chain guide the polypeptide to the endosomal/lysosomal compartments that were shown to improve MHCII presentation.
[0266] In some embodiments, the RNA vaccine comprises a 3’UTR. Certain untranslated sequences found 3’ to protein-coding sequences in mRNAs have been shown to improve RNA stability, translation, and protein expression. Polynucleotide sequences suitable for use as 3’ UTRs are described, for example, in PG Pub. No. US20190071682. In some embodiments, the 3’ UTR comprises the 3’ untranslated region of AES or a fragment thereof and/or the non-coding RNA of the mitochondrially encoded 12S RNA. The term “AES” relates to Amino-Terminal Enhancer Of Split and includes the AES gene (see, e.g., NCBI Gene ID: 166). The protein encoded by this gene belongs to the groucho/TLE family of proteins, can function as a homooligomer or as a heteroologimer with other family members to dominantly repress the expression of other family member genes. An exemplary AES mRNA sequence is provided in NCBI Ref. Seq. Accession NO. NM_198969. The term “MT RNRl” relates to Mitochondrially Encoded 12S RNA and includes the MT RNRl gene (see, e.g., NCBI Gene ID:4549). This RNA gene belongs to the Mt_rRNA class. Diseases associated with MT-RNR1 include restrictive cardiomyopathy and auditory neuropathy. Among its related pathways are Ribosome biogenesis in eukaryotes and CFTR translational fidelity (class I mutations). An exemplary MT RNRl RNA sequence is present within the sequence of NCBI Ref. Seq. Accession NO. NC_012920. In some embodiments, the 3’ UTR of the RNA vaccine comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCC GAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACU CACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO:33). In some embodiments, the 3’ UTR of the RNA vaccine comprises the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGG AAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO:35). In some embodiments, the 3’ UTR of the RNA vaccine comprises the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCC GAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACU CACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO:33) and the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGG AAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO:35). In some embodiments, the 3’ UTR of the RNA vaccine comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGG UACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGC CCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCA GCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACC UUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCA AUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID NO:31). In some embodiments, the 3’ UTR of the RNA vaccine is encoded by DNA comprising the sequence
CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGA GTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCC (SEQ ID NO:34). In some embodiments, the 3’ UTR of the RNA vaccine is encoded by DNA comprising the sequence
CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGA AACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAAC CCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (SEQ ID NO:36). In some embodiments, the 3’ UTR of the RNA vaccine is encoded by DNA comprising the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGA GTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCC (SEQ ID NO:34) and the sequence
CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGA AACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAAC CCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG (SEQ ID NO:36). In some embodiments, the 3’ UTR of the RNA vaccine is encoded by DNA comprising the sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGA GTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACG CTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAA ACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCC ACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO:32).
[0267] In some embodiments, the RNA vaccine comprises a poly(A) tail at its 3 ’end. In some embodiments, the poly(A) tail comprises more than 50 or more than 100 adenine nucleotides. For example, in some embodiments, the poly(A) tail comprises 120 adenine nucleotides. This poly(A) tail has been demonstrated to enhance RNA stability and translation efficiency (Holtkamp, S. et al. (2006) Blood 108:4009-4017). In some embodiments, the RNA comprising a poly(A) tail is generated by transcribing a DNA molecule comprising in the 5’ -> 3’ direction of transcription, a polynucleotide sequence that encodes at least 50, 100, or 120 adenine consecutive nucleotides and a recognition sequence for a type IIS restriction endonuclease. Exemplary poly(A) tail and 3’ UTR sequences that improve translation are found, e.g., in U.S. Pat. No. 9,476,055.
[0268] In some embodiments, an RNA vaccine or molecule of the present disclosure comprises the general structure (in the 5’->3’ direction): (1) a 5’ cap; (2) a 5’ untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding at least a portion of a transmembrane and cytoplasmic domain of a major histocompatibility complex (MHC) molecule; (5) a 3’ UTR comprising: (a) a 3’ untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (6) a poly(A) sequence. In some embodiments, an RNA vaccine or molecule of the present disclosure comprises, in the 5’->3’ direction: the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAU GAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCC UGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19); and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU GACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCU UUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUC CCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGC ACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACA GCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACC CCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCU AGCCGCGUCGCU (SEQ ID NO:20). Advantageously, RNA vaccines comprising this combination and orientation of structures or sequences are characterized by one or more of: improved RNA stability, enhanced translational efficiency, improved antigen presentation and/or processing (e.g., by DCs), and increased protein expression. [0269] In some embodiments, an RNA vaccine or molecule of the present disclosure comprises the sequence (in the 5’->3’ direction) of SEQ ID NO:42. In some embodiments, N refers to a polynucleotide sequence encoding at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 different neoepitopes. In some embodiments, N refers to a polynucleotide sequence encoding one or more linker-epitope modules (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 different linkerepitope modules). In some embodiments, N refers to a polynucleotide sequence encoding one or more linker-epitope modules (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 different linkerepitope modules) and an additional amino acid linker at the 3’ end.
[0270] In some embodiments, the RNA vaccine or molecule further comprises a polynucleotide sequence encoding at least one neoepitope; wherein the polynucleotide sequence encoding the at least one neoepitope is between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule in the 5’->3’ direction. In some embodiments, the RNA molecule comprises a polynucleotide sequence encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 different neoepitopes.
[0271] In some embodiments, the RNA vaccine or molecule further comprises, in the 5’->3’ direction: a polynucleotide sequence encoding an amino acid linker; and a polynucleotide sequence encoding a neoepitope. In some embodiments, the polynucleotide sequences encoding the amino acid linker and the neoepitope form a linker-neoepitope module (e.g., a continuous sequence in the 5’->3’ direction in the same open-reading frame). In some embodiments, the polynucleotide sequences forming the linker-neoepitope module are between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule, or between the sequences of SEQ ID NO: 19 and SEQ ID NO:20, in the 5’->3’ direction. In some embodiments, the RNA vaccine or molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 linker-epitope modules. In some embodiments, each of the linkerepitope modules encodes a different neoepitope. In some embodiments, the RNA vaccine or molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linkerepitope modules, and the RNA vaccine or molecule comprises polynucleotides encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 different neoepitopes. In some embodiments, the RNA vaccine or molecule comprises 5, 10, or 20 linker-epitope modules. In some embodiments, each of the linkerepitope modules encodes a different neoepitope. In some embodiments, the linker-epitope modules form a continuous sequence in the 5 ’->3’ direction in the same open-reading frame. In some embodiments, the polynucleotide sequence encoding the linker of the first linkerepitope module is 3’ of the polynucleotide sequence encoding the secretory signal peptide. In some embodiments, the polynucleotide sequence encoding the neoepitope of the last linkerepitope module is 5’ of the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule.
[0272] In some embodiments, the RNA vaccine is at least 800 nucleotides, at least 1000 nucleotides, or at least 1200 nucleotides in length. In some embodiments, the RNA vaccine is less than 2000 nucleotides in length. In some embodiments, the RNA vaccine is at least 800 nucleotides but less than 2000 nucleotides in length, at least 1000 nucleotides but less than 2000 nucleotides in length, at least 1200 nucleotides but less than 2000 nucleotides in length, at least 1400 nucleotides but less than 2000 nucleotides in length, at least 800 nucleotides but less than 1400 nucleotides in length, or at least 800 nucleotides but less than 2000 nucleotides in length. For example, the constant regions of an RNA vaccine comprising the elements described above are approximately 800 nucleotides in length. In some embodiments, an RNA vaccine comprising 5 tumor-specific neoepitopes (e.g., each encoding 27 amino acids) is greater than 1300 nucleotides in length. In some embodiments, an RNA vaccine comprising 10 tumor-specific neoepitopes (e.g., each encoding 27 amino acids) is greater than 1800 nucleotides in length.
[0273] In some embodiments, the one or more polynucleotides of the RNA vaccine are formulated with one or more lipids. In some embodiments, the RNA vaccine is formulated as a lipid nanoparticle, wherein the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipid nanoparticle. In some embodiments, the RNA vaccine is formulated as a lipoplex, wherein the one or more polynucleotides of the RNA vaccine and one or more lipids form the lipoplex. In some embodiments, the lipoplex comprises one or more lipids that form a multilamellar structure that encapsulates the one or more polynucleotides of the RNA vaccine. In some embodiments, a lipoplex formulation for the RNA (RNA-Lipoplex) is used to enable IV delivery of an RNA vaccine of the present disclosure. In some embodiments, a lipoplex formulation for the RNA cancer vaccine comprising the synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleyloxy-l-propanaminium chloride (DOTMA) and the phospholipid l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) is used, e.g., to enable IV delivery. The DOTMA/DOPE liposomal component has been optimized for IV delivery and targeting of antigen-presenting cells in the spleen and other lymphoid organs.
[0274] In some embodiments, the lipid nanoparticles or the lipoplexes comprise at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule, e.g., a molecule which comprises at least one hydrophilic and lipophilic moiety is a cationic lipid within the meaning of the present invention. In one embodiment, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the RNA. In one embodiment, the lipid nanoparticle or lipoplex comprise at least one helper lipid. The helper lipid may be a neutral or an anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In one embodiment, the cationic lipid and/or the helper lipid is a bilayer forming lipid. [0275] In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl- 3 -trimethylammonium propane (DOTMA) or analogs or derivatives thereof and/or 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP) or analogs or derivatives thereof.
[0276] In one embodiment, the at least one helper lipid comprises l,2-di-(9Z- octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE) or analogs or derivatives thereof, cholesterol (Choi) or analogs or derivatives thereof and/or l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) or analogs or derivatives thereof.
[0277] In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9: 1 to 3:7, 4: 1 to 1 :2, 4: 1 to 2:3, 7:3 to 1 : 1, or 2: 1 to 1 : 1, preferably about 1 : 1. In one embodiment, in this ratio, the molar amount of the cationic lipid results from the molar amount of the cationic lipid multiplied by the number of positive charges in the cationic lipid.
[0278] In one embodiment, the lipid is comprised in a vesicle encapsulating said RNA. The vesicle may be a multilamellar vesicle, an unilamellar vesicle, or a mixture thereof. The vesicle may be a lipoplex or lipid nanoparticle.
[0279] RNA vaccine formulations with one or more lipids described herein can be formed by adjusting a positive to negative charge, depending on the (+/-) charge ratio of a cationic lipid to RNA and mixing the RNA and the cationic lipid. The +/- charge ratio of the cationic lipid to the RNA in the lipid nanoparticles or the lipoplexes described herein can be calculated by the following equation. (+/- charge rati o)= [(cationic lipid amount (mol))*(the total number of positive charges in the cationic lipid)]: [(RNA amount (mol))*(the total number of negative charges in RNA)]. The RNA amount and the cationic lipid amount can be easily determined by one skilled in the art in view of a loading amount upon preparation of the nanoparticles or lipoplexes. For further descriptions of exemplary nanoparticles and lipoplexes, see, e.g., PG Pub. No. US20150086612.
[0280] In one embodiment, the overall charge ratio of positive charges to negative charges in the lipid nanoparticle (e.g., at physiological pH) is between 1.4: 1 and 1 :8, preferably between 1.2: 1 and 1 :4, e.g. between 1 : 1 and 1 :3 such as between 1 : 1.2 and 1 :2, 1 : 1.2 and 1 : 1.8, 1 :1.3 and 1 : 1.7, in particular between 1 : 1.4 and 1 : 1.6, such as about 1 : 1.5. In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipid nanoparticles is between 1 : 1.2 (0.83) and 1 :2 (0.5). In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipid nanoparticle is between 1.6:2 (0.8) and 1 :2 (0.5) or between 1.6:2 (0.8) and 1.1 :2 (0.55). In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipid nanoparticle is 1.3:2 (0.65). In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipid nanoparticle is not lower than 1.0:2.0. In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipid nanoparticle is not higher than 1.9:2.0. In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipid nanoparticle is not lower than 1.0:2.0 and not higher than 1.9:2.0.
[0281] In another embodiment, the overall charge ratio of positive charges to negative charges in the lipoplex (e.g., at physiological pH) is between 1.4: 1 and 1 :8, preferably between 1.2: 1 and 1 :4, e.g. , between 1 : 1 and 1 :3 such as between 1 : 1.2 and 1 :2, 1 : 1.2 and 1 : 1.8, 1 :1.3 and 1 : 1.7, in particular between 1 : 1.4 and 1 : 1.6, such as about 1 : 1.5. In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipoplex is between 1 : 1.2 (0.83) and 1 :2 (0.5). In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipoplex is between 1.6:2 (0.8) and 1 :2 (0.5) or between 1.6:2 (0.8) and 1.1 :2 (0.55). In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipoplex is 1.3:2 (0.65). In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipoplex is not lower than 1.0:2.0. In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipoplex is not higher than 1.9:2.0. In some embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the lipoplex is not lower than 1.0:2.0 and not higher than 1.9:2.0.
[0282] In one embodiment, the lipoplexes or lipid nanoparticles comprise DOTMA and DOPE in a molar ratio of 10:0 to 1 :9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1 :2, even more preferably 1.4:2 to 1.1 :2 and even more preferably about 1.2:2. In one embodiment, the lipoplexes or lipid nanoparticles comprise DOTMA and Cholesterol in a molar ratio of 10:0 to 1 :9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5, wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1 :2, even more preferably 1.4:2 to 1.1 :2 and even more preferably about 1.2:2. In one embodiment, the lipoplexes or lipid nanoparticles comprise DOTAP and DOPE in a molar ratio of 10:0 to 1 :9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5, wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1 :2, even more preferably 1.4:2 to 1.1 :2 and even more preferably about 1.2:2. In one embodiment, the lipoplexes or lipid nanoparticles comprise DOTMA and DOPE in a molar ratio of 2: 1 to 1 :2, preferably 2: 1 to 1 : 1, wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4: 1 or less. In one embodiment, the lipoplexes or lipid nanoparticles comprise DOTMA and cholesterol in a molar ratio of 2: 1 to 1 :2, preferably 2: 1 to 1 : 1, wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4: 1 or less. In one embodiment, the lipoplexes or lipid nanoparticles comprise DOTAP and DOPE in a molar ratio of 2: 1 to 1 :2, preferably 2: 1 to 1 : 1, wherein the charge ratio of positive charges in DOTAP to negative charges in the RNA is 1.4: 1 or less.
[0283] In one embodiment, the zeta potential of the lipoplexes or lipid nanoparticles is -5 or less, -10 or less, -15 or less, -20 or less or -25 or less. In various embodiments, the zeta potential of the lipoplexes or lipid nanoparticles is -35 or higher, -30 or higher or -25 or higher. In one embodiment, the lipoplexes or lipid nanoparticles have a zeta potential from 0 mV to -50 mV, preferably 0 mV to -40 mV or -10 mV to -30 mV.
[0284] In some embodiments, the poly dispersity index of the lipoplexes or lipid nanoparticles is 0.5 or less, 0.4 or less, or 0.3 or less, as measured by dynamic light scattering.
[0285] In some embodiments, the lipoplexes or lipid nanoparticles have an average diameter in the range of about 50 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 200 nm to about 600 nm, from about 250 nm to about 700 nm, or from about 250 nm to about 550 nm, as measured by dynamic light scattering.
[0286] In some embodiments, the individualized cancer vaccine is administered intravenously, for example, wherein the RNA vaccine is administered to the human patient at doses of 15 pg, 21 pg, 21.3 pg, 25 pg, 38 pg, or 50 pg. In some embodiments, 15 pg, 21 pg, 21.3 pg, 25 pg, 38 pg, or 50 pg of RNA is delivered per dose (z.e., dose weight reflects the weight of RNA administered, not the total weight of the formulation administered). In some embodiments, the RNA vaccine is administered to the human patient at a dose of about 25 pg. In some embodiments, the RNA vaccine is administered to the human patient at a dose of about 21 pg. In some embodiments, the RNA vaccine is administered to the human patient at a dose of about 21.3 pg. More than one individualized cancer vaccine may be administered to a subject, e.g., subject is administered one individualized cancer vaccine with a combination of neoepitopes and also administered a separate individualized cancer vaccine with a different combination of neoepitopes. In some embodiments, a first individualized cancer vaccine with five neoepitopes is administered in combination with a second individualized cancer vaccine with five alternative epitopes. In some embodiments, a first individualized cancer vaccine with ten neoepitopes is administered in combination with a second individualized cancer vaccine with ten alternative epitopes.
[0287] In some embodiments, the individualized cancer vaccine is administered such that it is delivered to the spleen. For example, the individualized cancer vaccine can be administered such that one or more antigen(s) (e.g., tumor-specific neo-antigens) are delivered to antigen presenting cells (e.g., in the spleen). [0288] Any of the individualized cancer vaccines or RNA vaccines of the present disclosure may find use in the methods described herein. For example, in some embodiments, a PD-1 axis binding antagonist of the present disclosure is administered in combination with an individualized cancer vaccine (ICV), e.g., an RNA vaccine described herein.
[0289] Further provided herein are DNA molecules encoding any of the RNA vaccines of the present disclosure. For example, in some embodiments, a DNA molecule of the present disclosure comprises the general structure (in the 5’->3’ direction): (1) a polynucleotide sequence encoding a 5’ untranslated region (UTR); (2) a polynucleotide sequence encoding a secretory signal peptide; (3) a polynucleotide sequence encoding at least a portion of a transmembrane and cytoplasmic domain of a major histocompatibility complex (MHC) molecule; (4) a polynucleotide sequence encoding a 3’ UTR comprising: (a) a 3’ untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (5) a polynucleotide sequence encoding a poly(A) sequence. In some embodiments, a DNA molecule of the present disclosure comprises, in the 5’->3’ direction: the polynucleotide sequence GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATG AGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGA CAGAGACATGGGCCGGAAGC (SEQ ID NO:40); and the polynucleotide sequence ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCC GTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGC TACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACA GCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCG TCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCC ACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAA TGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTA ACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCA ATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT (SEQ ID NO:41).
[0290] In some embodiments, the DNA molecule further comprises, in the 5’->3’ direction: a polynucleotide sequence encoding an amino acid linker; and a polynucleotide sequence encoding a neoepitope. In some embodiments, the polynucleotide sequences encoding the amino acid linker and the neoepitope form a linker-neoepitope module (e.g., a continuous sequence in the 5’->3’ direction in the same open-reading frame). In some embodiments, the polynucleotide sequences forming the linker-neoepitope module are between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule, or between the sequences of SEQ ID NO:40 and SEQ ID NO:41, in the 5’->3’ direction. In some embodiments, the DNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 linker-epitope modules, and each of the linker-epitope modules encodes a different neoepitope. In some embodiments, the DNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linker-epitope modules, and the DNA molecule comprises polynucleotides encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 different neoepitopes. In some embodiments, the DNA molecule comprises 5, 10, or 20 linker-epitope modules. In some embodiments, each of the linker-epitope modules encodes a different neoepitope. In some embodiments, the linker-epitope modules form a continuous sequence in the 5’->3’ direction in the same open-reading frame. In some embodiments, the polynucleotide sequence encoding the linker of the first linker-epitope module is 3’ of the polynucleotide sequence encoding the secretory signal peptide. In some embodiments, the polynucleotide sequence encoding the neoepitope of the last linker-epitope module is 5’ of the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule.
[0291] Also provided herein are methods of producing any of the RNA vaccine of the present disclosure, comprising transcribing (e.g., by transcription of linear, double-stranded DNA or plasmid DNA, such as by in vitro transcription) a DNA molecule of the present disclosure. In some embodiments, the methods further comprise isolating and/or purifying the transcribed RNA molecule from the DNA molecule.
[0292] In some embodiments, an RNA or DNA molecule of the present disclosure comprises a type IIS restriction cleavage site, which allows RNA to be transcribed under the control of a 5' RNA polymerase promoter and which contains a polyadenyl cassette (poly(A) sequence), wherein the recognition sequence is located 3' of the poly(A) sequence, while the cleavage site is located upstream and thus within the poly(A) sequence. Restriction cleavage at the type IIS restriction cleavage site enables a plasmid to be linearized within the poly(A) sequence, as described in U.S. Pat. Nos. 9,476,055 and 10,106,800. The linearized plasmid can then be used as template for in vitro transcription, the resulting transcript ending in an unmasked poly(A) sequence. Any of the type IIS restriction cleavage sites described in U.S. Pat. Nos. 9,476,055 and 10,106,800 may be used.
[0293] In some embodiments of the methods provided herein, the RNA vaccine includes one or more polynucleotides encoding 5-20 (e.g., any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen. In certain embodiments, the RNA vaccine is formulated with one or more lipids. In certain embodiments, the one or more polynucleotides of the RNA vaccine and the one or more lipids form a lipoplex. In certain embodiments, the lipoplex includes one or more lipids that form a multilamellar structure that encapsulates the one or more polynucleotides of the RNA vaccine. In certain embodiments, the one or more lipids include at least one cationic lipid and at least one helper lipid. In certain embodiments, the one or more lipids include (R)-N,N,N-trimethyl-2,3-dioleyloxy-l-propanaminium chloride (DOTMA) and l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In certain embodiments, at physiological pH the overall charge ratio of positive charges to negative charges of the liposome is 1.3:2 (0.65).
[0294] In certain embodiments, the RNA vaccine includes an RNA molecule including, in the 5’->3’ direction: (1) a 5’ cap; (2) a 5’ untranslated region (UTR); (3) a polynucleotide sequence encoding a secretory signal peptide; (4) a polynucleotide sequence encoding the one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; (5) a polynucleotide sequence encoding at least a portion of a transmembrane and cytoplasmic domain of a major histocompatibility complex (MHC) molecule; (6) a 3’ UTR including: (a) a 3’ untranslated region of an Amino-Terminal Enhancer of Split (AES) mRNA or a fragment thereof; and (b) non-coding RNA of a mitochondrially encoded 12S RNA or a fragment thereof; and (7) a poly(A) sequence.
[0295] In certain embodiments, the RNA molecule further includes a polynucleotide sequence encoding an amino acid linker; wherein the polynucleotide sequences encoding the amino acid linker and a first of the one or more neoepitopes form a first linker-neoepitope module; and wherein the polynucleotide sequences forming the first linker-neoepitope module are between the polynucleotide sequence encoding the secretory signal peptide and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule in the 5’->3’ direction. In certain embodiments, the amino acid linker includes the sequence GGSGGGGSGG (SEQ ID NO: 39). In certain embodiments, the polynucleotide sequence encoding the amino acid linker includes the sequence GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC (SEQ ID NO: 37).
[0296] In certain embodiments, the RNA molecule further includes, in the 5’->3’ direction: at least a second linker-epitope module, wherein the at least second linker-epitope module includes a polynucleotide sequence encoding an amino acid linker and a polynucleotide sequence encoding a neoepitope; wherein the polynucleotide sequences forming the second linker-neoepitope module are between the polynucleotide sequence encoding the neoepitope of the first linker-neoepitope module and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule in the 5’->3’ direction; and wherein the neoepitope of the first linker-epitope module is different from the neoepitope of the second linker-epitope module. In certain embodiments, the RNA molecule includes 5 linker-epitope modules, wherein the 5 linker-epitope modules each encode a different neoepitope. In certain embodiments, the RNA molecule includes 5 linker-epitope modules, wherein the 5 linker-epitope modules each encode a different neoepitope. In certain embodiments, the RNA molecule includes 10 linker-epitope modules, wherein the 10 linkerepitope modules each encode a different neoepitope. In certain embodiments, the RNA molecule includes 20 linker-epitope modules, wherein the 20 linker-epitope modules each encode a different neoepitope.
[0297] In certain embodiments, the RNA molecule further includes a second polynucleotide sequence encoding an amino acid linker, wherein the second polynucleotide sequence encoding the amino acid linker is between the polynucleotide sequence encoding the neoepitope that is most distal in the 3’ direction and the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule.
[0298] In certain embodiments, the 5’ cap includes a DI diastereoisomer of the structure:
[0299] In certain embodiments, the 5’ UTR includes the sequence
UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO:23). In certain embodiments, the 5’ UTR includes the sequence
GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO:21).
[0300] In certain embodiments, the secretory signal peptide includes the amino acid sequence MRVMAPRTLILLLS GAL ALTET WAGS (SEQ ID NO:27). In certain embodiments, the polynucleotide sequence encoding the secretory signal peptide includes the sequence
AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGC CCUGACAGAGACAUGGGCCGGAAGC (SEQ ID NO:25).
[0301] In certain embodiments, the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule includes the amino acid sequence IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO:30). In certain embodiments, the polynucleotide sequence encoding the at least portion of the transmembrane and cytoplasmic domain of the MHC molecule includes the sequence
AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU GACAGCC (SEQ ID NO:28).
[0302] In certain embodiments, the 3’ untranslated region of the AES mRNA includes the sequence
CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCC GAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACU CACCACCUCUGCUAGUUCCAGACACCUCC (SEQ ID NO:33). In certain embodiments, the non-coding RNA of the mitochondrially encoded 12S RNA includes the sequence CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGG AAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG (SEQ ID NO:35). In certain embodiments, the 3’ UTR includes the sequence
CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGG UACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGC CCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCA GCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACC UUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCA AUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU (SEQ ID N0:31).
[0303] In certain embodiments, the poly(A) sequence includes 120 adenine nucleotides.
[0304] In certain embodiments, the RNA vaccine includes an RNA molecule including, in the 5’->3’ direction: the polynucleotide sequence GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAU GAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCC UGACAGAGACAUGGGCCGGAAGC (SEQ ID NO: 19); a polynucleotide sequence encoding the one or more neoepitopes resulting from cancer-specific somatic mutations present in the tumor specimen; and the polynucleotide sequence AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU GACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCU UUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUC CCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGC ACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACA GCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACC CCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCU AGCCGCGUCGCU (SEQ ID NO:20).
IV PD-1 Axis Binding Antagonists
[0305] In some embodiments, an individualized cancer vaccine (e.g., an RNA vaccine) of the present disclosure is administered in combination with a PD-1 axis binding antagonist. [0306] For example, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PDL1 binding antagonist and a PDL2 binding antagonist. Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.
[0307] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner(s). In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner(s). In a specific aspect, PDL1 binding partner(s) are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner(s). In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0308] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
[0309] In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab (Bristol-Myers Squibb/Ono), also known as MDX-1106- 04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168. In some embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain sequence, wherein:
(a) the heavy chain comprises the amino acid sequence:
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 11), and
(b) the light chain comprises the amino acid sequence:
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12). [0310] In some embodiments, the anti-PD-1 antibody comprises the six HVR sequences from SEQ ID NO: 11 and SEQ ID NO: 12 (e.g., the three heavy chain HVRs from SEQ ID NO: 11 and the three light chain HVRs from SEQ ID NO: 12). In some embodiments, the anti- PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 11 and the light chain variable domain from SEQ ID NO: 12.
[0311] In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009/114335. In some embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain sequence, wherein:
(a) the heavy chain comprises the amino acid sequence: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GV
HTFP AVLQ S SGL YSLS S VVT VP S S SLGTKT YTCNVDHKP SNTKVDKRVESK YGPPCPP CP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 13), and
(b) the light chain comprises the amino acid sequence:
EIVLTQSPAT
LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPS VF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14).
[0312] In some embodiments, the anti-PD-1 antibody comprises the six HVR sequences from SEQ ID NO: 13 and SEQ ID NO: 14 (e.g., the three heavy chain HVRs from SEQ ID NO: 13 and the three light chain HVRs from SEQ ID NO: 14). In some embodiments, the anti- PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 13 and the light chain variable domain from SEQ ID NO: 14.
[0313] In some embodiments, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.
[0314] In some embodiments, the anti-PD-1 antibody is PDR001 (CAS Registry No. 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PDl antibody that blocks the binding of PDL1 and PDL2 to PD-1.
[0315] In some embodiments, the anti-PD-1 antibody is REGN2810 (Regeneron).
REGN2810 is a human anti-PDl antibody also known as LIBTAYO® and cemiplimab-rwlc. [0316] In some embodiments, the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).
[0317] In some embodiments, the anti-PD-1 antibody is JS-001 (Shanghai Junshi). JS-001 is a humanized anti-PDl antibody.
[0318] In some embodiments, the anti-PD-1 antibody is STI-A1110 (Sorrento). STI-A1110 is a human anti-PDl antibody.
[0319] In some embodiments, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR- 1210 is a human IgG4 anti-PDl antibody.
[0320] In some embodiments, the anti-PD-1 antibody is PF-06801591 (Pfizer).
[0321] In some embodiments, the anti-PD-1 antibody is TSR-042 (also known as ANB011;
Tesaro/AnaptysBio).
[0322] In some embodiments, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).
[0323] In some embodiments, the anti-PD-1 antibody is ENUM 244C8 (Enumeral
Biomedical Holdings). ENUM 244C8 is an anti-PDl antibody that inhibits PD-1 function without blocking binding of PDL1 to PD-1.
[0324] In some embodiments, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PDl antibody that competitively inhibits binding of PDL1 to PD-1.
[0325] In some embodiments, the PD-1 antibody comprises the six HVR sequences (e.g., the three heavy chain HVRs and the three light chain HVRs) and/or the heavy chain variable domain and light chain variable domain from a PD-1 antibody described in WO2015/112800 (Applicant: Regeneron), WO2015/112805 (Applicant: Regeneron), WO2015/112900 (Applicant: Novartis), US20150210769 (Assigned to Novartis), WO2016/089873 (Applicant: Celgene), W02015/035606 (Applicant: Beigene), WO2015/085847 (Applicants: Shanghai Hengrui Pharmaceutical/Jiangsu Hengrui Medicine), W02014/206107 (Applicants: Shanghai Junshi Biosciences/Junmeng Biosciences), WO2012/145493 (Applicant: Amplimmune), US9205148 (Assigned to Medlmmune), WO2015/119930 (Applicants: Pfizer/Merck), WO2015/119923 (Applicants: Pfizer/Merck), WO2016/032927 (Applicants: Pfizer/Merck), WO2014/179664 (Applicant: AnaptysBio), W02016/106160 (Applicant: Enumeral), and WO2014/194302 (Applicant: Sorrento).
[0326] In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (c.g, an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. AMP-224 (CAS Registry No. 1422184-00-6; GlaxoSmithKline/Medlmmune), also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
[0327] In some embodiments, the PD-1 binding antagonist is a peptide or small molecule compound. In some embodiments, the PD-1 binding antagonist is AUNP-12 (PierreFabre/Aurigene). See, e.g., WO2012/168944, WO2015/036927, WO2015/044900, W02015/033303, WO2013/144704, WO2013/132317, and WO2011/161699.
[0328] In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PD-1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1 and VISTA. In some embodiments, the PDL1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1 and TIM3. In some embodiments, the small molecule is a compound described in W02015/033301 and WO2015/033299.
[0329] In some embodiments, the PD-1 axis binding antagonist is an anti-PDLl antibody. A variety of anti-PDLl antibodies are contemplated and described herein. In any of the embodiments herein, the isolated anti-PDLl antibody can bind to a human PDL1, for example a human PDL1 as shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof. In some embodiments, the anti-PDLl antibody is capable of inhibiting binding between PDL1 and PD-1 and/or between PDL1 and B7-1. In some embodiments, the anti-PDLl antibody is a monoclonal antibody. In some embodiments, the anti-PDLl antibody is an antibody fragment selected from the group consisting of Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments. In some embodiments, the anti-PDLl antibody is a humanized antibody. In some embodiments, the anti-PDLl antibody is a human antibody. Examples of anti-PDLl antibodies useful for the methods of this invention, and methods for making thereof are described in PCT patent application WO 2010/077634 Al and US Patent No. 8,217,149, which are incorporated herein by reference.
[0330] In some embodiments, the anti-PDLl antibody comprises a heavy chain variable region and a light chain variable region, wherein:
(a) the heavy chain variable region comprises an HVR-H1, HVR-H2, and HVR- H3 sequence of GFTFSDSWIH (SEQ ID NO:1), AWISPYGGSTYYADSVKG (SEQ ID NO:2) and RHWPGGFDY (SEQ ID NO:3), respectively, and
(b) the light chain variable region comprises an HVR-L1, HVR-L2, and HVR-L3 sequence of RASQDVSTAVA (SEQ ID NO:4), SASFLYS (SEQ ID NO:5) and QQYLYHPAT (SEQ ID NO:6), respectively. [0331] In some embodiments, the anti-PDLl antibody is MPDL3280A, also known as atezolizumab and TECENTRIQ® (CAS Registry Number: 1422185-06-5), with a WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Proposed INN: List 112, Vol. 28, No. 4, published January 16, 2015 (see page 485) described therein. In some embodiments, the anti-PDLl antibody comprises a heavy chain and a light chain sequence, wherein:
(a) the heavy chain variable region sequence comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSS (SEQ ID NO: 7), and
(b) the light chain variable region sequence comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 8).
[0332] In some embodiments, the anti-PDLl antibody comprises a heavy chain and a light chain sequence, wherein:
(a) the heavy chain comprises the amino acid sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NOV), and
(b) the light chain comprises the amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).
[0333] In some embodiments, the anti-PDLl antibody is avelumab (CAS Registry Number: 1537032-82-8). Avelumab, also known as MSB0010718C, is a human monoclonal IgGl anti- PDL1 antibody (Merck KGaA, Pfizer). In some embodiments, the anti-PDLl antibody comprises a heavy chain and a light chain sequence, wherein:
(a) the heavy chain comprises the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGI TFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15), and
(b) the light chain comprises the amino acid sequence: QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRP SGVSNRFSGSKSGNTASLTISGLQAEDEAD YYC S S YTS S STRVFGTGTKVTVLGQPKA NPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 16). [0334] In some embodiments, the anti-PDLl antibody comprises the six HVR sequences from SEQ ID NO: 15 and SEQ ID NO: 16 (e.g., the three heavy chain HVRs from SEQ ID NO: 15 and the three light chain HVRs from SEQ ID NO: 16). In some embodiments, the anti- PDLl antibody comprises the heavy chain variable domain from SEQ ID NO: 15 and the light chain variable domain from SEQ ID NO: 16.
[0335] In some embodiments, the anti-PDLl antibody is durvalumab (CAS Registry Number: 1428935-60-7). Durvalumab, also known as MEDI4736, is an Fc optimized human monoclonal IgGl kappa anti-PDLl antibody (Medlmmune, AstraZeneca) described in WO201 1/066389 and US2013/034559. In some embodiments, the anti-PDLl antibody comprises a heavy chain and a light chain sequence, wherein:
(a) the heavy chain comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDG SEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPASIEI< TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17), and
(b) the light chain comprises the amino acid sequence:
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18).
[0336] In some embodiments, the anti-PDLl antibody comprises the six HVR sequences from SEQ ID NO: 17 and SEQ ID NO: 18 (e.g., the three heavy chain HVRs from SEQ ID NO: 17 and the three light chain HVRs from SEQ ID NO: 18). In some embodiments, the anti- PDLl antibody comprises the heavy chain variable domain from SEQ ID NO: 17 and the light chain variable domain from SEQ ID NO: 18.
[0337] In some embodiments, the anti-PDLl antibody is MDX-1105 (Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PDLl antibody described in W02007/005874.
[0338] In some embodiments, the anti-PDLl antibody is LY3300054 (Eli Lilly).
[0339] In some embodiments, the anti-PDLl antibody is STI-A1014 (Sorrento). STI-
A1014 is a human anti-PDLl antibody.
[0340] In some embodiments, the anti-PDLl antibody is KN035 (Suzhou Alphamab). KN035 is single-domain antibody (dAB) generated from a camel phage display library.
[0341] In some embodiments, the anti-PDLl antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by a protease in the tumor microenvironment), activates an antibody antigen binding domain to allow it to bind its antigen, e.g., by removing a nonbinding steric moiety. In some embodiments, the anti-PDLl antibody is CX-072 (CytomX Therapeutics).
[0342] In some embodiments, the PDL1 antibody comprises the six HVR sequences (e.g., the three heavy chain HVRs and the three light chain HVRs) and/or the heavy chain variable domain and light chain variable domain from a PDL1 antibody described in US20160108123 (Assigned to Novartis), W02016/000619 (Applicant: Beigene), WO2012/145493 (Applicant: Amplimmune), US9205148 (Assigned to Medlmmune), WO2013/181634 (Applicant: Sorrento), and W02016/061142 (Applicant: Novartis).
[0343] In a still further specific aspect, the antibody further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a still further specific aspect, the human constant region is IgGl. In a still further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a still further aspect, the murine constant region if IgG2A.
[0344] In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect, the minimal effector function results from an “effector-less Fc mutation” or aglycosylation mutation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some embodiments, the isolated anti-PDLl antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5- hydroxylysine may also be used. Removal of glycosylation sites form an antibody is conveniently accomplished by altering the amino acid sequence such that one of the abovedescribed tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site another amino acid residue (e.g., glycine, alanine or a conservative substitution).
[0345] In a still further embodiment, the present disclosure provides for compositions comprising any of the above described anti-PDLl antibodies in combination with at least one pharmaceutically-acceptable carrier.
[0346] In a still further embodiment, the present disclosure provides for a composition comprising an anti-PDLl, an anti-PD-1, or an anti-PDL2 antibody or antigen binding fragment thereof as provided herein and at least one pharmaceutically acceptable carrier. In some embodiments, the anti-PDLl, anti-PD-1, or anti-PDL2 antibody or antigen binding fragment thereof administered to the individual is a composition comprising one or more pharmaceutically acceptable carrier. Any of the pharmaceutically acceptable carriers described herein or known in the art may be used. [0347] In some embodiments, the PD-1 axis binding antagonist is administered intravenously to the human patient. In some embodiments, the anti-PD-Ll antibody is administered to the human patient at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, 1200 mg or about 1680 mg, for example about any of 1100 mg, 1150 mg, 1200 mg, 1250 mg, or 1300 mg or about any of 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 me, 1680 mg, 1690 mg, 1700 mg, or more. In some embodiments, the anti-PD-Ll antibody is nivolumab, and the nivolumab is administered intravenously to the human patient at a dose of about 480 mg.
V. Pharmaceutical Compositions and Formulations
[0348] Also provided herein are pharmaceutical compositions and formulations, e.g., for the treatment of MIUC. In some embodiments, the pharmaceutical compositions and formulations further comprise a pharmaceutically acceptable carrier.
[0349] After preparation of the antibody of interest (e.g., techniques for producing antibodies which can be formulated as disclosed herein are elaborated herein and are known in the art), the pharmaceutical formulation comprising said antibody is prepared. In certain embodiments, the antibody to be formulated has not been subjected to prior lyophilization, and the formulation of interest herein is an aqueous formulation. In certain embodiments, the antibody is a full-length antibody. In one embodiment, the antibody in the formulation is an antibody fragment, such as an F(ab')2. The therapeutically effective amount of antibody present in the formulation is determined by taking into account the desired dose volumes and mode(s) of administration, for example. From about 25 mg/mL to about 150 mg/mL, or from about 30 mg/mL to about 140 mg/mL, or from about 35 mg/mL to about 130 mg/mL, or from about 40 mg/mL to about 120 mg/mL, or from about 50 mg/mL to about 130 mg/mL, or from about 50 mg/mL to about 125 mg/mL, or from about 50 mg/mL to about 120 mg/mL, or from about 50 mg/mL to about 110 mg/mL, or from about 50 mg/mL to about 100 mg/mL, or from about 50 mg/mL to about 90 mg/mL, or from about 50 mg/mL to about 80 mg/mL, or from about 54 mg/mL to about 66 mg/mL is an exemplary antibody concentration in the formulation. In some embodiments, an anti-PDLl antibody described herein (such as atezolizumab) is administered at a dose of about 1200mg. In some embodiments, an anti-PDl antibody described herein (such as pembrolizumab) is administered at a dose of about 200mg. In some embodiments, an anti-PDl antibody described herein (such as nivolumab) is administered at a dose of about 240mg (e.g., every 2 weeks) or 480mg (e.g., every 4 weeks). In some embodiments, an anti-PDl antibody described herein is nivolumab and is administered at a dose of about 480mg every 4 weeks.
[0350] In some embodiments, an RNA vaccine described herein is administered at a dose of about 15 pg, about 21 pg, about 21.3 pg, about 25 pg, about 38 pg, or about 50 pg. For example, in some embodiments, the RNA vaccine is administered to the human patient at a dose of about 21 pg, about 21.3 pg, or about 25 pg.
[0351] In some embodiments, a pharmaceutical composition described herein is provided in a formulation that is “ready-to-use”, e.g., formulated for direct administration to a human subject without requiring dilution, such as, for example, as described in U.S. Patent Application No. 18/246,533 and in PCT Publication No. WO2022069632A1, each of which is hereby incorporated by reference in its entirety . In some embodiments, a pharmaceutical composition and/or formulation described herein is liquid and is formulated for direct administration to a human subject without dilution. In some embodiments, a pharmaceutical composition and/or formulation described herein is a liquid, is formulated for direct administration to a human subject without dilution, and comprises RNA lipoplex particles comprising: RNA; at least one cationic lipid, and at least one additional lipid; sodium chloride at a concentration of about 10 mM or less; a stabilizer at a concentration of more than about 10% weight by volume percent (% w/v) and less than about 15% weight by volume percent (% w/v); and a buffer. In some embodiments, the sodium chloride is at a concentration from about 5 mM to about 10 mM. In some embodiments, the sodium chloride is at a concentration of about 8.2 mM. In some embodiments, the stabilizer is a carbohydrate selected from a monosaccharide, a disaccharide, a trisaccharide, a sugar alcohol, an oligosaccharide or its corresponding sugar alcohol, and a straight chain polyalcohol. In some embodiments, the stabilizer is sucrose or trehalose. In some embodiments, the stabilizer is sucrose at a concentration from about 12 to about 14% (w/v). In some embodiments, the buffer is selected from the group consisting of 2-[4-(2-hydroxyethyl)piperazin-l- yl]ethanesulfonic acid (HEPES), histidine, acetic acid/sodium acetate, and MES (2-(N- morpholino)ethanesulfonic acid). In some embodiments, the buffer is HEPES. In some embodiments, the composition has a pH from 6.0 to 7.5, from 6.5 to 7.5, from 6.5 to 7.3, from 6.5 to 7.2, from 6.7 to 7.2, or from 6.5 to 7.0. In some embodiments, the composition has a pH of about 6.7. In some embodiments, the buffer is HEPES at a concentration of about 5 mM or less with a pH of about 6.7. In some embodiments, the at least one cationic lipid comprises l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA) and the at least one additional lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphoethanolamine (DOPE). In some embodiments, the RNA lipoplex particles comprise DOTMA and DOPE in a molar ratio of from about 10:0 to 1 :9, from about 4: 1 to 1 :2, from about 3 : 1 to about 1 : 1, or about 2: 1. In some embodiments, the composition further comprises a chelating agent. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In some embodiments, the EDTA is at a concentration of about 3.5 mM or less, or from about 0.25 mM to about 3.5 mM, or about 0.25 mM to about 2.5 mM. In some embodiments, the RNA encodes a peptide or protein comprising at least one epitope, wherein the ratio of positive charges to negative charges in the composition is from about 1 :2 to about 1.9:2, or about 1.3:2.0.
[0352] In some embodiments, a pharmaceutical composition and/or formulation described herein is a liquid, is formulated for direct administration to a human subject without dilution, and comprises RNA lipoplex particles comprising: RNA encoding a peptide or protein comprising at least one epitope, and DOTMA and DOPE in a molar ratio of about 2: 1, wherein the ratio of positive charges to negative charges in the composition is about 1.3:2.0, sodium chloride at a concentration of about 8.2 mM, sucrose at a concentration of about 13% (w/v), HEPES at a concentration of about 5 mM with a pH of about 6.7, and EDTA at a concentration of about 2.5 mM. In some embodiments, the RNA lipoplex particles have an average diameter that ranges from about 200 to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In some embodiments, the amount of RNA in the composition is from about 0.01 mg/mL to about 1 mg/mL, about 0.05 mg/mL to about 0.5 mg/mL, or about 0.025 mg/mL. In some embodiments, the composition is in a liquid, frozen or dehydrated state. In some embodiments, the frozen composition is stable at a temperature of about -15°C for at least six months. In some embodiments, the composition is in a liquid state which can be administered directly to a subject. In some embodiments, the composition is formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. Further provided in some embodiments are a method of preparing a liquid composition for direct administration to a subject comprising RNA lipoplex particles comprising (i) providing a ready -to-use composition described herein as a frozen composition and thawing the frozen composition to provide a liquid composition or (ii) providing a ready - to-use composition described herein as a dehydrated composition and dissolving the dehydrated composition to provide a liquid composition of an embodiment described herein. In some embodiments, the ready -to-use liquid composition has a pH from 6.5 to 7.2.
[0353] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington ’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
[0354] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer. [0355] The composition and formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. [0356] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington ’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0357] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. [0358] Pharmaceutical formulations of nivolumab, atezolizumab, and pembrolizumab are commercially available. For example, nivolumab is known under the trade name (as described elsewhere herein) OPDIVO®. Atezolizumab is known under the trade name (as described elsewhere herein) TECENTRIQ®. Pembrolizumab is known under the trade name (as described elsewhere herein) KEYTRUDA®. In some embodiments, nivolumab and the RNA vaccine, atezolizumab and the RNA vaccine, or pembrolizumab and the RNA vaccine, are provided in separate containers. In some embodiments, nivolumab, atezolizumab, and/or pembrolizumab are used and/or prepared for administration to an individual as described in the prescribing information available with the commercially available product.
VI. Articles of Manufacture or Kits
[0359] Further provided herein is an article of manufacture or kit comprising an RNA vaccine of the present disclosure. Further provided herein is an article of manufacture or a kit comprising a PD-1 axis binding antagonist (such as nivolumab). In some embodiments, the article of manufacture or kit further comprises package insert comprising instructions for using the RNA vaccine and/or PD-1 axis binding antagonist (e.g., in conjunction with the RNA vaccine) to treat or delay progression of MIUC in an individual. Also provided herein is an article of manufacture or a kit comprising a PD-1 axis binding antagonist (such as nivolumab) and an RNA vaccine.
[0360] In some embodiments, there is provided a kit comprising an individualized RNA vaccine, for use in a method for treating a UC in a human in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC tumor specimen obtained from the human. In some embodiments, the kit comprises a PD-1 axis binding antagonist for use in a method for treating a UC in a human in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UC tumor specimen obtained from the human. In some embodiments, there is provided a kit comprising an individualized RNA vaccine, for use in a method for treating a MIBC in a human in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a MIBC tumor specimen obtained from the human. In some embodiments, the kit comprises a PD-1 axis binding antagonist for use in a method for treating a MIBC in a human in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a MIBC tumor specimen obtained from the human. In some embodiments, there is provided a kit comprising an individualized RNA vaccine, for use in a method for treating a UTUC in a human in need thereof, wherein the RNA vaccine is to be administered in combination with a PD-1 axis binding antagonist according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UTUC tumor specimen obtained from the human. In some embodiments, the kit comprises a PD-1 axis binding antagonist for use in a method for treating a UTUC in a human in need thereof, wherein the PD-1 axis binding antagonist is to be administered in combination with an individualized RNA vaccine according to the method described herein, wherein the RNA vaccine comprises one or more polynucleotides encoding one or more neoepitopes resulting from cancer-specific somatic mutations present in a UTUC tumor specimen obtained from the human. [0361] In some embodiments, the PD-1 axis binding antagonist and the RNA vaccine are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agents include, for example, bottles, vials, bags and syringes. [0362] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES
[0363] The present disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
Example 1: A randomized phase II, double-blind, multicenter study of individualized cancer vaccine plus nivolumab versus placebo plus nivolumab as adjuvant therapy in patients with high-risk muscle-invasive urothelial carcinoma
[0364] This Example describes a randomized, double-blind, Phase II multicenter study designed to evaluate the efficacy and safety of adjuvant treatment with an individualized cancer vaccine (in this case, autogene cevumeran) plus nivolumab compared with placebo plus nivolumab in patients with resected muscle-invasive urothelial carcinoma (MIUC) who are at high risk for recurrence and have no evidence of disease after surgery. Thus, in this study, the individualized cancer vaccine and nivolumab are adjuvant to surgical resection.
[0365] The study consists of two screening phases (Part A and Part B), a treatment period consisting of priming, boost, and post-nivolumab boost phases, and a follow-up period. Since the individualized cancer vaccine is designed and manufactured for each individual patient, screening Part A preferably begin as soon as possible (ideally, prior to TURBT resection). For Part A, patients provide a tumor specimen sample (TURBT specimens strongly preferred, though some samples from radical cystectomy or radical nephroureterectomy may be acceptable as outlined below) and matching blood sample to begin upstream manufacturing of autogene cevumeran. Part B screening begins after pathological confirmation of disease status from radical cystectomy or radical nephroureterectomy and includes eligibility criteria screening in parallel with downstream manufacturing of the individualized cancer vaccine.
[0366] This study aims to identify a more effective adjuvant therapy for MIUC because, although the use of adjuvant nivolumab in this patient population is increasing, to date, no adjuvant therapies have shown improved survival in UC, and many MIUC patients are ineligible to receive cisplatin-based chemotherapy. Further, there continues to be an unmet need as more than half of patients treated with nivolumab relapse within 24 months, and adjuvant nivolumab has yet to demonstrate a survival benefit.
Study Objectives
[0367] The objectives of this study are to evaluate the efficacy and safety of adjuvant individualized cancer vaccine plus nivolumab as compared with placebo plus nivolumab in patients with high-risk MIUC.
Study Rationale
[0368] Neoadjuvant cisplatin based therapy is the standard of care in patients with MIUC that have a high risk of recurrence prior to cystectomy based on randomized trials demonstrating small survival benefit. In patients who do not receive neoadjuvant chemotherapy, adjuvant cisplatin therapy is an option. However, many patients are not eligible to receive cisplatin-based chemotherapy due to co-morbidities such as hearing loss, peripheral neuropathy, or impaired renal function. Adjuvant therapy with nivolumab has been approved based on results from the CheckMate 274 trial, which demonstrated an improvement in its primary endpoint of disease-free survival (DFS) with nivolumab versus placebo in both the intent-to-treat (ITT) population and in patients whose tumor cells express PD LI ≥ 1% (Bajorin et al. Adjuvant nivolumab versus placebo in muscle-invasive urothelial caminoma. New Engl J Med 2021;384:2102-14). However, there continues to be an unmet need as more than half of patients treated with nivolumab in CheckMate 274 will relapse within 24 months (median DFS of 22 months in the ITT population). In addition, adjuvant nivolumab has yet to demonstrate an overall survival (OS) benefit.
[0369] Autogene cevumeran is an individualized neoantigen-specific immunotherapy (i.e., an individualized cancer vaccine), which is a therapeutic mRNA platform that has been shown to induce antigen-specific T-cell responses to the neoantigens encoded (Braiteh et al. A phase la study to evaluate RO7198457, an individualized neoantigen specific immunotherapy (iNeST), in patients with locally advanced or metastatic solid tumors [abstract]. Cancer Res 2020;80 (Suppl 16):CT169, Lopez et al. A phase lb study to evaluate RO7198457, an individualized neoantigen-specific immunotherapy (iNeST), in combination with atezolizumab, in patients with locally advanced or metastatic solid tumors [abstract]. Cancer Res 2020;80 (Suppl 16):CT301, Rojas et al. Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature 2023;618: 144-50). Autogene cevumeran is designed by using immunogenic neoantigen epitopes (neoepitopes) that are predicted from somatic mutations identified by paired next-generation sequencing (NGS) of a patient’s peripheral blood and tumor tissue and quantified by RNA sequencing. It is hypothesized that these therapeutic vaccines may synergize with checkpoint inhibitors (CPIs), such as nivolumab, to increase antitumor activity in patients with disease that has a high risk of recurrence. Indeed, anti-PD-l/PD-Ll therapy is most often effective in patients who have a preexisting immune response against cancer neoantigens (Herbst et al. Predictive correlates of response to the anti-PD-Ll antibody MPDL3280A in cancer patients. Nature 2014;515:563-7; Tumeh et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014;515:568-71; Garon et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 2015;372:2018-28; Rosenberg et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum -based chemotherpay: a single-arm, multicentre, Phase 2 trial. Lancet 2016;387: 1909-20) and checkpoint blockade works mainly by restoring the activity of preexisting immune responses (Sun et al. Regulation and Function of the PD-L1 Checkpoint. Immunity 2018;48(3):434-52). Only a small percentage of patients (10%-30%) have the adequate preexisting T-cell responses to benefit from this treatment. Autogene cevumeran may synergize with checkpoint CPIs, such as nivolumab, to increase antitumor activity in patients with disease with a high risk of recurrence and together may improve the efficacy of PD-1/PD-L1 treatment by expanding the tumor-specific T-cell repertoire through, for example, the priming of de novo immune responses and further expansion of preexisting immune response.
[0370] Without wishing to be bound by theory, it is believed that the RNA vaccines described herein, such as autogene cevumeran, induce and increase the magnitude and breadth of the tumor-specific T-cell responses. Blocking the PD-1 pathway with a PD-1 axis binding antagonist such as nivolumab may augment the activity of the RNA vaccine by enhancing the initial priming or reactivation of T cells upon antigen encounter and/or improving the activity of dysfunctional T cells after persistent antigen exposure in the tumor. Hence, the combination of, for example, autogene cevumeran with nivolumab may result in more robust anti-tumor immune responses, leading to improved clinical efficacy. This hypothesis is supported in nonclinical models, where induction of antigen-specific immunity combined with concomitant blockade of PD-1 pathways demonstrated superior efficacy over the respective single-agent inhibitors of these pathways, even in models in which a singleagent vaccine had limited activity (Fu et al. (2014) Preclinical evidence that PD1 blockade cooperates with cancer vaccine TEGVAX to elicit regression of established tumors. Cancer Res 2014;74:4042-52; Duraiswamy et al. (2013) Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors. Cancer Res 2013;73:3591-603).
[0371] PD-1 expression in cancer cells correlates with responsiveness to immune checkpoint inhibitors. A PD-1 -adaptive upregulation can be induced by cancer vaccine- induced T cells and the release of interferon-y. Hence, priming the immune system with the individualized cancer vaccine, and the resulting priming of T cells as well as PD-1 upregulation, may increase the response to checkpoint blockade. The priming and first round of substantial expansion of vaccine-induced T cells typically takes about 2-3 weeks. Checkpoint inhibitors, such as an anti -PD-1 agent, may synergistically enhance the activity of cancer vaccine-induced T cells within the tumor microenvironment after their development. On the other hand, it has been suggested that administering checkpoint inhibitors before a cancer vaccine might negatively impact vaccine response by activating CD38+PD1+ immune suppressive T cells (Verma et al. Nat Immunol. 2019; 20, 1231-1243). Accordingly, the Priming Phase of the study described herein includes 2 priming doses of the individualized cancer vaccine administered prior to nivolumab, which may lead to improved immunogenicity and clinical activity.
[0372] In this study, TURBT specimens from patients will be prospectively tested for PD- L1 expression by a central laboratory during the screening period. The study will enroll individuals regardless of PD-L1 status; however, PD-L1 status (H4C score of <1% vs. ≥1%) will be used as one of the stratification factors, as discussed above.
Study Design
[0373] This study enrolls approximately 362 patients (including up to 12 participants enrolled in the safety run in) across approximately 110 sites in a global enrollment phase. As shown in FIGS. 1A-1B, the Phase II study includes i) a two-part screening period (Part A and Part B; shown in FIG. 1); ii) a treatment period consisting of two treatment arms (experimental and control), each with three phases (priming, boost, and post-nivolumab boost phases); and iii) a follow-up period. The total duration of study participation for each patient is expected to be approximately 6 years.
[0374] Patients who have histologically confirmed muscle-invasive MIBC or UTUC (i.e., renal pelvis or ureters) are eligible. Patients with UTUC will be limited to no more than approximately 10% of the study population. Patients with MIBC as the site of primary involvement must have undergone radical cystectomy with lymph node dissection. Patients with UTUC as the site of primary involvement must have undergone radical nephroureterectomy (RNU) with excision of the bladder cuff regardless of the location of the tumor in the upper urinary tract, and the RNU must include lymph node dissection.
[0375] Patients who have received prior neoadjuvant chemotherapy are eligible and must have tumor staging of ypT3-4a or ypN+ (for patients with MIBC; ypT3-4 or ypN+ for patients with UTUC) at pathological examination of resected specimen and M0 radiographically. Patients who have not received neoadjuvant chemotherapy must be ineligible for or declined treatment with cisplatin-based adjuvant chemotherapy and have tumor staging of pT3-4a or pN+ (for patients with MIBC; pT3-4 or pN+ for patients with UTUC) and M0.
[0376] Screening occurs in two parts, termed Part A and Part B (FIG. 1, Table 4). During screening Part A, participants will undergo limited screening for eligibility. During Part A, transurethral resection of the bladder tumor (TURBT) and paired whole blood samples from patients diagnosed with MIBC (cT3-T4 or N+) are preferably submitted for upstream manufacturing of individualized cancer vaccine (including, e.g., for Whole Exome Sequencing (WES), RNA sequencing, and neoepitope identification). In addition to TURBT, MIBC patients also undergo bladders resection (cystectomy), while UTUC patients also undergo kidney/ureter resection (nephroureterectomy). Preferably, TURBT samples are submitted for upstream manufacturing prior to radical cystectomy (in the case of patients with MIBC) or prior to radical nephroureterectomy (RNU; in the case of patients with UTUC) in order to ensure that vaccine manufacturing can be completed within an acceptable timeframe and does not lead to a delay in the administration of adjuvant therapy. Collection and submission of blood for manufacturing of autogene cevumeran is recommended to be performed at least 6 weeks prior to the planned randomization date and prior to cystectomy /nephroureterectomy to avoid delays in administration of adjuvant therapy.
[0377] In cases where TURBT samples are not available, a surgical resection specimen (e.g., from a cystectomy or from a nephroureterectomy) may be submitted. For example, for patients diagnosed with urinary tract urothelial cancer (UTUC), a surgical resection specimen obtained from RNU may be submitted for individualized cancer vaccine manufacturing, or for patients diagnosed with MIBC, a surgical resection specimen obtained from radical cystectomy may be submitted for individualized cancer vaccine manufacturing. However, TURBT samples are strongly preferred to be submitted to reduce the risk of individualized cancer vaccine manufacturing delays. WES obtained from upstream manufacturing may also be used for subsequent ctDNA testing. Leftover tissue from upstream manufacturing may be used for exploratory biomarker testing.
[0378] The RNA vaccine is manufactured as described in Rojas et al. (Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature 618, 144-150 (2023). https://doi.org/10.1038/s41586-023-06063-y), which is hereby incorporated by reference in its entirely. Briefly, for every patient, individualized mRNA neoantigen vaccines are manufactured under good manufacturing practice conditions containing two uridine-based mRNA strands with noncoding sequences optimized for superior translational performance (see, e.g., Holtkamp et al., Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009- 4017 (2006); Kreiter et al., Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692-696 (2015)). Each mRNA strand encodes up to 10 MHCI and MHCII neoepitopes, formulated in approximately 400 nm diameter lipoplex nanoparticles (e.g., as described in Kranz et al., Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534, 396-401 (2016)) comprising the synthetic cationic lipid (R)-N,N,N-trimethyl-2,3-dioleyloxy-l-propanaminium chloride (DOTMA) and the phospholipid l,2-dioleoyl-sn-glycero-3- phosphatidylethanolamine (DOPE) to enable intravenous delivery.
[0379] Part B screening begins after pathological confirmation of disease status from radical cystectomy or radical nephroureterectomy, and includes eligibility criteria screening in parallel with individualized cancer vaccine downstream manufacturing. During Part B, results of standard-of-care tests or examinations performed prior to obtaining informed consent and within the specified time window prior to initiation of study treatment on Day 1 may be used; such tests do not need to be repeated for screening.
[0380] Downstream manufacturing of individualized cancer vaccine (including drug design, quality control, and manufacture and drug release) begins once pathology from surgical resection specimen is confirmed and at least 5 cancer-specific neoepitopes (NEs) have been identified. At least 5 cancer-specific NEs are required for eligibility.
[0381] Patients who are within 120 days post-surgical resection may be included in the study and undergo screening assessments in parallel to downstream manufacturing of individualized cancer vaccine. Tumor specimens from surgical resection (i.e., radical cystectomy, RNU, or lymph node dissection), or from TURBT if needed, from patients being screened for inclusion will be evaluated for PD-L1 expression by H4C (Dako PD-L1 H4C 28- 8 pharmDx). Only patients whose tumors have sufficient amounts of viable tumor and are evaluable for PD-L1 expression as confirmed by a central pathology laboratory prior to enrollment of the patient in the study will be eligible.
Table 4: Schedule of screening activities
[0382] Concomitant medications include medication (e.g., prescription drugs, over-the- counter drugs, vaccines, herbal or homeopathic remedies, nutritional supplements) used by a participant in addition to protocol-mandated treatment (including prophylactic treatment for autogene cevumeran/placebo administration and medications resulting from an adverse event) from 7 days prior to screening Part B to the treatment discontinuation visit. After informed consent has been obtained but prior to initiation of study treatment, only serious adverse events caused by a protocol -mandated intervention should be reported. After initiation of study treatment, all adverse events will be reported until 90 days after the final dose of study treatment or until initiation of another systemic anti-cancer therapy, whichever occurs first. After this period, all deaths, regardless of cause, should be reported. [0383] Initially, patients are enrolled in a safety run-in phase where up to 12 patients receive individualized cancer vaccine + nivolumab. An Internal Monitoring Committee (IMC) reviews safety data after the first 6 patients in the safety run-in phase complete Cycle 1 Day 28 (C1D28). An additional 6 participants may be enrolled in the safety run-in if determined to be necessary by the IMC. Randomization will begin after all participants in the safety run-in have been enrolled to receive the individualized cancer vaccine and nivolumab. [0384] After the safety run-in phase has been enrolled, approximately 350 participants globally will be randomized to one of the following arms in a 1 : 1 ratio: the experimental arm (Arm A: to receive individualized cancer vaccine + nivolumab) or the control arm (Arm B; to receive placebo + nivolumab). A stratified, permuted-block randomization scheme is used to obtain an approximately 1 : 1 ratio between the two treatment arms. Randomization will be temporarily suspended if a study randomization cap of 40 patients total is reached prior to the IMC final safety review. If enrollment is suspended as a result of reaching the 40-patient randomization cap, randomization resumes after IMC review has determined the combination to be sufficiently safe. Subsequent safety reviews occur with an independent Data Monitoring Committee (iDMC) approximately every 6 months thereafter until the study data are unblinded or the study is terminated. Patients in the safety run-in phase will remain unblinded throughout and continue on the study, and will therefore be included in the safety analysis population.
[0385] Randomization is stratified by pathological nodal staging (N± vs. NO), PD-L1 IHC status (tumor cell score ≥1% vs. <1% or indeterminate), and whether prior neoadjuvant chemotherapy was administered (yes vs. no). Randomization occurs within 120 days after surgical resection of the primary tumor. Study drug administration begins within 3 calendar days after randomization; if possible, patients should receive their first dose of the individualized cancer vaccine on the day of randomization. Participants must have no evidence of disease (NED) by imaging within 28 days prior to randomization in order to be eligible.
[0386] The dosing regimen used in the study includes a priming phase and a boost phase (also referred to herein as a booster phase) given over 21 cycles, a discontinuation visit (DV), and follow-up, as shown in FIGS. 2A-2B.
[0387] Patients in the experimental arm (Arm A) receive the individualized cancer vaccine individualized cancer vaccine (also referred to herein as cevu) and nivolumab (also referred to herein as nivo) given over the priming and booster phases of treatment, while patients in the control arm (Arm B) receive the placebo and nivolumab given over the same time period as the administrations in Arm A. In each Arm, imaging is performed at baseline and every 3 months starting from C1D1 (including in, e.g., Weeks 60 and 84), and treatment is administered according to the schedules described below, an overview of which is shown in FIGS. 2A-2B. On treatment days when the individualized cancer vaccine (or placebo) and nivolumab are both administered, both half-doses (i.e., individualized cancer vaccine- A/placebo-A and individualized cancer vaccine-B/placebo-B) will be administered first, followed by nivolumab approximately 30 minutes after completion of the second half-dose (i.e., 30 minutes after individualized cancer vaccine-B/placebo-B administration). The individualized cancer vaccine/placebo should not be dosed more frequently than 48 hours apart.
Priming Phase
• The priming phase is named in reference to “priming” doses of the individualized cancer vaccine (or placebo), though also overlaps with administration of nivolumab. Without wishing to be bound by theory, the individualized cancer vaccine may act to “prime” neoantigen-specific T cells. 2-3 doses of nivolumab are administered during the priming phase in addition to individualized cancer vaccine. Specifically, in the priming phase, 8 priming doses of individualized cancer vaccine (or placebo) are administered over the first 9 weeks, in addition to at least the first 2 doses of nivolumab (which is administered every 4 weeks (Q4W) beginning Week 2), as follows:
• Cycle 1 (28 Days): Individualized cancer vaccine 25 pg IV or placebo on Days 1, 8, 15, and 22 (individualized cancer vaccine (or placebo) priming doses 1, 2, 3, and 4, respectively), for a total of 4 individualized cancer vaccine (or placebo) priming doses within the 28 days of Cycle 1; and nivolumab 480 mg IV on Day 9 (nivolumab dose 1), for a total of 1 dose of nivolumab within the 28 days of Cycle 1, such that two doses of the individualized cancer vaccine (or placebo) are administered before starting nivolumab. Preferably, the first dose of nivolumab is administered 24 or more hours after administration of the second priming dose of the individualized cancer vaccine (or placebo).
• Cycle 2 (28 Days): Individualized cancer vaccine 25 pg IV or placebo on Days 1, 8, and 15 (individualized cancer vaccine (or placebo) priming doses 5, 6, and 7, respectively), for a total of 3 individualized cancer vaccine (or placebo) priming doses within the 28 days of Cycle 2; and nivolumab 480 mg IV on Day 8 (nivolumab dose 2), for a total of 1 dose of nivolumab within the 28 days of Cycle 2.
• Cycle 3 (28 Days): Individualized cancer vaccine 25 pg IV or placebo on Day 1 (individualized cancer vaccine (or placebo) priming dose 8), for a total of 1 individualized cancer vaccine (or placebo) priming dose within the 28 days of Cycle 3. The priming phase is considered to conclude with the last (i.e., 8th) individualized cancer vaccine (or placebo) priming dose. The third dose of nivolumab (480 mg IV) is then administered one week later, ideally on Cycle 3 Day 8, such that individualized cancer vaccine (or placebo) priming dose 8 occurs 1 week before nivolumab dose 3, for a total of 1 dose of nivolumab within the 28 days of Cycle 3.
• Make-up doses of individualized cancer vaccine, placebo, and/or nivolumab in the event of toxicity are permitted during the priming phase. Make-up priming doses of the individualized cancer vaccine, if necessary, should be administered no more frequently than weekly (± 2 days) and may be administered at an unscheduled visit (UV).
• If there are dose delays (inadvertently or, for example, due to occurrence of an adverse event) such that, for example, the 8 priming phase doses of individualized cancer vaccine (or placebo) are administered over the course of, for example, the first 4 months instead of only over the first 9 weeks, then the priming phase may overlap with additional doses of nivolumab (i.e., beyond the first 2 doses of nivolumab), such as, for example, at least the third dose of nivolumab, due to the regular Q4W administration of nivolumab during, e.g., Cycles 2-4. Thus, the third dose of nivolumab is ideally administered after conclusion of the priming phase (and before commencement of the booster phase) but may in some circumstances be included in the priming phase (i.e., before the last priming dose of individualized cancer vaccine or placebo).
• A schedule of activities in the priming is provided in Table 5. Unscheduled visits (UVs) may be performed if clinically indicated. Participants will undergo the specified assessments, and additional assessments may be performed if clinically indicated, as determined by the investigator. Participants who discontinue treatment during the priming phase must complete a treatment discontinuation visit, as detailed in Table 6. • Patient-reported outcome (PRO) assessments (EORTC QLQ-C30, PRO- Common
Terminology Criteria for Adverse Events (CTCAE), European Organisation for Research and Treatment of Cancer (EORTC) IL46, and EQ-5D-5L questionnaires) will be completed (in that order) before the participant receives any information on disease status and prior to the performance of non-PRO assessments and the administration of study treatment. In scenarios where laboratory assessments (e.g., blood draws) are done at a different location than the one providing treatment or when they are done on an earlier day than study treatment administration, laboratory assessments can be completed before the completion of PRO instruments as long as results have not been discussed with the participant. Study personnel should verify that all PRO instruments have been completed before the participant leaves the investigational site, and a reason should be recorded for any missed PRO instruments. In the event that a dose delay occurs after PRO assessments are conducted at a particular visit, PRO assessments will not be repeated at the subsequent make-up visit.
• Weight, ECOG performance status, limited physical examination, and local hematology and chemistry panels may be obtained within 72 hours before a scheduled treatment and/or the treatment discontinuation visit, as applicable.
• Limited, symptom-directed physical examination should be performed at specified visits and as clinically indicated. Examination will be included as part of the evaluation for recurrence of MIUC.
Booster Phase
• The booster phase is named in reference to “booster” doses of the individualized cancer vaccine (or placebo), though also overlaps with administration of nivolumab. Without wishing to be bound by theory, the individualized cancer vaccine may act to “boost” (increase the number of) neoantigen specific T cells as quantified in peripheral blood. The booster phase contains a first portion, comprising ten 28-Day Cycles (Cycles 4-13), in which nivolumab and the first and second booster doses of the individualized cancer vaccine (or placebo) and are administered; and a second portion (referenced herein as the booster phase post- nivolumab), in which the third and fourth booster doses of the individualized cancer vaccine (or placebo) and are administered, as follows: • Cycles 4-13: Individualized cancer vaccine (25 pg IV) or placebo on Day 8 of Cycles 4 and 10, for a total of two booster doses over Cycles 4-13 (booster doses 1 and 2, respectively); and nivolumab 480 mg IV on Day 8 of each cycle, for a total of 10 nivolumab doses over Cycles 4-13, with booster doses 1 and 2 administered on the same day as the dose of nivolumab occurring in Weeks 14 and 38, respectively.
• Booster phase post-nivolumab: Individualized cancer vaccine 25 pg IV or placebo booster dose 3 is administered approximately 15 months post-initiation of nivolumab, after review of an imaging assessment from approximately Week 60. Individualized cancer vaccine 25 pg IV or placebo booster dose 4 is administered approximately 21 months post-initiation of nivolumab, after review of an imaging assessment from approximately Week 84 imaging assessment. Thus, visits for Cycles 15 and 21 will be scheduled at approximately Weeks 60 and 84, respectively, and the individualized cancer vaccine/placebo should be administered following review of tumor assessments required at Weeks 60 and 84, respectively. Thus, it is recommended that the tumor assessments be performed earlier in the visit window to allow for timely individualized cancer vaccine /placebo dosing. The window for the autogene cevumeran/placebo booster doses on these two visits is ±7 days.
• Make-up doses of individualized cancer vaccine, placebo, and/or nivolumab in the event of toxicity, missed priming doses, and/or missed booster doses may be permitted during the booster phase.
• A schedule of activities from the booster phase through the follow-up period is provided in Table 6. Unscheduled visits (UVs) may be performed if clinically indicated.
• PRO assessments (EORTC QLQ-C30, PRO-CTCAE, EORTC IL46, and EQ-5D- 5L questionnaires) will be completed (in that order) before the participant receives any information on disease status and prior to the performance of non-PRO assessments and the administration of study treatment. In scenarios where laboratory assessments (e.g., blood draws) are done at a different location than the one providing treatment or when they are done on an earlier day than study treatment administration, laboratory assessments can be completed before the completion of PRO instruments as long as results have not been discussed with the participants. Participants will complete the EORTC QLQ-C30 and EQ 5D 5L questionnaires at 2 subsequent survival follow-up visits (i.e., at Months 3 and 6 after treatment discontinuation). Study personnel should verify that all PRO instruments have been completed before the participant leaves the investigational site, and a reason should be recorded for any missed PRO instruments. In the event that a dose delay occurs after PRO assessments are conducted at a particular visit, PRO assessments will not be repeated at the subsequent make-up visit.
• Weight, ECOG performance status, limited physical examination, and local hematology and chemistry panels may be obtained within 72 hours before each scheduled treatment and the treatment discontinuation visit, as applicable.
• Limited, symptom-directed physical examination should be performed at specified visits and as clinically indicated. Examination will be included as part of the evaluation for recurrence of MIUC.
[0388] Table 5: Schedule of priming phase activities
Table 6: Schedule of Activities (Boost to Follow-up)
[0389] Treatment in either arm is discontinued in the event of disease recurrence, unacceptable toxicity, withdrawal of consent, or study termination. Serum samples will be obtained at least at screening for potential autoantibody testing (e.g., screening for antibodies binding to nivolumab), and optionally at later timepoints for autoantibody testing as clinically indicated. Serum samples will also be collected to monitor biomarker subsets, ctDNA clearance, and T-cell response. A sample of archival tumor tissues, as well as serum and plasma samples, will be collected for future exploratory biomarker assessments.
[0390] Participants who complete study treatment in full or who discontinue study treatment prior to receiving all planned doses will return to the clinic 30 (±7) days after the final dose of study treatment for a discontinuation visit (DV) and will then receive follow-up assessments. Tumor assessment scans and collection of blood sample for PBMC-I performed within 6 weeks prior to the treatment discontinuation visit do not need to be repeated. The visit at which an assessment confirms recurrence of MIUC may be used as the treatment discontinuation visit and all indicated assessments should be performed. Determination of disease recurrence should be based on presence of unequivocal disease. [0391] All patients will undergo scheduled assessments for tumor recurrence at baseline, within 28 days of enrollment, every 12 weeks (± 1 week; e.g., weeks 12, 24, 36, and 48) for the first year, and every 12 weeks (each ± 2 weeks; e.g., weeks 60, 72, 84, 96) for year 2. Subsequently, surveillance for tumor recurrence is performed every 16 weeks (± 2 weeks) for year 3 (e.g., weeks 112, 128, and 144), and every 24 weeks (± 2 weeks) for years 4-5 (e.g., weeks 168, 192, 216, and 240), and then concluding at year 6. In the absence of an investigator-assessed disease-free survival (DFS) event (e.g., local (pelvic) recurrence of UC; urinary tract recurrence of UC; distant metastasis of UC; or death from any cause), surveillance for tumor recurrence continues regardless of whether patients start new anticancer therapy, until withdrawal of consent, loss to follow-up, or study termination, whichever occurs first. Unscheduled assessments for tumor recurrence may be performed at any time if clinically indicated. Assessments will continue on the above-mentioned schedule regardless of dose delays, or until radiographic disease recurrence.
[0392] Patients undergo imaging and other cancer-related assessments at screening and scheduled intervals for the duration of the study as outlined above. Cancer-related assessments include imaging (e.g., radiographic assessments), clinical evaluation for signs and symptoms of disease, and/or tumor assessments. Serum samples will be collected to monitor biomarker subsets, ctDNA clearance, and T-cell response. Blood samples for exploratory biomarker analyses, pharmacokinetic analyses, and immunogenicity analyses are collected at various timepoints. Plasma samples are collected at every scheduled imaging assessment for tumor recurrence to evaluate ctDNA clearance.
[0393] Assessments occurring during the treatment period are performed within 5 days of the scheduled visit, unless otherwise specified, and assessments on treatment days are performed prior to dosing, unless otherwise specified.
[0394] All patients are closely monitored in safety assessments for the incidence, nature, and severity of adverse events, changes in vital signs, and laboratory abnormalities graded per National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), Version 5.0. Plasma samples are collected at every scheduled imaging assessment for tumor recurrence to evaluate ctDNA clearance.
[0395] For patients with muscle-invasive bladder cancer (MIBC), surveillance for tumor recurrence can include physical examination, laboratory evaluation, and imaging studies of the chest, abdomen, upper urinary tracts, and pelvis. Disease recurrence is determined by the investigator based on radiographic evidence, and whenever possible supported/confirmed by biopsy results. For patients with UTUC, surveillance for tumor recurrence can include physical examination, cystoscopy, urine cytology and imaging studies of the chest, abdomen, and pelvis. A confirmatory tumor biopsy is encouraged if feasible and at the clinical judgement of the investigator at the time of disease recurrence for both MIBC and UTUC. Cases for which biopsy results definitively rule out recurrence of UC are not considered to be disease recurrence for the purposes of this study. After the primary analysis, a confirmatory tumor biopsy is optional at the time of disease recurrence.
[0396] For assessments of MIUC disease recurrence after screening, the following will be evaluated at each post baseline time point: the evolution of each pre-existing lesion, the appearance of new lesions, and an evaluation of previously noted suspicious findings. Recurrence will be assessed in accordance with the guidance published by Apolo AB et al. 2019 (Apolo, AB, Milowsky MI, Kim L, et al. Eligibility and Radiologic Assessment in Adjuvant Clinical Trials in Bladder Cancer. JAMA Oncol 2019;5(12): 1790-8) or in accordance with a modification thereof, as described herein. During follow-up, participants will continue to undergo assessments for tumor recurrence and blood sample collections for biomarkers every 3 months until investigator-assessed recurrence of MIUC.
[0397] The end of this study is defined as the date at which the last data required for all study analyses have been collected. The end of the study is expected to occur approximately 74 months after the first patient is randomized.
Dosing and Dose Modifications
[0398] A standard dose of nivolumab is 480 mg. Modification of the nivolumab dose is not permitted.
[0399] A standard dose of the individualized cancer vaccine (or placebo) is 25 pg. For each individualized cancer vaccine administration, two RNA drug products (individualized cancer vaccine-A and individualized cancer vaccine-B) will be infused sequentially at a half-dose each, each via manual IV push, with an approximately 5-minute observation period between infusions. Placebo administration will follow the same method of administration. For the first infusion of individualized cancer vaccine (or placebo), participants should be observed for approximately 6 hours after the end of the infusion. For each subsequent infusion of individualized cancer vaccine (or placebo) administered, participants should be observed for approximately 4-6 hours after the end of the infusion of autogene cevumeran/placebo. If treatment is interrupted, participants may be permitted to receive make-up doses of individualized cancer vaccine/placebo in order to receive a total of 8 “priming” doses and 4 “booster” doses.
[0400] The dose of the individualized cancer vaccine (or placebo) can be reduced once from 25 pg to 15 pg (but not below 15 pg) for management of drug-related toxi cities, in the event of systemic reactions after infusion. Dose modification rules for the individualized cancer vaccine (or placebo) are outlined in Table 7 and depend on the severity of the systemic reaction after infusion (graded according to NCI CTCAE v.5.0) and on an assessment of specific circumstances of the event. Systemic reactions after infusion of the individualized cancer vaccine commonly manifest as chills (rigors), pyrexia, fatigue, nausea, vomiting, headache, myalgia, and tachycardia. These systemic reactions usually occur 2-4 hours post-infusion.
Table 7: Dose Modification Guidance for Systemic Reactions After Infusion of Individualized Cancer Vaccine.
Methods of Radiographic Assessment and MIUC Assessment
[0401] Standard radiographic assessments conducted as part of this study (e.g., the Week 60 and Week 84 imaging assessments) may include any of the following: Contrast-enhanced CT (computed tomography) chest, contrast-enhanced CT abdomen, contrast-enhanced CT pelvis, and PET (positron emission tomography)-CT (if needed to clarify indeterminate or suspicious lesions seen on CT of the chest/abdomen/pelvis or MRI). Imaging of the upper urinary tracts is not required if covered by abdomen and pelvis scans. For patients with renal insufficiency or a contrast allergy, any of the following radiographic assessments may be used: Non-contrast CT chest, MRI with gadolinium abdomen, MRI with gadolinium pelvis, CT urography (recommended with reduced contrast), MRI urogram (recommended), ureteroscopy, IVP X-ray, or renal ultrasound with retrograde pyelogram (X-ray). For patients with an MRI contrast allergy, any of the following radiographic assessments may be used: Non-contrast MRI urogram with static fluid T2-weighted sequences, or ureteroscopy.
[0402] All eligible patients will undergo a contrast-enhanced CT scan of the chest, abdomen, and pelvis within 28 days prior to randomization. For patients with renal insufficiency or contrast allergy who cannot undergo CT imaging with contrast, non-contrast CT scan of the chest and MRI of abdomen and pelvis with gadolinium should be done. Tumor assessments performed as standard of care prior to obtaining informed consent and within 28 days prior to randomization do not have to be repeated at screening. Patients must have no evidence of disease by imaging as determined by an IRF within 28 days prior to randomization.
[0403] Surveillance for tumor recurrence must include imaging assessments of the chest, abdomen, upper urinary tracts, and pelvis. Imaging of the upper urinary tracts may include one or more of the following: IVP, CT urography, renal ultrasound with retrograde pyelogram, ureteroscopy, or MRI urogram. However, separate imaging of the upper urinary tracts during surveillance via one of these modalities is not required if the upper tracts are covered in the imaging of the abdomen and pelvis. Imaging for tumor assessments for UC recurrence during the study period should use the same imaging modality that was used at screening and be performed at the timepoints specified in Table 6, regardless of drug delays or interruptions.
[0404] At screening, determination of absence of residual disease and absence of metastasis will be made on the basis of radiographic evidence, supported/confirmed by biopsy results (from biopsies performed before or during the screening period) and/ or historical scans (if available). Disease-free status by imaging in order to meet eligibility requirements is defined as meeting all of the following criteria:
• Lymph nodes must be non-pathologic, which is defined as less than 1.0 cm in short axis;
• Absence of non nodal lesions (e.g., no skin or subcutaneous lesions and no presence of CNS metastases or leptomeningeal disease), with the exception of non nodal lesions that are diagnosed as histologically benign; non-nodal lesions measuring 1.0 cm or greater in long axis, which are not feasible for biopsy, must demonstrate radiographic stability across imaging tests at least 4 weeks apart;
• Presence of stable pulmonary nodules is acceptable, provided either of the following conditions are met: o Participants who have stable pulmonary nodules 6 mm or less are considered eligible for enrolment in the study provided that the pulmonary nodules are clinically not suspected of being malignant. Participants with multifocal nodules measuring <6 mm with typical radiographic features of miliary metastases, and/or metabolic features pathognomonic for metastatic disease are considered as having residual disease; o Pulmonary nodules greater than 6 mm in diameter require either evidence that the lesion is histologically benign or demonstration of radiographic stability across imaging tests at least 4 weeks apart. o Surgical bed: There is potential for some radiographic changes in the postoperative setting to be related to inflammation/scarring. If an indeterminate soft tissue lesion of any size is observed in the surgical bed, biopsy is recommended, if feasible. If biopsy is not feasible, lesions that are radiographically consistent with urothelial malignancy, or demonstrating growth over at least two post operative imaging tests (either CT or MRI) at least 4 weeks apart should be considered recurrence.
[0405] Pleural effusion should not be considered as disease presence unless there is a suspicion for underlying pulmonary/pleural malignancy based on CT or additional imaging. In the absence of coexisting nonmalignant clinical disease, indeterminate fluid should be sampled for histologic confirmation of malignancy.
[0406] After screening, determination of disease recurrence may include the following scenarios:
• If a new unequivocal lesion appears; no confirmatory scan needed.
• If an existing lesion that was >10 growths of 20% and ≥5 mm absolut ≥e to nadir; no confirmatory scan needed.
• If an existing lesion that was <10 mm growths of 5 mm to nad ≥ir, continue study treatment (if applicable) and perform confirmatory scan (unscheduled or at the next planned visit). If on this scan the observed lesion will turn unequivocal then recurrence should be declared from the first date a suspicious finding meets the criteria for unequivocal recurrence (not confirmatory scan)
• If a lesion calling recurrence does not show further interval growth (taking measurement errors into account) on subsequent scans, recurrence is unlikely and should be recalled. [0407] Recurrence will be assessed in accordance with the guidance published by Apolo AB et al. 2019, supra, which have been partially modified such that the following findings qualify as “unequivocal disease recurrence”:
• New lesion 1 ≥0 mm that was absent on baseline (in retrospect);
• Lesion present at baseline (in retrospect): o If <10 mm at nadir within preceding 12 months, demonstrating 5 mm ≥ absolute increase on 2 consecutive examinations; o If ≥ 10 mm at nadir within preceding 12 months, demonstrating 20% growth ≥ in size with 5 ≥ mm absolute increase on a single examination;
• Multifocal lesions measuring <10 mm that demonstrate geographic distribution or radiographic and/or metabolic features pathognomonic for metastatic disease (example miliary lung metastasis);
• Soft tissue means all solid tumors including soft tissue component of a bone lesion; and
• RD also includes any typical brain or leptomeningeal lesion of any size, particularly a lesion with ringlike enhancement that is hemorrhagic and/or associated with vasogenic edema.
Study Participants
Inclusion Criteria
[0408] Screening Part A inclusion criteria: Patients that meet the following criteria are included in Screening Part A of this study:
• Signed Informed Consent Form
• Age 18 years or older at the time of signing Informed Consent Form
• Planned to undergo radical surgical resection for MIUC, and medically eligible for radical cystectomy /nephroureterectomy and pelvic lymph node dissection o For patients identified prior to radical surgical resection: muscle- invasive UC of the bladder identified based on computed tomography (CT) or magnetic resonance imaging (MRI) scan of the pelvis, abdomen, and chest as cT3-T4 or N+. o For patients identified at the time of surgical resection, TNM classification (UICC/AJCC 7th edition) at pathological examination of surgical resection specimen as follows:
■ Tumor stage of (y)pT3-4a or (y)pN+ (for patients with MIBC) or (y)pT3-4 or (y)pN+ (for patients with UTUC) and MO regardless of whether or not prior neoadjuvant chemotherapy was administered
• Submission of a representative formalin-fixed paraffin-embedded (FFPE) tumor specimen from the pretreatment tumor biopsy (i.e., TURBT, or surgical resection specimen if patient is identified at surgical resection and TURBT samples are not available), preferably in paraffin blocks o This is used for upstream manufacturing of individualized cancer vaccine including WES and RNA sequencing o If an intact FFPE tumor block cannot be provided, then submission of unstained, serial thin sections derived from an FFPE tumor block (ideally about 20 slides, freshly cut from a single collection procedure) appropriately prepared and shipped is allowed, provided the tumor tissue is of good quality based on total and viable tumor content and contains a muscle invasive component (i.e., T2 or greater) of the tumor as verified by local pathology review.
• Submission of a matched blood sample for the identification of somatic mutations in tumor tissue for upstream manufacturing of the individualized cancer vaccine.
[0409] Screening Part B inclusion criteria: Patients that meet the Screening Part A inclusion criteria outlined above are eligible to be included in the study only if all of the following criteria apply:
• Age 18 years or older
• Histologically confirmed muscle-invasive UC (also termed TCC) of the bladder or upper urinary tract (i.e., renal pelvis, or ureters) o Patients with mixed or variant histologies are required to have a dominant (i.e., >50%) urothelial pattern
• TNM classification (UICC/AJCC 7th edition) at pathological examination of surgical resection specimen as follows: o Tumor stage of (y)pT3-4a or (y)pN+ (for patients with MIBC), or (y)pT3-4 or (y)pN+ (for patients with UTUC) and MO, regardless of whether or not prior neoadjuvant chemotherapy was administered.
• Surgical resection of muscle-invasive UC of the bladder (MIBC), or UTUC upper tract o For patients with MIBC, radical cystectomy may be performed by the open, laparoscopic, or robotic approach. Cystectomy must include bilateral lymph node dissection, the extent of which will be at the discretion of the treating urologist but optimally should extend at a minimum from the mid common iliac artery proximally to Cooper's ligament distally, laterally to the genitofemoral nerve, and inferiorly to the obturator nerve. The method of urinary diversion for patients undergoing cystectomy will be at the discretion of the urologist and choice of the patient
■ Patients with a negative surgical margin (i.e., RO resection) or with carcinoma in situ (CIS) at the distal ureteral or urethral margin will be eligible.
■ Patients with a positive R2 margin (i.e., a tumor identified at the inked perivesical fat margin surrounding the cystectomy specimen) or R1 margin (i.e., evidence of microscopic disease identified at the tumor margin), except for CIS at distal ureteral or urethral margin, will be excluded. o For patients with UTUC, an RNU with excision of the bladder cuff is required and may be performed by the open or laparoscopic approach. RNU must include lymph node dissection (LND), the extent of which will be at the discretion of the treating urologist but optimally should include the para-aortic, paracaval or interaortocaval nodes from the renal hilum to the inferior mesenteric artery in renal pelvis and proximal ureteral tumors, or nodes from the renal hilum to the bifurcation of the common iliac artery and ipsilateral pelvic nodes in mid and lower ureteral tumors, respectively.
■ Patients must have a negative surgical margin (i.e., RO resection). Patients with a positive R1 or R2 surgical margin will be excluded. • Patients who have or have not received prior platinum-based neoadjuvant chemotherapy (NAC) are eligible for the study. o Patients who have received at least three cycles of a platinum- containing regimen are considered to have received prior neoadjuvant chemotherapy. o Patients who have not received prior NAC should be ineligible to receive adjuvant cisplatin-based therapy either due to cisplatin ineligibility, patient refusal, or investigator decision
• Cisplatin ineligibility may be met by any of the following criteria: o Impaired renal function (glomerular filtration rate (GFR) < 60 mL/min); GFR should be assessed by direct measurement (i.e., creatinine clearance or ethyldediaminetetra-acetate) or, if not available, by calculation from serum/plasma creatinine (Cockcroft-Gault formula); o A hearing loss (measured by audiometry) of 25 dB at two contiguous frequencies; o Grade 2 or greater peripheral neuropathy (i.e., sensory alteration or parasthesis including tingling); o ECOG performance status of 2.
• Availability of a surgical tumor specimen that is suitable (e.g., adequate quality and quantity) for use in determining PD-L1 expression and for exploratory biomarker research assessed by central laboratory testing. o A representative FFPE tumor block must be submitted along with an associated pathology report. o If an intact FFPE tumor block cannot be provided, then submittal of approximately 20 slides containing unstained, freshly cut serial sections derived from an FFPE tumor block is allowed. o Tumor tissue should be of good quality, as determined on the basis of total and viable tumor content and must contain a muscle invasive component (i.e., T2) of the tumor as verified by local pathology review. Samples must contain a minimum of 50 viable tumor cells that preserve cellular context and tissue architecture regardless of needle gauge or retrieval method. Tumor tissue from bone metastases that have been decalcified is not acceptable. o In situations where multiple specimens were received from different sites or at different times, the score from the surgical resection of the primary tumor or lymph node dissection specimen will be used for both primary and secondary analyses. o In situations in which multiple specimens are received from different sites or at different times, the score from the surgical resection of the primary tumor or lymph node dissection specimen is used for both primary and secondary analyses.
• Tumor tissue from the most recently resected site of disease must be provided for PD-L1 IHC. In order to be randomized, the participant must have a PD-L1 expression level (IHC tumor cell score of 1%, <1% ≥, indeterminate) as determined by the central laboratory. If insufficient tumor tissue content is provided for analysis (e.g., unevaluable), acquisition of additional archived tumor tissue from the most recent resection or TURBT that yielded the initial muscle invasive diagnosis is required
• Absence of residual disease and absence of metastasis, as confirmed by a negative baseline computed tomography (CT) or magnetic resonance imaging (MRI) scan of the pelvis, abdomen, and chest no more than 28 days prior to randomization. o Confirmation of disease-free status will be assessed by independent central radiologic review of imaging data. o For patients with MIBC, if upper urinary tracts cannot be visualized on CT scan, imaging of the upper urinary tracts must include one or more of the following: intravenous pyelogram (IVP), CT urography, renal ultrasound with retrograde pyelogram, ureteroscopy or MRI urogram, and must be completed no more than 28 days prior to randomization o For patients with UTUC, cystoscopy and urine cytology must be completed no more than 28 days prior to randomization; upper tract imaging to confirm absence of contralateral disease is required. o For patients with both primary MIBC and primary UTUC, upper tract imaging and urine cytology to confirm absence of contralateral disease is required o Other examinations are performed as clinically indicated.
• At least 5 cancer-specific neoepitopes identified from analysis of blood and tumor specimens submitted for generation of individualized cancer vaccine.
• Full recovery from cystectomy or nephroureterectomy within 120 days following surgery
• ECOG performance status of 0 or 1. o However, patients that have not received cisplatin based neoadjuvant chemotherapy and are considered ineligible for cisplatin adjuvant chemotherapy may enter the study with ECOG PS 2.
• Adequate hematologic and end-organ function, as defined by the following laboratory results obtained within 14 days prior to the first study treatment: o ANC ≥ 1000 cell s/pL o WBC > 2000/pL o Platelet count 10 ≥0, 000/pL o Hemoglobin 9 ≥.0 g/dL (Patients may be transfused or receive erythropoietic treatment to meet this criterion) o AST, ALT < 3.0 x the upper limit of normal (ULN) o Serum bilirubin < 1.5 x ULN (however, patients with known Gilbert disease who have serum bilirubin level < 3 x ULN may be enrolled. o For patients who are not receiving therapeutic anti coagulation: PTT/PT < 1.5 x ULN or INR < 1.7 x ULN (patients who are receiving therapeutic anti coagulation should be on a stable dose) o Serum creatinine < 1.5 x ULN or creatinine clearance 30 mL/min, ≥ using the following Cockcroft-Gault formula: o Negative HIV test at screening, with the following exception: individuals with a positive HIV test at screening are eligible provided they are stable on anti-retroviral therapy, have a CD4 count 200/ pL, ≥ and have an undetectable viral load.
No evidence of active hepatitis B (i.e., having a negative hepatitis B surface antigen (HBsAg) test at screening) o Patients with past or resolved hepatitis B infection (e.g., having a negative HBsAg test and a positive total hepatitis B core antibody (HBcAb) test) are eligible; however, a hepatitis B virus (HBV) DNA test must be obtained prior to initiation of study drug and must demonstrate absence of active infection
• Negative hepatitis C virus (HCV) antibody test at screening, or a positive HCV antibody test followed by a negative HCV RNA test at screening o The HCV RNA test must be performed for individuals who have a positive HCV antibody test
• For female participants of childbearing potential: agreement to remain abstinent (refrain from heterosexual intercourse) or use contraception, as follows: o Female participants must remain abstinent or use contraceptive methods with a failure rate of < 1% per year during the treatment period and for 28 days after the final dose of individualized cancer vaccine and for 5 months after the final dose of nivolumab o A female participant is considered to be of childbearing potential if she meets the following criteria (which may be adapted for alignment with local guidelines or regulations): she is postmenarchal, has not reached a postmenopausal state (> 12 continuous months of amenorrhea with no identified cause other than menopause), and is not permanently infertile due to surgery (i.e., removal of ovaries, fallopian tubes, and/or uterus, though not including tubal ligation) or another cause as determined by the investigator (e.g., Mullerian agenesis)
• For male participants: agreement to remain abstinent (refrain from heterosexual intercourse) or use a condom, and agree to refrain from donating sperm, as follows: o With a female partner of childbearing potential or pregnant female partner, men must remain abstinent or use a condom during the treatment period and for 28 days after the final dose of individualized cancer vaccine and for 5 months after the final dose of nivolumab. Male participants must refrain from donating sperm during this same period.
Exclusion Criteria
[0410] Patients that meet the following criteria are excluded from this study:
• Pregnancy or breastfeeding, or intention of becoming pregnant during the study or within 28 days after the final dose of individualized cancer vaccine or 5 months after the final dose of nivolumab o Female participants of childbearing potential must have a negative serum pregnancy test result within 14 days prior to initiation of study treatment
• Partial cystectomy in the setting of bladder cancer primary tumor or partial nephrectomy in the setting of renal pelvis primary tumor
• Any approved anti-cancer therapy, including chemotherapy, or hormonal therapy, within 3 weeks prior to initiation of study treatment o Hormone-replacement therapy or oral contraceptives are allowed.
• Any prior neoadjuvant immunotherapy
• Adjuvant chemotherapy or radiation therapy for UC following surgical resection o Antegrade or retrograde instillation of chemotherapy or BCG is not allowed for patients with UTUC. However, a single dose of intravesical chemotherapy post nephroureterectomy is allowed.
• Treatment with any other investigational agent or participation in another clinical trial with therapeutic intent within 28 days or five half-lives of the drug, whichever is longer, prior to randomization
• Malignancies other than UC within 5 years prior to randomization, subject to the following: o Patients with high-risk UTUC (i.e., tumor stage (y)pT3-4 or ypN+) within 5 years prior to Cycle 1 Day 1 are ineligible if the UTUC limit of approximately 10% has been met. o Patients with localized low risk prostate cancer (Stage < T2b, Gleason score < 7, and prostate-specific antigen (PSA) at prostate cancer diagnosis < 20 ng/mL, if measured) treated with curative intent and without prostate-specific antigen (PSA) recurrence are eligible. o Patients with low-risk prostate cancer (Stage Tl/T2a, Gleason score <7 and PSA < 10 ng/mL) who are treatment-naive and undergoing active surveillance are eligible. o Patients with malignancies of a negligible risk of metastasis or death (e.g., risk of metastasis or death <5% at 5 years) are eligible if they meet all of the following criteria:
■ Malignancy treated with expected curative intent (such as adequately treated carcinoma in situ of the cervix, basal or squamous cell skin cancer, or ductal carcinoma in situ treated surgically with curative intent)
■ No evidence of recurrence or metastasis by follow-up imaging and any disease-specific tumor markers
• Absence of spleen (due to splenectomy, splenic injury/infarction, or functional asplenia)
• Major surgical procedure, other than for diagnosis or for resection of disease under current study, within < 6 weeks prior to initiation of study treatment, or anticipation of need for a major surgical procedure during the study o Placement of a central venous access catheter (e.g., port or similar) is not considered a major surgical procedure and is therefore permitted
• Significant cardiovascular disease (such as New York Heart Association Class II or greater cardiac disease, myocardial infarction, or cerebrovascular accident) within 3 months prior to initiation of study treatment, unstable arrhythmia, or unstable angina
• Clinically significant liver disease, including active viral, alcoholic, or other hepatitis, cirrhosis, and inherited liver disease, or current alcohol abuse as determined by the investigator
• Active or history of autoimmune disease or immune deficiency, including: Acute disseminated encephalomyelitis, Addison disease, Ankylosing spondylitis, Anti-phospholipid antibody syndrome, Aplastic anemia, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune hypoparathyroidism, Autoimmune hypophysitis, Autoimmune myelitis, Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune thrombocytopenic purpura, Behget disease, Bullous pemphigoid, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy, Churg- Strauss syndrome, Crohn disease, Dermatomyositis, Diabetes mellitus type 1, Dysautonomia, Epidermolysis bullosa acquisita, Gestational pemphigoid, Giant cell arteritis, Goodpasture syndrome, Graves disease, Guillain-Barre syndrome, Hashimoto disease, IgA nephropathy, Inflammatory bowel disease, Interstitial cystitis, Kawasaki disease, Lambert- Eaton myasthenia syndrome, Lupus erythematosus, Lyme disease (chronic), Meniere syndrome, Mooren ulcer, Morphea, Multiple sclerosis, Myasthenia gravis, Neuromyotonia, Opsoclonus myoclonus syndrome, Optic neuritis, Ord thyroiditis, Pemphigus, Pernicious anemia, Polyarteritis nodosa, Polyarthritis, Polyglandular autoimmune syndrome, Primary biliary cholangitis, Psoriasis, Reiter syndrome, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren syndrome, Stiff-Person syndrome, Takayasu arteritis, Ulcerative colitis, Vitiligo, Vogt-Koyanagi -Harada disease, or Granulomatosis with polyangiitis, with the following exceptions: o Patients with a history of autoimmune-related hypothyroidism who are on thyroid replacement hormone are eligible for the study; o Patients with controlled Type 1 diabetes mellitus who are on an insulin regimen are eligible for the study; o Participants with a medical history of such entities as atopic disease or childhood arthralgias where the clinical suspicion of autoimmune disease is low may be eligible; o Patients with eczema, psoriasis, lichen simplex chronicus, or vitiligo with dermatologic manifestations only (e.g., patients with psoriatic arthritis are excluded) are eligible for the study provided all of following conditions are met:
■ Rash must cover < 10% of body surface area;
■ Disease is well controlled at baseline and requires only low- potency topical corticosteroids; and
■ There has been no occurrence of acute exacerbations of the underlying condition requiring psoralen plus ultraviolet A radiation, methotrexate, retinoids, biologic agents, oral calcineurin inhibitors, or high potency or oral corticosteroids within the previous 12 months;
• Known primary immunodeficiencies, either cellular (e.g., DiGeorge syndrome, T negative severe combined immunodeficiency (SCID)) or combined T- and B-cell immunodeficiencies (e.g., T- and B-negative SCID, Wiskott-Aldrich syndrome, ataxia telangiectasia, common variable immunodefi ci ency ) ;
• Treatment with monoamine oxidase inhibitors (MAOIs) within 3 weeks prior to initiation of study treatment or requirement for ongoing treatment with MAOIs;
• Treatment with systemic immunostimulatory agents (including, but not limited to, interferon and IL-2) within 4 weeks or 5 drug-elimination half-lives (whichever is longer) prior to initiation of study treatment;
• Treatment with systemic immunosuppressive medication (including, but not limited to: corticosteroids, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents) within 2 weeks prior to initiation of study treatment, or anticipation of need for systemic immunosuppressive medication during study treatment, with the following exceptions: o Patients who received acute, low-dose systemic immunosuppressant medication or a one-time pulse dose of systemic immunosuppressant medication (e.g., 48 hours of corticosteroids for a contrast allergy) are eligible for the study; o Patients who received mineralocorticoids (e.g., fludrocortisone), inhaled or low dose corticosteroids (defined as < 10 mg oral prednisone per day or daily equivalent) for chronic obstructive pulmonary disease or asthma, or low-dose corticosteroids for orthostatic hypotension or adrenal insufficiency are eligible for the study;
• History of idiopathic pulmonary fibrosis, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced pneumonitis, or idiopathic pneumonitis, or evidence of active pneumonitis on screening chest CT scan;
• Known active or latent tuberculosis o If the investigator considers a potential patient to be at an increased risk for infection with Mycobacterium tuberculosis, latent tuberculosis diagnostic procedures must be followed according to local practice standards during the screening period.
• Recent acute infection, defined as severe infection within 4 weeks prior to initiation of study treatment, including, but not limited to, hospitalization for complications of infection, bacteremia, or severe pneumonia, or any active infection that could impact patient safety;
• Prior allogeneic stem cell or solid organ transplantation;
• Any other disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding that contraindicates the use of an investigational drug, may affect the interpretation of the results, or may render the patient at high risk from treatment complications;
• Treatment with a live, attenuated vaccine within 4 weeks prior to initiation of study treatment, or anticipation of need for such a vaccine during study treatment, or for 5 months after the final dose of study treatment; o Influenza vaccination should be given during influenza season only.
• Receipt of any mRNA vaccine (e.g., COVID-19 vaccine) within 7 days prior to start of study treatment;
• History of severe allergic anaphylactic reactions to chimeric or humanized antibodies or fusion proteins;
• Known hypersensitivity to Chinese hamster ovary cell products;
• Known hypersensitivity or allergy to any component of the individualized cancer vaccine or nivolumab products.
Study Outcome Measures
[0411] The primary outcome measures of this study include the following:
• DFS (disease-free survival) after randomization, defined as the time from randomization to: o The first recurrence of disease, or o death from any cause, (whichever occurs first). o Disease recurrence is determined by the investigator and is defined as any of the following: ■ Local (pelvic) recurrence of urothelial carcinoma (UC) (including soft tissue and regional lymph nodes);
■ Urinary tract recurrence of UC (including all pathological stages and grades); or
■ Distant metastasis of UC.
[0412] The secondary outcome measures of this study include the following:
• OS (overall survival) after randomization, defined as the time from randomization to death from any cause.
• Investigator-assessed DFS in PD-L1 1%, de ≥fined as the time from randomization to the first occurrence of a documented disease recurrence or death from any cause in participants with tumors expressing PD LI 1% as ≥ determined by immunohistochemistry (IHC) (Dako PD-L1 IHC 28-8 pharmDx).
• Investigator-assessed distant metastasis-free survival (DMFS), defined as the time from randomization to the date of diagnosis of distant (i.e., non locoregional) metastases.
• Incidence and severity of adverse events, with severity determined according to the NCI CTCAE v5.0.
• Change from baseline (defined as the last value prior to initiation of study treatment) in selected vital signs.
• Change from baseline (defined as the last value prior to initiation of study treatment) in selected clinical laboratory test results.
• Mean and mean changes from baseline (defined as the last value prior to initiation of study treatment) score (by Cycle) in patient-reported Pain, Physical Functioning, Role Functioning, and global health status (GHS)/ quality of life (QoL) scales, as assessed through use of the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30.
• Presence, frequency of occurrence, severity, and/or degree of interference with daily function of symptomatic treatment toxicities (fatigue, chills, nausea, vomiting, diarrhea, constipation, decreased appetite, swelling, itching, rash, headache, muscle pain, joint pain, general pain, cough, and shortness of breath) as assessed through the use of the National Cancer Institute (NCI) patient reported outcome (PRO) CTCAE. • Change from baseline in symptomatic treatment toxicities, as assessed through use of the NCI PRO CTCAE.
• Frequency of patients’ response of the degree they are troubled with treatment symptoms, as assessed through use of the single-item EORTC Item Library 46 (IL46).
[0413] The exploratory outcome measures of this study include the following:
• Relationship between biomarkers at baseline (defined as the last value prior to initiation of study treatment) or on-treatment in blood and tumor tissue and efficacy, safety, or other biomarker endpoints. o Exploratory biomarker research may include, but will not be limited to, analysis of circulating tumor DNA, genes or gene signatures associated with tumor immunobiology, lymphocytes, cytokines associated with T cell activation. Research may involve extraction of DNA, cell-free DNA, or RNA; analysis of mutations, single nucleotide polymorphisms, and other genomic variants; and genomic profiling. o Genomic research with a focus on somatic variants may be conducted by comparing DNA extracted from blood or PBMCs with DNA extracted from tissue to distinguish somatic variants from germline variants. Genomic profiling may include whole genome sequencing (WGS) or whole exome sequencing (WES) of blood samples, with a focus on somatic variants. WGS or WES of blood samples with a focus on germline variants may also be conducted at participating sites.
• Longitudinal changes from baseline in biomarkers of blood and tumor tissue.
• Changes or clearance of circulating tumor DNA (ctDNA) from baseline a levels upon study treatment.
• Changes from baseline in antigen-specific T-cell responses.
• Mean and mean changes from baseline score (by cycle) in all symptoms other than patient-reported Pain, Physical and Role Functioning, and GHS/QoL scales, as assessed through use of the EORTC QLQ-C30.
• Health utility and visual analogue scale (VAS) scores of the EQ-5D-5L questionnaire.
• Plasma concentrations of DOTMA ((R) N,N,N trimethyl 2,3 dioleyloxy-1- propanaminium chloride), blood concentration of mRNA, and serum concentrations of nivolumab at: Cycle 1 Day 1, Cycle 1 Day 8 (for DOTMA), Cycle 1 Day 9 (for nivolumab), Cycle 4 Day 8, Cycle 8 Day 8 (for nivolumab), Cycle 13 Day 8 (for nivolumab);
• Prevalence of anti-drug antibodies (AD As) to nivolumab at baseline and incidence of AD As to nivolumab during the study.
SEQUENCES
All polynucleotide sequences are depicted in the 5 ’->3’ direction. All polypeptide sequences are depicted in the N-terminal to C-terminal direction.
Anti-PDLl antibody HVR-H1 sequence (SEQ ID NO:1)
GFTFSDSWIH
Anti-PDLl antibody HVR-H2 sequence (SEQ ID NO:2)
AWISPYGGSTYYADSVKG
Anti-PDLl antibody HVR-H3 sequence (SEQ ID NO:3) RHWPGGFDY
Anti-PDLl antibody HVR-L1 sequence (SEQ ID NO:4)
RASQDVSTAVA
Anti-PDLl antibody HVR-L2 sequence (SEQ ID NO: 5)
SASFLYS
Anti-PDLl antibody HVR-L3 sequence (SEQ ID NO: 6)
QQYLYHPAT
Anti-PDLl antibody VH sequence (SEQ ID NO:7)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSS
Anti-PDLl antibody VL sequence (SEQ ID NO: 8)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR
Anti-PDLl antibody heavy chain sequence (SEQ ID NO: 9)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGL YSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Anti-PDLl antibody light chain sequence (SEQ ID NO: 10)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Nivolumab heavy chain sequence (SEQ ID NO: 11) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTL
VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
Nivolumab light chain sequence (SEQ ID NO: 12)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Pembrolizumab heavy chain sequence (SEQ ID NO: 13)
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG
INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYW
GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GV
HTFP AVLQ S SGL YSLS S VVT VP S S SLGTKT YTCNVDHKP SNTKVDKRVESK YGPPCPP CP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
Pembrolizumab light chain sequence (SEQ ID NO: 14)
EIVLTQSPAT
LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES
GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPS VF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Avelumab heavy chain sequence (SEQ ID NO: 15)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGI TFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQG
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGL YSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Avelumab light chain sequence (SEQ ID NO: 16)
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRP SGVSNRFSGSKSGNTASLTISGLQAEDEAD YYC S S YTS S STRVFGTGTKVTVLGQPKA NPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Durvalumab heavy chain sequence (SEQ ID NO: 17)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDG
SEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDY
WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD
KTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPASIEI<
TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Durvalumab light chain sequence (SEQ ID NO: 18)
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGI
PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Full ICV RNA 5’ constant sequence (SEQ ID NO: 19)
GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAU
GAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGCCC UGACAGAGACAUGGGCCGGAAGC
Full ICV RNA 3’ constant sequence (SEQ ID NO:20)
AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG
CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC
AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU
GACAGCCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCU
UUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUC
CCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGC
ACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACA
GCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACC CCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCU AGCCGCGUCGCU
Full ICV Kozak RNA (SEQ ID NO:21)
GGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC
Full ICV Kozak DNA (SEQ ID NO:22)
GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC short Kozak RNA (SEQ ID NO:23)
UUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC short Kozak DNA (SEQ ID NO:24)
TTC:T’rC’TGGI'CCC:CACAGACT'CAGAGAGAACCCGCC:ACC sec RNA (SEQ ID NO:25) AUGAGAGUGAUGGCCCCCAGAACCCUGAUCCUGCUGCUGUCUGGCGCCCUGGC CCUGACAGAGACAUGGGCCGGAAGC sec DNA (SEQ ID NO:26)
ATGAGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCC T G AC A GA GA C AT GGGC C GG A AGC sec protein (SEQ ID NO:27)
MRVMAPRTLILLLSGALALTETWAGS
MITD RNA (SEQ ID NO:28)
AUCGUGGGAAUUGUGGCAGGACUGGCAGUGCUGGCCGUGGUGGUGAUCGGAG CCGUGGUGGCUACCGUGAUGUGCAGACGGAAGUCCAGCGGAGGCAAGGGCGGC AGCUACAGCCAGGCCGCCAGCUCUGAUAGCGCCCAGGGCAGCGACGUGUCACU GACAGCC
MITD DNA (SEQ ID NO:29)
ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCC GTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGC TACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACA GCC
MITD protein (SEQ ID NO: 30)
IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA
Full ICV FI RNA (SEQ ID NO: 31)
CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGG UACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGC CCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCA GCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACC UUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCA AUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGUCGCUAGCCGCGUCGCU
Full ICV FI DNA (SEQ ID NO:32)
CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGA
GTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC CACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACG CTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAA ACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCC ACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT
F element RNA (SEQ ID NO:33)
CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCC GAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACU CACCACCUCUGCUAGUUCCAGACACCUCC
F element DNA (SEQ ID NO:34) CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGA
GTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCAC
CACCTCTGCTAGTTCCAGACACCTCC
I element RNA (SEQ ID NO: 35)
CAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGG
AAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCG
I element DNA (SEQ ID NO: 36)
CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGA
AACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAAC
CCCAGGGTTGGTCAATTTCGTGCCAGCCACACCG linker RNA (SEQ ID NO: 37)
GGCGGCUCUGGAGGAGGCGGCUCCGGAGGC linker DNA (SEQ ID NO: 38)
GGCGGCTCTGGAGGAGGCGGCTCCGGAGGC linker protein (SEQ ID NO: 39)
GGSGGGGSGG
Full ICV DNA 5’ constant sequence (SEQ ID NO:40)
GGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATG AGAGTGATGGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGA CAGAGACATGGGCCGGAAGC
Full ICV DNA 3’ constant sequence (SEQ ID NO:41)
ATCGTGGGAATTGTGGCAGGACTGGCAGTGCTGGCCGTGGTGGTGATCGGAGCC GTGGTGGCTACCGTGATGTGCAGACGGAAGTCCAGCGGAGGCAAGGGCGGCAGC TACAGCCAGGCCGCCAGCTCTGATAGCGCCCAGGGCAGCGACGTGTCACTGACA GCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCG TCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCC ACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAA TGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTA ACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCA
ATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGTCGCTAGCCGCGTCGCT
Full ICV RNA with 5’ GG from cap (SEQ ID NO: 42)
GGGGCGAACU AGUAUUCLTJC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACCAUGAGAG UGAUGGCCCC CAGAACCCTJG AUCCUGCUGC UGUCUGGCGC CCUGGCCCUG ACAGAGACAU GGGCCGGAAG CNAUCGUGGGA AUUGUGGCAG GACUGGCAGU GCUGGCCGUG GUGGUGAUCG GAGCCGUGGU GGCUACCGUG AUGUGCAGAC GGAAGUCCAG CGGAGGCAAG GGCGGCAGCU ACAGCCAGGC CGCCAGCUCU GAUAGCGCCC AGGGCAGCGA CGUGUCACUG ACAGCCUAGU AACUCGAGCU GGUACUGC AU GCACGCAAUG CUAGCUGCCC CUUUCCCGUC CUGGGUACCC CGAGUCUCCC CCGACCUCGG GUCCCAGGUA UGCUCCCACC UCCACCUGCC CCACUCACCA CCUCUGCUAG UUCCAGACAC CUCCCAAGCA CGCAGCAAUG CAGCUCAAAA CGCUUAGCCU AGCCACACCC CCACGGGAAA CAGCAGUGAU UAACCUUUAG CAAUA AACGA AAGUUUAACU AAGCUAUACU AACCCCAGGG UUGGTJCAAUU UCGUGCCAGC CACACCGAGA CCUGGUCCAG AGUCGCUAGC CGCGUCGCUA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA