Construction of aptamer conjugates

A 2′-fluoro-pyrimidine-modified dimeric 4–1BB RNA aptamer transcribedin vitro from a DNA template described (21) extended at the 3′ end with a linker sequence 5′-UCCCG-CUAUAAGUGUGCAUGAGAAC-3 was annealed to a chemically synthesized (IDT) VEGF aptamer (38) via a complementary linker sequence engineered at the 3′ end. Equimolar amounts of 4–1BB and VEGF aptamer was mixed, heated to 82°C, and cooled to room temperature. Annealing efficiency, monitored by agarose gel electrophoresis, was >90%. To preventin vivo annealing of the two aptamers, they were annealed separately with a 2-fold excess of complementary linker sequence before tail vein injection.

Tumor immunotherapy studies

The facilities at the University of Miami’s Division of Veterinary Resources are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and U.S. Drug Administration. An Office of Laboratory of Animal Welfare assurance is on file, ensuring that humane animal care and use practices, as outlined in the Guide for the Care and Use of Laboratory Animals, are followed. Of note, 5- to 6-week-old and Balb/c (H-2d)mice were purchased from The Jackson Laboratory and used within 1 to 3 weeks.

Subcutaneous tumor models

Six- to 8-week-old Balb/c mice were injected subcutaneously on the right flanks with 1.0 × 10⁵ 4T07 or CT26 tumors cells. Tumors were allowed to grow to about 75 to 100 mm³, at which point mice were anesthetized with a mixture of ketamine and xylazine (80–100 and 10 mg/kg, respectively) by intraperitoneal injection using an insulin syringe. Once anesthetized, animals were placed in a custom lead shielding apparatus to expose the tumor but minimally expose the rest of the animal. The apparatus was placed into and RS-2000 irradiator (RadSource) with 225 kV energy at an approximate rate of 1.8 Gy/minute where tumors were irradiated with a single fraction of 12 Gy. At days 6, 8, and 10 post irradiation, animals were administered with 100 pmol of aptamer conjugate or mixture of via tail vein, or with 800 pmol of 4–1BB (clone 3H3, BioXcell).

3-Methylcholanthrene-induced fibrosarcoma model

Balb/c mice were injected subcutaneously with 400 μg of 3-methylcholanthrine (MCA) in castor oil. When tumors reached an approximate size of 100 mm³, mice were irradiated as previously described with 20 Gy. As indicated, mice were injected with 100 pmol of aptamer conjugate or mixture of via tail vein three times, 3 days apart. Mice were sacrificed when tumor exceeded 1,200 mm³ or mice exhibited signs of morbidity.

Subcutaneous abscopal model

Balb/c mice were implanted subcutaneously on the right flank with 2.0 × 10⁴ 4T1 cells. Eight days later, when right flank tumor started to become palpable, 2.0 × 10⁴ 4T1 cells were injected subcutaneously on the left flank. At day 13 post right flank implantation, when tumors reached 75 to 100 mm³, the mice were anesthetized and irradiated as described above followed by 100 pmol administration of aptamer conjugate via tail vein injection on days 3, 6, and 9 postirradiation. Alternatively, 200 μg of CTLA-4 (clone 9H10, BioXcell) and/or 200 μg PD-1 antibody (clone RMP1–14, produced by HY) were injected intraperitoneally.

Metastatic abscopal model

Balb/c mice were implanted subcutaneously on the right flank with 2.0 × 10⁴ 4T1 cells. Eight days later, when right flank tumor started to become palpable, 2.0 × 10⁴ 4T1 cells were injected intravenously. At day 13 after right flank implantation, when tumors were roughly 75 to 100 mm³, the mice were anesthetized and irradiated as described above with a single dose of 12 Gy followed by 100 pmol administration of aptamer conjugate via tail vein injection on days 3, 6, and 9 post irradiation. At day 21 post intravenous injection, when untreated mice started to show signs of morbidity, mice were sacrificed and the weight of lungs was determined as a measure of tumor burden.

Toxicity studies

Balb/c mice bearing 4T07 tumors were irradiated and treated with 100 pmol of aptamer conjugate, 100 or 800 pmol of 4–1BB 3H3 agonistic antibody, or with 800 pmol of isotype. Mice were sacrificed 5 days after the last treatment. Lung, liver, and spleen weights were weighed, fixed in formalin, and embedded followed by staining with hematoxylin and eosin or homogenized to generate a single-cell suspension and CD8⁺ T cells were stained with fluorophore-labeled antibody for flow cytometry.

FACS analysis

Cells were incubated with fixable viability dye eFluor780 for 10 minutes at 4°C and washed twice in FACS buffer prior to staining in order distinguish live cells from the dead cells. Cells were incubated with Fc-blocking antibodies for 10 minutes at 4°C, then incubated for 30 minutes at 4°C with CD3 FITC, CD4 PerCP Cy5.5, CD25 PE-Cy7, CD62LeFluor450, FOXP3 PE, KLRG1APC, CD127 AF700, CD11B PerCp Cy5.5, CD11cAPC, GR1 PE, F4/80 PE-Cy7 from eBioscience, and CD8 BV605 and CD44 BV510 from Biolegend. For intracellular staining, cells were fixed and permeabilized with FOXp3 Staining Buffer Kit (eBioscience) according to manufacturer’s instructions. Cells were incubated for 30 minutes at room temperature in dark with Foxp3-PE and analyzed using the CytoFLex (BD Biosciences) and Kaluza software (Beckman Coulter).

Isolation of tumor-infiltrating T cells

Tumors were harvested 2 days after last treatment and weighed. Cells were isolated by dissecting tumor tissue into small fragments followed by digesting in 1 mg/mL of collagenase in complete RPMI media (10% FBS, 1% penicillin, streptomycin) prior to using Gentle MACS Dissociator. Cell suspension was passed through a 70 μm nylon strainer to obtain a single-cell population. Cells were washed twice with FACS buffer and stained as described above and analyzed by flow cytometry.

Cytokine analysis in the serum

Plasma levels of IFNγ, GM-CSF, TNFα, MCP-1, ILlα, ILlβ, IL10, and IL6 were analyzed by Multianalyte Flow Assay Kit (BioLegend) according to the manufacturer’s instructions. Mean fluorescent intensity (MFI) was plotted for each analyte in the serum sample. Data collection and analysis was done using CytoFlex (BD Biosciences).

Tumor homing

³²P-labeled VEGF aptamer was generated by T4 kinase end labeling using γP³² ATP and purified using G25 Microspin Columns (GE). A total of 2 × 10⁵ cpm of material was injected via tail vein into mice bearing both an irradiated and nonirradiated subcutaneous 4T07 tumor that were approximately 150 mm³. Forty-eight hours postinjection, mice were sacrificed, tumors were excised, placed in 500 μL of scintillation fluid, and counted with a Scintillation counter.

1HC of VEGF in tumor tissue

4T1, 4T07, and CT26 subcutaneously established tumors were resected and embedded in paraffin. Nonspecific immunoreactivity in slide-mounted tissue sections was blocked with Serum-Blocking Reagent D (R&D Systems), and incubated with goat polyclonal anti-mouse VEGF at 1:20 (R&D Systems) at 4°C overnight, washed with PBS, and incubated with biotinylated anti-goat secondary antibody (R&D Systems) for 40 minutes, followed by high-sensitivity streptavidin (HSS)-horseradish peroxidase (HRP; R&D Systems) for 30 minutes. Slides were washed with PBS and then incubated with an HRP-reactive DAB chromogen (R&D Systems) for 7 minutes. Slides were rinsed in H₂O, incubated in hematoxylin (Vector) at 1:5 for 3 minutes and incubated with 0.1% sodium bicarbonate as a bluing agent, at 1:5 for 3 minutes, followed by rinses with H₂O. Slides were dehydrated in increasing concentrations of alcohol (70%, 90%, 100%; 3 minutes each), briefly washed in xylene, and cover-slipped using mounting medium (Richard-Allan Scientific). Negative control slides for VEGF IHC were prepared by completing the above protocol in the absence of primary antibody.

Hematoxylin and eosin staining

Lungs, liver, and small intestines were fixed in 10% formalin overnight at room temperature and embedded in paraffin. Slides were stained with eosin (Across organics) for 5 minutes, followed by 10 minutes of hematoxylin staining (Vector), incubated with 0.1% sodium bicarbonate as a bluing agent, rinsed in H₂O, and then dehydrated in increasing concentrations of alcohol, as described earlier.

Intracellular granzyme B staining

CD8⁺ T cells were purified from splenocytes of treated animals using a CD8 Enrichment Kit (Miltenyi Biotec). Cells were incubated overnight in the presence of murine CD3 antibody. Eighteen hours later, cells were treated with Fix/Perm (Becton Dickinson) and stained with an antimurine granzyme B antibody (Becton Dickinson).

Radiation sensitizes VEGF^low-expressing tumors to VEGF-targeted 4–1BB costimulation

We have previously shown that VEGF-targeted 4–1BB costimulation using a systemically administered VEGF-4–1BB aptamer conjugate enhanced tumor immunity in multiple tumor models including the VEGF-expressing 4T1 breast carcinoma tumors. VEGF-targeted 4–1BB costimulation was, however, less effective against 4T07 tumors, a nonmetastatic sibling of 4T1 tumors that expresses low levels of VEGF (21). Given that radiation upregulates intratumoral VEGF expression, we tested the hypothesis that local tumor radiation will enhance the susceptibility of VEGF^low 4T07 tumors to VEGF-targeted 4–1BB costimulation. Using immunohistochemical analysis,Fig. 1A shows that radiation of subcutaneously implanted 4T07 tumors upregulates VEGF expression to levels comparable with that of 4T1 tumors. There was no detectable increase in VEGF levels in the circulation of 4T1 tumor-bearing mice that were or were not irradiated (data not shown). Using mice bearing two subcutaneous 4T07 tumors implanted at opposite flanks,Fig. 1B shows that intravenously administered VEGF, but not control, aptamer accumulate in the irradiated, but not in the nonirradiated, tumor lesion. The VEGF-4–1BB aptamer conjugate accumulated preferentially in irradiated or nonirradiated 4T1 tumors compared with normal organs (Supplementary Fig. S1).

To determine whether radiation-induced tumor accumulation of VEGF-4–1BB aptamer conjugate leads to control of tumor growth mice were treated systemically with the VEGF-4–1BB conjugates by tail vein injection. As shown inFig. 1C, mono-therapy with either radiation or VEGF-4–1BB conjugate had a modest effect on the rate of tumor growth but no appreciable impact on survival, whereas a combination of radiation and VEGF-4–1BB conjugate significantly inhibited tumor growth leading to the long-term survival of 50% of the treated mice. Consistent with the targeted nature of the action of VEGF-4–1BB aptamer conjugates, coadministration of unconjugated VEGF and 4–1BB aptamers did not potentiate the radiation effects on tumor growth, showing that the synergy between radiation and VEGF-4–1BB conjugate was not a result of the separate contributions of VEGF and/or 4–1BB aptamers.

We next compared the antitumor activity of the VEGF-targeted 4–1BB aptamer conjugate to that of an agonistic 4–1BB antibody when used in combination with irradiation. As shown inFig. 1D, both antibody and aptamer conjugate potentiated the ability of radiation to inhibit tumor growth, leading to the long-term survival of 50% to 60% of the treated mice in each group. On a molar basis, however, 8-fold more antibody was required to achieve a comparable antitumor effect. VEGF-targeted 4–1BB costimulation was also able to potentiate radiation-induced tumor immunity in the autochthonous MCA-induced fibrosarcoma model. As shown inSupplementary Fig. S2, combined radiation and VEGF-4–1BB conjugate treatment significantly enhanced the overall survival of the treated group, with 2 out of 6 mice exhibiting complete tumor regression, whereas either radiation orVEGF-4-1BB conjugate administration alone delayed tumor growth and enhanced survival though all mice eventually succumbed to the tumor and were sacrificed.

Therapeutic dose of 4–1BB antibody, but not VEGF-4–1BB aptamer conjugate, elicits nonspecific immune activation and inflammation

Therapeutic doses of 4–1BB antibody (18,19), but not VEGF-4–1BB conjugates elicited immune pathologies in mice (21), that were seen also in patients (20). Because combination therapies that lead to enhanced antitumor immune responses could exacerbate such autoimmune sequelae, we evaluated whether cotherapy of radiation and 4–1BB costimulation will induce and/or exacerbate immune pathology. In murine tumor studies, 4–1BB antibodies were shown to synergize with radiation but toxicities were not evaluated (39–41). In this study, the livers and spleens of mice treated with a therapeutic dose of the agonistic 4–1BB antibody (8×) shown inFig. 1D were enlarged (Fig. 2A andB), the proportion of CD8⁺ T cells in both organs was elevated (Fig. 2C andD), IFNγ levels were increased in the serum (Fig. 2E), and the splenic CD8⁺ T cells exhibited an activated-effector phenotype expressing granzyme B (Fig. 2F). In contrast, mice treated with a therapeutic dose ofVEGF-4-1BB aptamer conjugate (1×) did not exhibit changes in organ weight, CD8⁺ T-cell percentage, or activation status. It is noteworthy that even 8-fold less, subtherapeutic doses of antibody (1×) elicited measurable immune anomalies, increased liver and spleen weight (Fig. 2A andB), increased proportion of CD8⁺ T cells in the spleen (Fig. 2C), and a slight increase in granzyme B-expressing cells (Fig. 2F). Consistent with this, treatment with radiation and 4–1BB antibody, but not VEGF-4–1BB aptamer conjugate, led to significant increases in TNF, MCP-1, and IFNγ in the circulation, whereas increases in IL1α, IL1β, IL10, and IL6 exhibited a trend that did not reach statistical significance (Supplementary Fig. S3). Treatment of the tumor irradiated mice with the therapeutic dose of 4–1BB antibody (8×), but not VEGF-4–1BB (1×), was accompanied by a massive infiltration of leukocytes in the lung (Fig. 2G) and elicited characteristic inflammatory foci, local accumulation of leukocytes, in the livers of the treated mice (Fig. 2H). Hepatic toxicity was the major immune-related toxicity in patients treated with a 4–1BB antibody (20). Inflammatory foci could be also detected in mice treated with 8-fold lower subtherapeutic doses of antibody (arrows,Fig. 2H). Acute phase proteins alanine transaminase (ALT) and aspartate transaminase (AST) levels in the circulation, a measure of liver dysfunction, were elevated in tumor-bearing mice treated with radiation and 4–1BB antibody compared with that of mice treated with radiation and VEGF-4–1BB aptamer conjugates (Fig. 2I). Note that radiation and/or tumor alone increased AST and ALT levels, whereas cotreatment with VEGF-4–1BB aptamer conjugate prevented or reduced the increase, probably reflecting the reduced tumor volume in the aptamer conjugate-treated mice. In contrast, although cotreatment with 4–1BB antibody also led to a comparable decrease in tumor volume (Fig. 1D), the AST and ALT levels remained high. Overall, these experiments suggest that in combination with radiation, VEGF-targeted 4–1BB costimulation exhibit a more than 8-fold higher therapeutic index then the 4–1BB antibodies.

VEGF-targeted costimulation enhances radiation-induced abscopal effect

To determine whether VEGF-targeted 4–1BB costimulation with aptamer conjugates can potentiate the abscopal effect of radiation, 4T1 tumor cells were implanted subcutaneously in the right flank of a mouse and 8 days later 4T1 tumor cells were implanted also in the left flank. When tumors in the right flank reached about 75–100 mm³, they were irradiated and mice were treated systemically with either VEGF-4–1BB aptamer conjugates, CTLA-4 antibody, PD-1 antibody, or a combination of PD-1 and CTLA-4 antibodies. CTLA-4 and PD-1 antibodies were previously shown to induce abscopal effects in mice that underwent local tumor radiation (8,10). As shown inFig. 3A (tumor size at day 19 post irradiation, representative of other time points) andSupplementary Fig. S4A andS4B, time course of tumor growth), treatment of tumors by local radiation or systemic administration of antibodies or aptamer conjugate inhibited the growth of the irradiated tumor to a comparable extent, and combined radiation with either treatment was significantly enhanced.

The effects of the treatment combinations on the nonirradiated tumor are shown inFig. 3B andSupplementary Fig. S4C andS4D. Radiation alone had no effect, underscoring the challenging nature of inducing an abscopal effect, whereas treatment with antibodies or aptamer conjugate without irradiation reduced tumor growth to a comparable extent. When combined with radiation, CTLA-4 antibody and VEGF-4–1BB aptamer conjugate treatment significantly enhanced inhibition of tumor growth, whereas PD-1 antibody did not. Somewhat unexpectedly, whereas combination of CTLA-4 + PD-1 antibodies enhanced radiation effect on the irradiated tumor, as was previously reported except for using PD-L1 antibody and a different mouse tumor model (42), the abscopal effect was not enhanced. The reason for that is currently under investigation. Measuring prolongation of survival (Fig. 3C), which accounts for the combined contribution of both irradiated and nonirradiated tumor, radiation combined with PD-1 + CTLA-4 antibodies or with VEGF-4–1BB aptamer conjugates were most effective. Radiation with CTLA-4 antibody, or PD-1+ CTLA-4 antibody without irradiation were also effective but less so, while the remaining treatment groups showed a trend toward extended survival that did not reach statistical significance.

To assess the abscopal effect on lung metastasis, 4T1 tumor cells were administered intravenously at day 5 prior to radiation of the subcutaneously implanted tumors. Experiment was terminated when tumor-bearing mice in the untreated group had to be sacrificed. Lung metastasis was determined measuring lung weight. As shown inFig. 4C, radiation of the subcutaneous tumors followed by systemic therapy with the VEGF-4–1BB conjugate significantly inhibited lung metastasis, whereas radiation alone or radiation coadministered with unconjugated VEGF and 4–1BB aptamers had a modest effect. Note that radiation alone had a less pronounced effect on the nonirradiated lung métastasés (Fig 4C) compared with that of the irradiated tumor (Fig. 4A andB), underscoring the challenging nature eliciting an effective abscopal effect.

Combination of radiation and CTLA-4 antibody, but not VEGF-4–1BB aptamer conjugate, elicits organ-wide inflammation

In murine studies, treatment with CTLA-4 antibodies was shown to enhance radiation-induced antitumor immunity but toxicities were not evaluated (7,10,42). In human patients, treatment with CTLA-4 antibody (Ipilumimab) was accompanied by immune-related adverse effects (irAE) that are generally mild and reversible but occasionally can be severe and life threatening (13,14). Enterocolitis was the most frequent toxicity defined by grade 3/4 clinical presentation and/or biopsy documentation presents with diarrhea, pain, and sometimes bowel perforation requiring surgical intervention and can be lethal (43).

To determine whether CTLA-4 antibody or VEGF-4–1BB aptamer conjugate treatment in combination with radiation elicit irAEs in mice, we analyzed the small intestine, liver, and lungs of mice for inflammatory infiltrates. Recapitulating the scenario in human patients, immunohistochemical analysis of small intestine of mice treated with CTLA-4 antibody alone or in combination with radiation and/or PD-1 antibody exhibited a significant increase in leukocytic infiltrates and deformation of the crypt architecture (Fig. 5;Supplementary Fig. S5). Strikingly, radiation and/or VEGF-4–1BB aptamer conjugate showed no sign of increased leukocytic infiltrates or tissue damage. Similar pattern of inflammatory infiltrates was seen in the livers and lungs (Supplementary Figs. S6 andS7). Radiation in combination with CTLA-4 + PD-1 antibodies, and to a lesser combination with CTLA-4 antibody alone, but not with VEGF-4–1BB aptamer conjugate, elicited significant increases of MCP-1, IL6, and GM-CSF in the circulation, whereas radiation in combination with VEGF-4–1BB aptamer conjugates did not (Supplementary Fig. S8). VEGF-targeted 4–1BB costimulation, therefore, exhibited a significantly increased therapeutic index compared with CTLA-4 antibody or CTLA-4 and PD-1 antibody combinations, with or without radiation.

Treatment with VEGF-4–1BB aptamer conjugates enhances the infiltration of immune cells into irradiated tumors

To investigate the immunologic basis for the antitumor activity of radiation and VEGF-4–1BB aptamer conjugates, we evaluated the nature of tumor-infiltrating lymphocytes (TIL). As shown inFig. 6, radiation promotes the infiltrations of a diverse subsets of immune cells, including CD4⁺ and CD8⁺T cells, Ml-polarized macrophages, and B cells that was significantly enhanced by cotreatment with VEGF-4–1BB aptamer conjugates, whereas treatment with VEGF-4–1BB aptamer conjugate alone had a minimal effect at best. Conversely, radiation + VEGF-4–1BB aptamer conjugate treatment led to a reduction in tumor-infiltrating Treg and increase in the ratio of CD8⁺ T cells/foxp3+ Treg that has been associated with tumor immunogenicity (44). The immune-potentiating effects of cotreatment with VEGF-4–1BB aptamer conjugate was also indicated by a significant increase in overall cell death at the tumor site over that of radiation alone (Fig. 6). These findings are consistent with the immune-promoting properties of local tumor irradiation (2–4) and the enhanced antitumor activity of combination with VEGF-targeted 4–1BB costimulation (Figs. 1,3,4;Supplementary Figs. S3 andS4).

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