本申请要求申请日为2024/2/8的中国专利申请2024101775792的优先权。本申请引用上述中国专利申请的全文。This application claims priority to Chinese patent application No. 2024101775792, filed on February 8, 2024. This application incorporates the entirety of the aforementioned Chinese patent application.
本发明属于生物医药领域，具体涉及一种抗ILT2/4单抗的制剂、包含其的药盒及其应用。The present invention belongs to the field of biomedicine, and specifically relates to an anti-ILT2/4 monoclonal antibody preparation, a drug kit containing the same, and applications thereof.
免疫系统是恶性肿瘤发生发展的重要屏障，以免疫检查点阻滞(ICB)为代表的肿瘤免疫治疗近年在肿瘤治疗领域取得举世瞩目成就。但肿瘤免疫疗法在临床应用中依旧面临较大的挑战，如响应率偏低、原发性耐药、继发性耐药等问题，新的免疫检查点分子及联合治疗策略的开发将成为重塑T细胞的抗肿瘤活性、克服临床免疫耐药的关键。The immune system is a critical barrier to the development and progression of malignant tumors. Tumor immunotherapy, exemplified by immune checkpoint blockade (ICB), has achieved remarkable success in recent years. However, clinical application of tumor immunotherapy still faces significant challenges, such as low response rates, primary and secondary drug resistance. The development of new immune checkpoint molecules and combination therapy strategies will be key to remodeling T cell anti-tumor activity and overcoming clinical immune resistance.
ILT2全称是Immunoglobulin-like transcript 2，免疫球蛋白样转录因子2，也被称为淋巴细胞免疫球蛋白样受体B1(LILRB1)。ILT4全称是Immunoglobulin-like transcript 4，免疫球蛋白样转录因子4，也被称为淋巴细胞免疫球蛋白样受体B2(LILRB2)。它们是免疫球蛋白样转录子(ILTs)家族中的两种抑制性受体。二者有着相似的结构。其胞外段均含有4个Ig-like domain，胞内分别含有4个和3个免疫受体酪氨酸抑制基序(ITIM)结构域。它们通过这些ITIM结构域向胞内传递抑制信号，进而调控免疫细胞的功能。正是结构上的相似使得使用同一种抗体同时抑制ILT2和ILT4的功能成为可能。ILT2 stands for Immunoglobulin-like transcript 2, immunoglobulin-like transcription factor 2, also known as lymphocyte immunoglobulin-like receptor B1 (LILRB1). ILT4 stands for Immunoglobulin-like transcript 4, immunoglobulin-like transcription factor 4, also known as lymphocyte immunoglobulin-like receptor B2 (LILRB2). They are two inhibitory receptors in the immunoglobulin-like transcript (ILTs) family. The two have similar structures. Their extracellular segments contain 4 Ig-like domains and 4 and 3 immunoreceptor tyrosine-based inhibitory motif (ITIM) domains, respectively. They transmit inhibitory signals to the cell through these ITIM domains, thereby regulating the function of immune cells. It is precisely the structural similarity that makes it possible to use the same antibody to simultaneously inhibit the functions of ILT2 and ILT4.
ILT2和ILT4共同的配体主要包括：I型HLA和HLA-G、其中与HLA-G的亲和力高于其他I型HLA成员。当ILT2/4与HLA结合后，细胞内的ITIM可以招募含SH-2domain的SHP-1或SHP-2磷酸酶，抑制上述细胞中活化信号的传递及免疫功能。ILT2 and ILT4 share common ligands including class I HLA and HLA-G, with HLA-G having a higher affinity than other class I HLA members. When ILT2/4 bind to HLA, the intracellular ITIM recruits SH-2 domain-containing SHP-1 or SHP-2 phosphatases, inhibiting activation signaling and immune function in these cells.
生理条件下，ILT4主要在单核细胞、巨噬细胞、树突状细胞和中性粒细胞等髓系细胞表面表达。ILT2除了在这些细胞表达外还表达于部分NK、T、B细胞。在肿瘤微环境中，ILT2或ILT4在肿瘤相关巨噬细胞(TAMs)、髓源性抑制细胞(MDSCs)以及DC细胞中高表达，推动肿瘤微环境向免疫抑制方向转化，从而促进肿瘤生长。Under physiological conditions, ILT4 is primarily expressed on the surface of myeloid cells, including monocytes, macrophages, dendritic cells, and neutrophils. In addition to being expressed on these cells, ILT2 is also expressed on some NK, T, and B cells. Within the tumor microenvironment, ILT2 or ILT4 is highly expressed in tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and DCs, driving the transformation of the tumor microenvironment toward an immunosuppressive state, thereby promoting tumor growth.
重组全人源抗ILT2/4 S228P IgG4κ单克隆抗体，命名为抗ILT2/4，能以类似的效力结合细胞表面的ILT2/ILT4分子，从而将肿瘤相关的髓系细胞重新编程为促炎状态。由此产生的促炎症反应可能更好地刺激抗肿瘤T细胞反应(特别是当与PD-1/PD-L1抗体联用时)，从而达到激活肿瘤免疫微环境进而治疗肿瘤的目的。The recombinant fully human anti-ILT2/4 S228P IgG4κ monoclonal antibody, designated anti-ILT2/4, binds to cell surface ILT2/ILT4 molecules with similar potency, thereby reprogramming tumor-associated myeloid cells into a pro-inflammatory state. The resulting pro-inflammatory response may better stimulate anti-tumor T cell responses (especially when combined with PD-1/PD-L1 antibodies), thereby activating the tumor immune microenvironment and ultimately treating tumors.
作为生物大分子，容易受到物理(如冻融、振荡、光照、温度等)或化学作用(如pH、有机溶剂、微生物污染等)因素的影响，而导致聚集、降解、纯度降低、活性降低等现象，导致该分子临床疗效降低，甚至可能会在临床使用时危害病人健康。As biological macromolecules, they are easily affected by physical (such as freeze-thaw, oscillation, light, temperature, etc.) or chemical (such as pH, organic solvents, microbial contamination, etc.) factors, which may lead to aggregation, degradation, reduced purity, reduced activity, etc., resulting in reduced clinical efficacy of the molecule and may even endanger patient health during clinical use.
为了解决抗ILT2/4单抗容易受到物理(如冻融、振荡、光照、温度等)或化学作用(如pH、有机溶剂、微生物污染等)因素的影响，而导致聚集、降解、纯度降低、活性降低的问题，本发明提供了一种抗ILT2/4单抗的制剂、包含其的药盒及其应用。本发明的发明人通过对抗ILT2/4的制剂处方进行了筛选和研究，最终获得了一种针对抗ILT2/4单抗最为适用的、且能稳定保存所述单抗的溶液制剂，该制剂能够充分抑制抗ILT2/4蛋白聚集、降解、沉淀等，从而保持其有效组分的生物学活性，便于长期储存及临床使用，大大降低由于药物质量变化导致的临床使用不良事件。In order to solve the problem that anti-ILT2/4 monoclonal antibodies are easily affected by physical (such as freeze-thaw, oscillation, light, temperature, etc.) or chemical factors (such as pH, organic solvents, microbial contamination, etc.), resulting in aggregation, degradation, reduced purity, and reduced activity, the present invention provides a preparation of anti-ILT2/4 monoclonal antibodies, a drug kit containing the same, and its use. The inventors of the present invention screened and studied the formulation of anti-ILT2/4 preparations and ultimately obtained a solution preparation that is most suitable for anti-ILT2/4 monoclonal antibodies and can stably preserve the monoclonal antibodies. The preparation can fully inhibit the aggregation, degradation, precipitation, etc. of anti-ILT2/4 proteins, thereby maintaining the biological activity of its effective components, facilitating long-term storage and clinical use, and greatly reducing adverse events in clinical use caused by changes in drug quality.
为了解决上述技术问题，本发明第一方面提供了一种抗ILT2/4单抗的制剂，所述抗ILT2/4单抗包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:29和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，其中，X1为N、A或T，X2为G、A或S，和氨基酸序列分别如SEQ ID NO:4～6所示的LCDR1、LCDR2和LCDR3。In order to solve the above technical problems, the first aspect of the present invention provides a preparation of an anti-ILT2/4 monoclonal antibody, wherein the anti-ILT2/4 monoclonal antibody comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 29 and SEQ ID NO: 3, respectively, whereinX1 is N, A or T, X2 is G, A or S, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 4 to 6, respectively.
在本发明的一些实施方案中，所述抗ILT2/4单抗包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:7和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4～6所示的LCDR1、LCDR2和LCDR3。In some embodiments of the present invention, the anti-ILT2/4 monoclonal antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4 to 6, respectively.
在本发明的一些实施方案中，所述抗ILT2/4单抗包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4～6所示的LCDR1、LCDR2和LCDR3。In some embodiments of the present invention, the anti-ILT2/4 monoclonal antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:4 to 6, respectively.
在本发明的一些实施方案中，所述抗ILT2/4单抗包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:8和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4～6所示的LCDR1、LCDR2和LCDR3。In some embodiments of the present invention, the anti-ILT2/4 monoclonal antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4 to 6, respectively.
在本发明的一些实施方案中，所述抗ILT2/4单抗包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:9和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4～6所示的LCDR1、LCDR2和LCDR3。In some embodiments of the present invention, the anti-ILT2/4 monoclonal antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4 to 6, respectively.
在本发明的一些实施方案中，所述抗ILT2/4单抗包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:10和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和氨基酸序列分别如SEQ ID NO:4～6所示的LCDR1、LCDR2和LCDR3。In some embodiments of the present invention, the anti-ILT2/4 monoclonal antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 10 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 4 to 6, respectively.
在本发明的一些实施方案中，所述抗ILT2/4单抗的VH包含如SEQ ID NO:30所示或与SEQ ID NO:30具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，其中，X为N、A或T，X2为G、A或S；所述抗ILT2/4单抗的VL包含如SEQ ID NO:12所示或与SEQ ID NO:12具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。In some embodiments of the present invention, the VH of the anti-ILT2/4 monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO:30 or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30, wherein X is N, A or T, and X2 is G, A or S; the VL of the anti-ILT2/4 monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO:12 or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12.
在本发明的一些实施方案中，所述抗ILT2/4单抗的VH包含如SEQ ID NO:13所示的氨基酸序列；所述抗ILT2/4单抗的VL包含如SEQ ID NO:12所示的氨基酸序列。In some embodiments of the present invention, the VH of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:13; the VL of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:12.
在本发明的一些实施方案中，所述抗ILT2/4单抗的VH包含如SEQ ID NO:11所示的氨基酸序列；所述抗ILT2/4单抗的VL包含如SEQ ID NO:12所示的氨基酸序列。In some embodiments of the present invention, the VH of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:11; the VL of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:12.
在本发明的一些实施方案中，所述抗ILT2/4单抗的VH包含如SEQ ID NO:14所示的氨基酸序列；所述抗ILT2/4单抗的VL包含如SEQ ID NO:12所示的氨基酸序列。In some embodiments of the present invention, the VH of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:14; the VL of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:12.
在本发明的一些实施方案中，所述抗ILT2/4单抗的VH包含如SEQ ID NO:15所示的氨基酸序列；所述抗ILT2/4单抗的VL包含如SEQ ID NO:12所示的氨基酸序列。In some embodiments of the present invention, the VH of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:15; the VL of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:12.
在本发明的一些实施方案中，所述抗ILT2/4单抗的VH包含如SEQ ID NO:16所示的氨基酸序列；所述抗ILT2/4单抗的VL包含如SEQ ID NO:12所示的氨基酸序列。In some embodiments of the present invention, the VH of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:16; the VL of the anti-ILT2/4 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:12.
在本发明的一些较佳实施方案中，所述抗ILT2/4单抗的HC包含如SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:20、SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列，或与SEQ ID NO:19、SEQ ID NO:17或SEQ ID NO:20具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗ILT2/4单抗的LC包含如SEQ ID NO:18所示或与SEQ ID NO:18具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。In some preferred embodiments of the present invention, the HC of the anti-ILT2/4 monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 19, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19, SEQ ID NO: 17 or SEQ ID NO: 20; the LC of the anti-ILT2/4 monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 18 or a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18.
在本发明的一些更佳实施方案中，所述制剂还包含缓冲液、稳定剂和/或表面活性剂。In some more preferred embodiments of the present invention, the formulation further comprises a buffer, a stabilizer and/or a surfactant.
在本发明的一些实施方案中，所述缓冲液选自醋酸-醋酸钠缓冲液和PB缓冲液(磷酸二氢钠-磷酸氢二钠缓冲液)。In some embodiments of the present invention, the buffer is selected from acetic acid-sodium acetate buffer and PB buffer (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer).
在本发明的一些优选实施方案中，所述缓冲液为醋酸-醋酸钠缓冲液。In some preferred embodiments of the present invention, the buffer is acetic acid-sodium acetate buffer.
在本发明的一些较佳实施方案中，所述缓冲液的浓度不低于10mM，例如为10mM、20mM、30mM、40mM或50mM。In some preferred embodiments of the present invention, the concentration of the buffer is not less than 10 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM.
在本发明的一些优选实施方案中，所述缓冲液的浓度为10mM。In some preferred embodiments of the present invention, the concentration of the buffer is 10 mM.
在本发明的一些实施方案中，所述制剂的pH范围为pH4～6。In some embodiments of the present invention, the pH range of the formulation is pH 4-6.
在本发明的一些优选实施方案中，所述制剂的pH范围为pH4.5～5.5。In some preferred embodiments of the present invention, the pH range of the preparation is pH 4.5 to 5.5.
在本发明的一些具体实施方案中，所述制剂的pH范围为pH4.0、4.5、5.0或5.5。In some embodiments of the present invention, the pH range of the formulation is pH 4.0, 4.5, 5.0 or 5.5.
在本发明的一些实施方案中，所述稳定剂为蔗糖和/或海藻糖。In some embodiments of the present invention, the stabilizer is sucrose and/or trehalose.
在本发明的一些优选实施方案中，所述稳定剂为海藻糖。In some preferred embodiments of the present invention, the stabilizer is trehalose.
在本发明的一些较佳实施方案中，所述稳定剂的浓度范围为1％～20％。In some preferred embodiments of the present invention, the concentration of the stabilizer ranges from 1% to 20%.
在本发明的一些优选实施方案中，所述稳定剂的浓度范围为7％～9％。In some preferred embodiments of the present invention, the concentration of the stabilizer ranges from 7% to 9%.
在本发明的一些具体实施方案中，所述稳定剂的浓度为7％、8％或9％。In some embodiments of the present invention, the concentration of the stabilizer is 7%, 8% or 9%.
在本发明的一些实施方案中，所述表面活性剂选自聚山梨酯20、聚山梨酯80和泊洛沙姆188中的一种或多种。In some embodiments of the present invention, the surfactant is selected from one or more of polysorbate 20, polysorbate 80 and poloxamer 188.
在本发明的一些优选实施方案中，所述表面活性剂为聚山梨酯20。In some preferred embodiments of the present invention, the surfactant is polysorbate 20.
在本发明的一些较佳实施方案中，所述表面活性剂的浓度范围为0.001％～0.1％。In some preferred embodiments of the present invention, the concentration of the surfactant is in the range of 0.001% to 0.1%.
在本发明的一些优选实施方案中，所述表面活性剂的浓度范围为0.01～0.1％。In some preferred embodiments of the present invention, the concentration of the surfactant is in the range of 0.01 to 0.1%.
在本发明的一些更优选实施方案中，所述表面活性剂的浓度范围为0.02％～0.06％。In some more preferred embodiments of the present invention, the concentration of the surfactant is in the range of 0.02% to 0.06%.
在本发明的一些具体实施方案中，所述表面活性剂的浓度为0.02％、0.04％或0.06％。In some specific embodiments of the present invention, the concentration of the surfactant is 0.02%, 0.04% or 0.06%.
在本发明的一些实施方案中，所述抗ILT2/4单抗的浓度为1～30mg/mL。In some embodiments of the present invention, the concentration of the anti-ILT2/4 monoclonal antibody is 1 to 30 mg/mL.
在本发明的一些优选实施方案中，所述抗ILT2/4单抗的浓度为10～30mg/mL。In some preferred embodiments of the present invention, the concentration of the anti-ILT2/4 monoclonal antibody is 10-30 mg/mL.
在本发明的一些更优选实施方案中，所述抗ILT2/4单抗的浓度为15～25mg/mL。In some more preferred embodiments of the present invention, the concentration of the anti-ILT2/4 monoclonal antibody is 15-25 mg/mL.
在本发明的一些具体实施方案中，所述抗ILT2/4单抗的浓度为15mg/mL、20mg/mL或25mg/mL。In some specific embodiments of the present invention, the concentration of the anti-ILT2/4 monoclonal antibody is 15 mg/mL, 20 mg/mL or 25 mg/mL.
在本发明的一些实施方案中，所述制剂为液体制剂。In some embodiments of the present invention, the formulation is a liquid formulation.
在本发明的一些具体实施方案中，所述抗ILT2/4单抗的制剂的pH为5.0，所述抗ILT2/4单抗的浓度为20mg/mL，聚山梨酯20含量为0.04％，海藻糖含量为8％；或In some specific embodiments of the present invention, the pH of the anti-ILT2/4 monoclonal antibody formulation is 5.0, the concentration of the anti-ILT2/4 monoclonal antibody is 20 mg/mL, the polysorbate 20 content is 0.04%, and the trehalose content is 8%; or
pH为4.5，所述抗ILT2/4单抗的浓度为15mg/mL，聚山梨酯20含量为0.02％，海藻糖含量为7％；或，The pH is 4.5, the concentration of the anti-ILT2/4 monoclonal antibody is 15 mg/mL, the polysorbate 20 content is 0.02%, and the trehalose content is 7%; or,
pH为5.5，所述抗ILT2/4单抗的浓度为15mg/mL，聚山梨酯20含量为0.06％，海藻糖含量为7％；或，The pH is 5.5, the concentration of the anti-ILT2/4 monoclonal antibody is 15 mg/mL, the polysorbate 20 content is 0.06%, and the trehalose content is 7%; or,
pH为5.5，所述抗ILT2/4单抗的浓度为25mg/mL，聚山梨酯20含量为0.02％，海藻糖含量为7％；或，The pH is 5.5, the concentration of the anti-ILT2/4 monoclonal antibody is 25 mg/mL, the polysorbate 20 content is 0.02%, and the trehalose content is 7%; or,
pH为4.5，所述抗ILT2/4单抗的浓度为15mg/mL，聚山梨酯20含量为0.06％，海藻糖含量为9％；或，The pH is 4.5, the concentration of the anti-ILT2/4 monoclonal antibody is 15 mg/mL, the polysorbate 20 content is 0.06%, and the trehalose content is 9%; or,
pH为5.5，所述抗ILT2/4单抗的浓度为25mg/mL，聚山梨酯20含量为0.06％，海藻糖含量为9％；或，The pH is 5.5, the concentration of the anti-ILT2/4 monoclonal antibody is 25 mg/mL, the polysorbate 20 content is 0.06%, and the trehalose content is 9%; or,
pH为4.5，所述抗ILT2/4单抗的浓度为25mg/mL，聚山梨酯20含量为0.02％，海藻糖含量为9％；或，The pH is 4.5, the concentration of the anti-ILT2/4 monoclonal antibody is 25 mg/mL, the polysorbate 20 content is 0.02%, and the trehalose content is 9%; or,
pH为5.5，所述抗ILT2/4单抗的浓度为15mg/mL，聚山梨酯20含量为0.02％，海藻糖含量为9％；或，The pH is 5.5, the concentration of the anti-ILT2/4 monoclonal antibody is 15 mg/mL, the polysorbate 20 content is 0.02%, and the trehalose content is 9%; or,
pH为4.5，所述抗ILT2/4单抗的浓度为25mg/mL，聚山梨酯20含量为0.06％，海藻糖含量为7％。其中％为质量体积比。The pH is 4.5, the concentration of the anti-ILT2/4 monoclonal antibody is 25 mg/mL, the polysorbate 20 content is 0.06%, and the trehalose content is 7%, where % represents the mass-to-volume ratio.
为了解决上述技术问题，本发明第二方面提供了一种包含如发明第一方面所述的制剂的药盒，其特征在于，所述药盒还包括容器。In order to solve the above technical problems, the second aspect of the present invention provides a medicine box containing the preparation as described in the first aspect of the invention, characterized in that the medicine box also includes a container.
在本发明的一些较佳实施方案中，所述容器为聚碳酸酯材质瓶、聚丙烯瓶(PP瓶)、超低密度聚乙烯袋子(ULDPE袋子)或乙烯-醋酸乙烯酯共聚物材质袋子(EVA袋子)。In some preferred embodiments of the present invention, the container is a polycarbonate bottle, a polypropylene bottle (PP bottle), an ultra-low density polyethylene bag (ULDPE bag) or an ethylene-vinyl acetate copolymer bag (EVA bag).
为了解决上述技术问题，本发明第三方面提供了如发明第一方面所述的制剂在制备诊断、治疗和/或预防ILT4和/或ILT2介导的疾病或病症的产品中的应用。In order to solve the above technical problems, the third aspect of the present invention provides the use of the preparation according to the first aspect of the invention in the preparation of products for diagnosing, treating and/or preventing diseases or conditions mediated by ILT4 and/or ILT2.
在本发明的一些较佳实施方案中，所述的疾病或病症为癌症。In some preferred embodiments of the present invention, the disease or condition is cancer.
在本发明的一些更佳实施方案中，所述癌症为ILT4阳性和/或ILT2阳性癌症。In some more preferred embodiments of the present invention, the cancer is ILT4-positive and/or ILT2-positive cancer.
在本发明的一些进一步更佳实施方案中，所述癌症为实体癌或血液癌；In some further preferred embodiments of the present invention, the cancer is a solid cancer or a blood cancer;
所述血液癌为例如B细胞白血病、淋巴瘤、急性骨髓性白血病、伯基特淋巴瘤、套细胞淋巴瘤、急性淋巴母细胞性白血病、慢性淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤或者边缘区淋巴瘤；The blood cancer is, for example, B-cell leukemia, lymphoma, acute myeloid leukemia, Burkitt's lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or marginal zone lymphoma;
所述实体癌为例如头颈部鳞状细胞癌、肾细胞癌、卵巢癌、尿路上皮癌、弥漫性大B细胞癌、非小细胞肺癌、黑色素瘤、转移性肾细胞癌、转移性结直肠癌、小细胞肺癌、转移性非小细胞肺癌、乳腺癌、肺癌、头颈部癌、结直肠癌、前列腺癌、皮肤癌、胃癌、肠癌、宫颈癌、子宫癌、子宫内膜癌、膀胱癌、脑癌、食道癌、肝癌、肾癌、睾丸癌、间皮瘤、恶性胶质瘤、胆管癌或胰腺癌。The solid cancer is, for example, head and neck squamous cell carcinoma, renal cell carcinoma, ovarian cancer, urothelial carcinoma, diffuse large B-cell carcinoma, non-small cell lung cancer, melanoma, metastatic renal cell carcinoma, metastatic colorectal cancer, small cell lung cancer, metastatic non-small cell lung cancer, breast cancer, lung cancer, head and neck cancer, colorectal cancer, prostate cancer, skin cancer, stomach cancer, intestinal cancer, cervical cancer, uterine cancer, endometrial cancer, bladder cancer, brain cancer, esophageal cancer, liver cancer, kidney cancer, testicular cancer, mesothelioma, glioblastoma, bile duct cancer, or pancreatic cancer.
为了解决上述技术问题，本发明第四方面提供了一种免疫检测或者测定ILT4和/或ILT2的方法，所述方法包括将如本发明第一方面所述的制剂与样品接触，通过结合情况判断样品中是否存在ILT4和/或ILT2。In order to solve the above technical problems, the fourth aspect of the present invention provides a method for immunodetection or determination of ILT4 and/or ILT2, which comprises contacting the preparation described in the first aspect of the present invention with a sample, and determining whether ILT4 and/or ILT2 are present in the sample based on the combined situation.
在本发明的一些优选实施方案中，所述检测为非诊断目的，所述非诊断目的的适用场景可为实验室研发或环境中免疫检测或测定等。In some preferred embodiments of the present invention, the detection is for non-diagnostic purposes, and the applicable scenarios of the non-diagnostic purposes can be laboratory research and development or immune detection or determination in the environment, etc.
为了解决上述技术问题，本发明第五方面提供了一种治疗和/或预防ILT4和/或ILT2介导的疾病或病症的方法，所述方法包括向有需要的患者施用治疗有效量的如发明第一方面所述的制剂。In order to solve the above technical problems, the fifth aspect of the present invention provides a method for treating and/or preventing diseases or conditions mediated by ILT4 and/or ILT2, which comprises administering a therapeutically effective amount of the preparation as described in the first aspect of the invention to a patient in need.
在本发明的一些较佳实施方案中，所述的疾病或病症为癌症。In some preferred embodiments of the present invention, the disease or condition is cancer.
在本发明的一些更佳实施方案中，所述癌症为ILT4阳性和/或ILT2阳性癌症。In some more preferred embodiments of the present invention, the cancer is ILT4-positive and/or ILT2-positive cancer.
在本发明的一些进一步更佳实施方案中，所述癌症为实体癌或血液癌；In some further preferred embodiments of the present invention, the cancer is a solid cancer or a blood cancer;
所述血液癌为例如B细胞白血病、淋巴瘤、急性骨髓性白血病、伯基特淋巴瘤、套细胞淋巴瘤、急性淋巴母细胞性白血病、慢性淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤或者边缘区淋巴瘤；The blood cancer is, for example, B-cell leukemia, lymphoma, acute myeloid leukemia, Burkitt's lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or marginal zone lymphoma;
所述实体癌为例如头颈部鳞状细胞癌、肾细胞癌、卵巢癌、尿路上皮癌、弥漫性大B细胞癌、非小细胞肺癌、黑色素瘤、转移性肾细胞癌、转移性结直肠癌、小细胞肺癌、转移性非小细胞肺癌、乳腺癌、肺癌、头颈部癌、结直肠癌、前列腺癌、皮肤癌、胃癌、肠癌、宫颈癌、子宫癌、子宫内膜癌、膀胱癌、脑癌、食道癌、肝癌、肾癌、睾丸癌、间皮瘤、恶性胶质瘤、胆管癌或胰腺癌。The solid cancer is, for example, head and neck squamous cell carcinoma, renal cell carcinoma, ovarian cancer, urothelial carcinoma, diffuse large B-cell carcinoma, non-small cell lung cancer, melanoma, metastatic renal cell carcinoma, metastatic colorectal cancer, small cell lung cancer, metastatic non-small cell lung cancer, breast cancer, lung cancer, head and neck cancer, colorectal cancer, prostate cancer, skin cancer, stomach cancer, intestinal cancer, cervical cancer, uterine cancer, endometrial cancer, bladder cancer, brain cancer, esophageal cancer, liver cancer, kidney cancer, testicular cancer, mesothelioma, glioblastoma, bile duct cancer, or pancreatic cancer.
为了解决上述技术问题，本发明第六方面提供了一种联合疗法，其包括分别向有需要的患者施用如发明第一方面所述的制剂；和第二治疗剂；所述第二治疗剂较佳地包含其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。In order to solve the above technical problems, the sixth aspect of the present invention provides a combination therapy, which comprises administering the preparation as described in the first aspect of the invention to patients in need; and a second therapeutic agent; the second therapeutic agent preferably comprises other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies, and/or one or more of a group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.
为了解决上述技术问题，本发明第七方面提供了如本发明第一方面所述的制剂，其用于治疗和/或预防ILT4和/或ILT2介导的疾病或病症。In order to solve the above technical problems, the seventh aspect of the present invention provides a preparation as described in the first aspect of the present invention, which is used to treat and/or prevent diseases or conditions mediated by ILT4 and/or ILT2.
在本发明的一些较佳实施方案中，所述的疾病或病症为癌症。In some preferred embodiments of the present invention, the disease or condition is cancer.
在本发明的一些更佳实施方案中，所述癌症为ILT4阳性和/或ILT2阳性癌症。In some more preferred embodiments of the present invention, the cancer is ILT4-positive and/or ILT2-positive cancer.
在本发明的一些进一步更佳实施方案中，所述癌症为为实体癌或血液癌；In some further preferred embodiments of the present invention, the cancer is a solid cancer or a blood cancer;
所述血液癌为例如B细胞白血病、淋巴瘤、急性骨髓性白血病、伯基特淋巴瘤、套细胞淋巴瘤、急性淋巴母细胞性白血病、慢性淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤或者边缘区淋巴瘤；The blood cancer is, for example, B-cell leukemia, lymphoma, acute myeloid leukemia, Burkitt's lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, or marginal zone lymphoma;
所述实体癌为例如头颈部鳞状细胞癌、肾细胞癌、卵巢癌、尿路上皮癌、弥漫性大B细胞癌、非小细胞肺癌、黑色素瘤、转移性肾细胞癌、转移性结直肠癌、小细胞肺癌、转移性非小细胞肺癌、乳腺癌、肺癌、头颈部癌、结直肠癌、前列腺癌、皮肤癌、胃癌、肠癌、宫颈癌、子宫癌、子宫内膜癌、膀胱癌、脑癌、食道癌、肝癌、肾癌、睾丸癌、间皮瘤、恶性胶质瘤、胆管癌或胰腺癌。The solid cancer is, for example, head and neck squamous cell carcinoma, renal cell carcinoma, ovarian cancer, urothelial carcinoma, diffuse large B-cell carcinoma, non-small cell lung cancer, melanoma, metastatic renal cell carcinoma, metastatic colorectal cancer, small cell lung cancer, metastatic non-small cell lung cancer, breast cancer, lung cancer, head and neck cancer, colorectal cancer, prostate cancer, skin cancer, stomach cancer, intestinal cancer, cervical cancer, uterine cancer, endometrial cancer, bladder cancer, brain cancer, esophageal cancer, liver cancer, kidney cancer, testicular cancer, mesothelioma, glioblastoma, bile duct cancer, or pancreatic cancer.
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are commercially available.
本发明的积极进步效果在于：本发明提供的制剂能够提高抗ILT2/4抗体的储存稳定性，充分抑制抗ILT2/4蛋白聚集、降解、沉淀等，能够在长期储存期间各质量指标不发生较大变化，从而保持其有效组分的生物学活性，便于临床使用，大大降低由于药物质量变化导致的临床使用不良事件。The positive progress of the present invention is that the preparation provided by the present invention can improve the storage stability of anti-ILT2/4 antibodies, fully inhibit the aggregation, degradation, precipitation, etc. of anti-ILT2/4 proteins, and can maintain the biological activity of its effective components during long-term storage without significant changes in various quality indicators, thereby facilitating clinical use and greatly reducing adverse events caused by changes in drug quality during clinical use.
图1为检测ILT4抗体与人/猴ILT4-ECD蛋白的结合能力。FIG1 is a graph showing the binding ability of ILT4 antibodies to human/monkey ILT4-ECD proteins.
图2为基于ELISA测定法检测ILT-4抗体与ILT2-ECD蛋白的结合能力。FIG2 is an ELISA assay to detect the binding ability of ILT-4 antibodies to ILT2-ECD protein.
图3为基于ELISA检测ILT4抗体对巨噬细胞的激活作用。FIG3 shows the activation effect of ILT4 antibodies on macrophages detected by ELISA.
图4为基于ELISA检测ILT4抗体对外周血单核细胞的激活作用。FIG4 shows the activation effect of ILT4 antibodies on peripheral blood mononuclear cells detected by ELISA.
图5为基于ELISA检测ILT4抗体在混合淋巴细胞反应中的激活作用。FIG5 is an ELISA-based assay for the activation of ILT4 antibodies in mixed lymphocyte reaction.
图6为ILT4抗体在SU8686小鼠肿瘤模型内的药效学实验。FIG6 is a pharmacodynamics experiment of ILT4 antibody in SU8686 mouse tumor model.
图7为DOE软件分析所得40℃2周加速样品聚体与蛋白浓度、pH的影响趋势图。Figure 7 is a trend diagram of the effects of protein concentration and pH on aggregates of samples accelerated at 40°C for 2 weeks obtained by DOE software analysis.
图8为DOE软件分析所得40℃2周加速样品CE-SDS(NR)小分子组分含量与蛋白浓度、pH的影响趋势图。W代表week，40度2W代表40℃2周。Figure 8 shows the influence of protein concentration and pH on the content of small molecule components in CE-SDS(NR) samples accelerated at 40°C for 2 weeks, as analyzed by DOE software. W stands for week, and 40°C 2W stands for 40°C for 2 weeks.
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.
实施例1Example 1
实施例1抗体亲和力检测实验Example 1 Antibody affinity detection experiment
使用huILT4-ECD-Fc蛋白(UniprotKB NO.Q8N423胞外区1-460位)免疫Harbour H2L2转基因小鼠(Harbour Antibodies BV，该小鼠是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域，并通过IgG亚型的转变，PTM优化筛选获得如表1所示序列地抗体。将按照表1中的抗体通过生物膜干涉(Biolayerinterferometry，BLI)的方法(Fortebio octet RED 96e)，检测其对于人ILT4-ECD蛋白的结合动力学。Harbour H2L2 transgenic mice (Harbour Antibodies BV, a transgenic mouse carrying a human immunoglobulin immune library) were immunized with huILT4-ECD-Fc protein (UniprotKB NO. Q8N423 extracellular region 1-460). The antibodies produced by the mice have complete human antibody variable domains and rat constant domains. Through IgG subtype conversion and PTM optimization screening, the antibodies with the sequences shown in Table 1 were obtained. The antibodies listed in Table 1 were tested for their binding kinetics to human ILT4-ECD protein by biolayer interferometry (BLI) (Fortebio octet RED 96e).
将待测抗体稀释至终浓度6μg/mL，直接固化到AHC biosensor上，进行动力学测量，将抗原蛋白(HuILT4，UniprotKB NO.Q8N423)用0.02％PBST20分别稀释至100nM、50nM、25nM 3个浓度，进样70s，结合时间为300s，解离时间为300s，10mM glycine-HCl(pH1.5)再生15s。使用简单一对一Languir结合模型(Octet Red 96数据分析软件)，计算结合速率(kon)和解离速率(kdis)，平衡解离常数(kD)以比率kdis/kon计算。抗体亲和力检测结果如下表2所示。The test antibody was diluted to a final concentration of 6 μg/mL and directly immobilized onto the AHC biosensor for kinetic measurements. The antigen protein (HuILT4, UniprotKB No. Q8N423) was diluted in 0.02% PBST20 to three concentrations: 100 nM, 50 nM, and 25 nM. The sample was injected for 70 s, with an association time of 300 s and a dissociation time of 300 s. Regeneration was performed in 10 mM glycine-HCl (pH 1.5) for 15 s. The association rate (k₁₀) and dissociation rate (k₁₀) were calculated using a simple one-to-one Languir binding model (Octet Red 96 data analysis software). The equilibrium dissociation constant (k₁₀) was calculated as the ratio k₁₀/k₁₀. The antibody affinity test results are shown in Table 2 below.
表1抗体序列
Table 1 Antibody sequences
表2抗体亲和力检测结果
Table 2 Antibody affinity test results
从表2结果中可以看出，PR302264、PR302264-2和PR302264-3抗体亲和力均比对照抗体PR001795(Merck：US20180298096)要好。As can be seen from the results in Table 2, the affinity of PR302264, PR302264-2 and PR302264-3 antibodies is better than that of the control antibody PR001795 (Merck: US20180298096).
实施例2抗体在蛋白水平与ILT4-ECD的结合能力检测Example 2 Detection of the binding ability of antibodies to ILT4-ECD at the protein level
基于ELISA测定法检测表1中抗体与ILT4-ECD蛋白的结合能力。通过比较不同抗体与ILT4-ECD蛋白的结合曲线来测定其结合能力。具体实验过程为：先将人ILT4-ECD和食蟹猴ILT4-ECD蛋白依次稀释成1μg/mL浓度，加入96孔板中，每孔100μL，放置于4℃过夜。96孔板用PBST溶液洗三遍后，加入2％BSA的PBS溶液于37℃放置1h。将待测抗体进行浓度梯度稀释后(100nM，10-fold-dilution)，加入96孔板中，37℃孵育1h。PBST溶液洗三遍后，加入anti-human IgG Fc-HRP二抗(5000×稀释使用)于37℃孵育30-60min。PBST溶液洗三遍后，加入TMB显色液5-15min，然后加入终止液终止显色。The binding ability of the antibodies listed in Table 1 to the ILT4-ECD protein was tested using an ELISA assay. The binding ability of different antibodies to the ILT4-ECD protein was determined by comparing their binding curves. The specific experimental procedure was as follows: Human ILT4-ECD and cynomolgus macaque ILT4-ECD proteins were diluted sequentially to a concentration of 1 μg/mL, added to a 96-well plate, and incubated at 4°C overnight. The 96-well plate was washed three times with PBST solution, then added with a 2% BSA solution in PBS and incubated at 37°C for 1 hour. The test antibody was diluted in a concentration gradient (100 nM, 10-fold dilution) and added to the 96-well plate. The plate was incubated at 37°C for 1 hour. After washing three times with PBST solution, an anti-human IgG Fc-HRP secondary antibody (5000× dilution) was added and incubated at 37°C for 30-60 minutes. After washing three times with PBST solution, TMB color development solution was added for 5-15 minutes, and then color development was terminated by adding stop solution.
表3抗体在蛋白水平与ILT4-ECD的结合能力检测结果
Table 3 Results of antibody binding ability test with ILT4-ECD at protein level
检测结果如表3和图1所示，PR302264抗体具有和人ILT4-ECD蛋白结合的能力，且与对照抗体PR301366(Five Prime：WO2020014132)相当；且PR302264抗体还具有和猴ILT4-ECD蛋白结合的能力。The test results are shown in Table 3 and Figure 1. The PR302264 antibody has the ability to bind to the human ILT4-ECD protein, and is comparable to the control antibody PR301366 (Five Prime: WO2020014132); and the PR302264 antibody also has the ability to bind to the monkey ILT4-ECD protein.
实施例3抗体在蛋白水平与ILT2-ECD结合能力的检测Example 3 Detection of Antibody Binding Ability to ILT2-ECD at the Protein Level
基于ELISA测定法检测表1中的抗体与ILT2-ECD(UniprotKB NO.Q8NHL6)蛋白的结合能力。通过比较不同抗体与ILT2-ECD蛋白的结合曲线来测定其结合能力。具体实验过程为：先将人ILT2-ECD蛋白(UniprotKB NO.Q8NHL6)依次稀释成1μg/mL浓度，加入96孔板中，每孔100μL，放置于4℃过夜。96孔板用PBST溶液洗三遍后，加入2％BSA的PBS溶液于37℃放置1h。将待测抗体进行浓度梯度稀释后(100nM，10-fold-dilution)，加入96孔板中，37℃孵育1h。PBST溶液洗三遍后，加入anti-human IgG Fc-HRP二抗(5000×稀释使用)于37℃孵育30-60min。PBST溶液洗三遍后，加入TMB显色液5-15min，然后加入终止液终止显色。检测结果如图2所示，PR302264-2和PR302264-3抗体与Human ILT2有较强的结合能力。The binding ability of the antibodies listed in Table 1 to the ILT2-ECD (UniprotKB NO. Q8NHL6) protein was tested using an ELISA assay. Binding abilities of different antibodies to the ILT2-ECD protein were determined by comparing their binding curves. The specific experimental procedure was as follows: Human ILT2-ECD protein (UniprotKB NO. Q8NHL6) was diluted sequentially to a concentration of 1 μg/mL and added to a 96-well plate (100 μL per well). The plate was incubated at 4°C overnight. The plate was washed three times with PBST solution, then a 2% BSA solution in PBS was added and incubated at 37°C for 1 hour. The antibody to be tested was serially diluted (100 nM, 10-fold dilution) and added to the 96-well plate. The plate was incubated at 37°C for 1 hour. After washing three times with PBST solution, an anti-human IgG Fc-HRP secondary antibody (5000× dilution) was added and incubated at 37°C for 30-60 minutes. After washing three times with PBST, TMB colorimetric solution was added for 5-15 minutes, and then the color development was terminated by adding stop solution. As shown in Figure 2, the PR302264-2 and PR302264-3 antibodies strongly bound to Human ILT2.
实施例4抗体与ILT4其他家族蛋白的交叉反应Example 4 Cross-reaction of antibodies with other ILT4 family proteins
取100μL浓度为1μg/mL的人LILRA1(R&D System，Cat:9226-T4-050)，LILRA2(R&D System，Cat：9040-T4-050)，LILRA3(R&D System，Cat：9517-T4-100)，LILRA4(R&D System，Cat：8914-T4-050)，LILRA5(R&D System，Cat：8956-T4-100)，LILRA6(R&D System，Cat:9088-T4-050)，LILRB3(R&D System，Cat：9159-T5-050)，LILRB4(R&D System，Cat：8488-T4-025)，LILRB5(R&D System，Cat：8478-T4-025)蛋白进行ELISA包被，4℃过夜。用PBST清洗4遍，取200μL溶于PBST(PBS+0.05％吐温20)+2％BSA进行封闭，37℃，1小时。用PBST清洗4遍。向ELISA板孔中分别加入100μL浓度为100nM的ILT4抗体，以及各个蛋白的阳性对照抗体(LILRA1 Antibody：R&D System，Cat：MAB30851；LILRA2 Antibody：R&D System，Cat：MAB6364；LILRA3Antibody：R&D System，Cat：MAB2574；LILRA4 Antibody：R&D System，Cat：MAB6287；LILRA5 Antibody：R&D System，Cat：MAB6754；LILRA6 Antibody：R&D System，Cat：MAB8656；LILRB3 Antibody：R&D System，Cat：MAB1806-100；LILRB4 Antibody：R&DSystem，Cat：MAB24251；LILRB5 Antibody：R&D System，Cat：MAB3065)。37℃孵育1小时。用PBST清洗4遍。加入100μl Goat Anti-Human IgG，(Fab)-HRP(1:5000，Jackson，Cat:109-035-098)，37℃孵育1小时。用PBST清洗4遍。加入100μl TMB底物(Biopanda，Cat:TMB-S-003)，室温孵育5分钟，随后加入50μL终止缓冲液(Solarbio，Cat#:C1058)来终止反应。用酶标仪(Molecular Devices，Spectra max 384plus)记录OD 450nm读值。对照抗体PR304683(NGM：WO2021127200)。Take 100 μL of 1 μg/mL human LILRA1 (R&D System, Cat: 9226-T4-050), LILRA2 (R&D System, Cat: 9040-T4-050), LILRA3 (R&D System, Cat: 9517-T4-100), LILRA4 (R&D System, Cat: 8914-T4-050), LILRA5 (R&D System ELISA was performed using proteins from R&D System, Cat: 8956-T4-100), LILRA6 (R&D System, Cat: 9088-T4-050), LILRB3 (R&D System, Cat: 9159-T5-050), LILRB4 (R&D System, Cat: 8488-T4-025), and LILRB5 (R&D System, Cat: 8478-T4-025) at 4°C overnight. The cells were washed four times with PBST and blocked with 200 μL of a solution of PBST (PBS + 0.05% Tween 20) + 2% BSA at 37°C for 1 hour. The cells were then washed four times with PBST. 100 μL of 100 nM ILT4 antibody and positive control antibodies of each protein (LILRA1 Antibody: R&D System, Cat: MAB30851; LILRA2 Antibody: R&D System, Cat: MAB6364; LILRA3 Antibody: R&D System, Cat: MAB2574; LILRA4 Antibody: R&D System, Cat: MAB6284) were added to the ELISA plate wells. 7; LILRA5 Antibody: R&D System, Cat: MAB6754; LILRA6 Antibody: R&D System, Cat: MAB8656; LILRB3 Antibody: R&D System, Cat: MAB1806-100; LILRB4 Antibody: R&D System, Cat: MAB24251; LILRB5 Antibody: R&D System, Cat: MAB3065). Incubate at 37°C for 1 hour. Wash four times with PBST. Add 100 μl of Goat Anti-Human IgG, (Fab)-HRP (1:5000, Jackson, Cat: 109-035-098) and incubate at 37°C for 1 hour. Wash four times with PBST. 100 μL of TMB substrate (Biopanda, Cat: TMB-S-003) was added and incubated at room temperature for 5 minutes. The reaction was terminated by adding 50 μL of stop buffer (Solarbio, Cat#: C1058). OD values at 450 nm were recorded using a microplate reader (Molecular Devices, Spectramax 384plus). The control antibody PR304683 (NGM: WO2021127200) was used.
表4抗体与ILT4其他家族蛋白的交叉反应结果
Table 4 Cross-reaction results of antibodies with other ILT4 family proteins
结果如表4所示，除了结合ILT4，PR302264-2与LIR6和ILT2有交叉结合能力。The results are shown in Table 4. In addition to binding to ILT4, PR302264-2 has cross-binding ability with LIR6 and ILT2.
实施例5抗体对巨噬细胞激活作用的检测-细胞因子TNF-α分泌Example 5 Detection of Antibody Activation Effect on Macrophages - Cytokine TNF-α Secretion
使用人CD14细胞分选试剂盒(Miltenyi，130-050-021)从人PBMC(上海赛笠生物科技有限公司、XFB-HP050A)中分离出CD14阳性细胞。使用1640完全培养基将分离的CD14阳性细胞重悬稀释到8*105/mL，取96孔细胞培养板，每孔加入100μL细胞悬液；取50μL梯度稀释(5倍稀释，起始终浓度50nM)的抗体至96孔细胞板中；将MCSF(R&D System，216-MC-025/CF)稀释至400ng/mL，每孔加入50μL。于37℃，CO2培养箱中孵育，第三天时进行换液，保持抗体和MCSF浓度不变。第六天时再进行换液，保持抗体和MCSF浓度不变，同时再加入终浓度为50ng/mL的LPS，24h后收取细胞上清，使用ELISA试剂盒(Ebioscience，88-7346-88)检测TNF-α的含量。结果如图3所示，PR302264抗体对巨噬细胞都有很强的激活作用。CD14-positive cells were isolated from human PBMCs (Shanghai Saili Biotechnology Co., Ltd., XFB-HP050A) using a human CD14 cell isolation kit (Miltenyi, 130-050-021). The isolated CD14-positive cells were resuspended in 1640 complete medium to a concentration of 8 x 105 /mL. 100 μL of the cell suspension was added to each well of a 96-well cell culture plate. 50 μL of serially diluted (5-fold dilutions, starting and ending at 50 nM) antibody was added to each well of the 96-well plate. MCSF (R&D System, 216-MC-025/CF) was diluted to 400 ng/mL and added to each well. The plates were incubated at 37°C in aCO2 incubator. The medium was changed on the third day, maintaining constant antibody and MCSF concentrations. On the sixth day, the medium was changed again, maintaining the same antibody and MCSF concentrations. LPS was added to a final concentration of 50 ng/mL. After 24 hours, the cell supernatant was collected and TNF-α levels were measured using an ELISA kit (Ebioscience, 88-7346-88). As shown in Figure 3, the PR302264 antibody had a strong activating effect on macrophages.
实施例6抗体对外周血单核细胞的作用检测-细胞因子TNF-α分泌Example 6 Detection of the Effect of Antibodies on Peripheral Blood Mononuclear Cells - Cytokine TNF-α Secretion
使用健康人PBMC，用1640完全培养基将细胞重悬稀释到2*106/mL，取96孔细胞培养板，每孔加入100μL细胞悬液；取50μl梯度稀释(最高终浓度为30nM，稀释到1nM，再5倍稀释)的抗体至96孔细胞板中；将LPS稀释至400ng/mL，每孔加入50μL。于37℃，CO2培养箱中孵育三天后，取细胞上清，ELISA试剂盒检测TNF-α的含量。结果如图4所示，抗体PR302264-2、PR302264-3、对PBMC都有很强的激活作用。Healthy human PBMCs were resuspended in 1640 complete medium and diluted to 2 x 106 /mL. 100 μL of the cell suspension was added to each well of a 96-well cell culture plate. 50 μL of serially diluted antibodies (maximum final concentration: 30 nM, diluted to 1 nM, and then diluted five-fold) were added to the 96-well plate. LPS was diluted to 400 ng/mL and 50 μL was added to each well. After three days of incubation at 37°C in aCO2 incubator, the cell supernatant was collected and assayed for TNF-α levels using an ELISA kit. As shown in Figure 4, antibodies PR302264-2 and PR302264-3 all showed strong activation effects on PBMCs.
实施例7抗体在混合淋巴细胞反应中的作用检测-细胞因子IFN-γ分泌Example 7: Detection of the Effect of Antibodies in Mixed Lymphocyte Reaction - Cytokine IFN-γ Secretion
使用人CD14细胞分选试剂盒(Miltenyi，130-050-021)从人PBMC中分离出CD14阳性细胞。使用1640完全培养基将分离的CD14阳性细胞重悬稀释到1.5*105/mL，取96孔细胞培养板，每孔加入100μL细胞悬液；取50μL梯度稀释(5倍稀释，起始终浓度50nM)的抗体至96孔细胞板中；将MSCF(R&D System，216-MC-025/CF)稀释至400ng/mL，每孔加入50μL。于37℃，CO2培养箱中孵育，第三天时进行换液，保持抗体和MCSF浓度不变。第六天时再进行换液，保持抗体和MCSF浓度不变，同时再加入终浓度为50ng/mL的LPS。第七天时，取另一个人PBMC，用人CD3分选试剂盒(Miltenyi，130-045-501)分选出CD3阳性细胞，使用1640完全培养基将分离的CD3阳性细胞重悬稀释到1.5*106/mL。将96孔细胞培养板中的上清去除，然后加入100μL的CD3阳性细胞悬液，以及稀释好的抗体50μL，最后再补液至200μL。共孵育72h后，收集细胞上清，ELISA试剂盒检测IFN-γ的含量。结果如图5所示，抗体PR302264-2在混合淋巴细胞反应实验中具有很好的激活作用。CD14-positive cells were isolated from human PBMCs using a human CD14 cell isolation kit (Miltenyi, 130-050-021). The isolated CD14-positive cells were resuspended in 1640 complete medium to a concentration of 1.5 x 105 /mL. 100 μL of the cell suspension was added to each well of a 96-well cell culture plate. 50 μL of serially diluted (5-fold dilutions, starting at a final concentration of 50 nM) antibody was added to each well of the 96-well plate. MSCF (R&D System, 216-MC-025/CF) was diluted to 400 ng/mL and 50 μL was added to each well. The cells were incubated at 37°C in aCO2 incubator. On the third day, the medium was changed, maintaining the same antibody and MCSF concentrations. On the sixth day, the medium was changed again, maintaining the same antibody and MCSF concentrations, and LPS was added to a final concentration of 50 ng/mL. On day 7, another human PBMC was isolated and CD3-positive cells were isolated using a human CD3 isolation kit (Miltenyi, 130-045-501). The isolated CD3-positive cells were resuspended and diluted to 1.5 x 106 /mL in 1640 complete medium. The supernatant from the 96-well cell culture plate was removed, and 100 μL of the CD3-positive cell suspension and 50 μL of the diluted antibody were added, and the volume was finally refilled to 200 μL. After 72 hours of co-incubation, the cell supernatant was collected and the IFN-γ content was measured by ELISA kit. The results, as shown in Figure 5, show that the antibody PR302264-2 has a good activation effect in the mixed lymphocyte reaction assay.
实施例8抗体在体内的药效学实验Example 8 In vivo pharmacodynamics experiments of antibodies
本实验利用SU8686小鼠肿瘤模型研究抗人ILT4抗体的抗肿瘤活性。研究了PR302264-2抗体单独给药(20mg/kg)在SU8686小鼠模型中的药效学。人胰腺癌SU8686细胞(ATCC)皮下接种到雌性hHSC-NOG-EXL人源化小鼠(维通利华)。抗体组处理方法为：肿瘤细胞接种后，待肿瘤体积生长到100mm3的时候，开始第一次给药，在接下来的第3天，第7天，第10天，第14天和第17天给各组小鼠腹腔注射相应的抗体，并同时测量肿瘤体积，之后对小鼠实施安乐死。结果图6所示，本申请的抗人ILT4抗体PR302264-2与阴性对照相比，有明细抑制肿瘤生长作用，其中PR302264-2抗体对肿瘤的抑制效果要好于对照抗体PR001795(Merck：US20180298096)和PR304683(NGM：WO2021127200)。This study used the SU8686 mouse tumor model to investigate the antitumor activity of anti-human ILT4 antibodies. The pharmacodynamics of PR302264-2 antibody administered alone (20 mg/kg) in the SU8686 mouse model were investigated. Human pancreatic cancer SU8686 cells (ATCC) were subcutaneously inoculated into female hHSC-NOG-EXL humanized mice (Vitamin B). The antibody group was treated as follows: after tumor cell inoculation, the first dose was initiated when the tumor volume grew to 100 mm3. On the following days, the corresponding antibody was intraperitoneally injected into each group of mice on the 3rd, 7th, 10th, 14th, and 17th day. The tumor volume was measured at the same time, and the mice were euthanized. The results are shown in Figure 6. Compared with the negative control, the anti-human ILT4 antibody PR302264-2 of the present application has a clear inhibitory effect on tumor growth, among which the inhibitory effect of PR302264-2 antibody on tumors is better than that of the control antibodies PR001795 (Merck: US20180298096) and PR304683 (NGM: WO2021127200).
实施例9 pH筛选Example 9 pH screening
为筛选抗ILT2/4抗体(PR302264-2)最优pH范围，发明人对该分子进行了pH梯度筛选，考察了抗ILT2/4抗体在pH4至pH8范围内的高温加速、冻融、光照条件下的沉淀、分子排阻色谱法(SEC)纯度、非还原十二烷基磺酸钠毛细管电泳(CE-SDS(NR))、全柱成像毛细管等电聚焦(icIEF)技术所测酸性、碱性峰及主峰纯度变化以及结合活性变化，而确定抗ILT2/4适宜的处方pH范围。结果见表5-表9。To identify the optimal pH range for the anti-ILT2/4 antibody (PR302264-2), the inventors conducted a pH gradient screening of the molecule. This screening examined the antibody's purity across the pH range of 4 to 8, including high-temperature accelerated, freeze-thaw, and light-induced precipitation, as well as changes in size exclusion chromatography (SEC), purity of the acidic and basic peaks, and the main peak measured by capillary electrophoresis with sodium dodecyl sulfate (CE-SDS(NR)), and whole-column imaging capillary isoelectric focusing (icIEF), as well as changes in binding activity. The results were summarized in Tables 5-9.
分子排阻色谱法(SEC)：Size Exclusion Chromatography (SEC):
实验条件设置：Waters 2695液相色谱仪(Waters公司，美国)；TSKgelG3000SWXL串联色谱柱(购自Waters公司，美国)；流动相为0.2M磷酸盐缓冲液，0.25M氯化钾，pH6.2，0.5mL/分钟，进样量为100μg，检测波长为280nm。Experimental conditions were set up as follows: Waters 2695 liquid chromatograph (Waters, USA); TSKgelG3000SWXL tandem chromatographic columns (purchased from Waters, USA); the mobile phase was 0.2 M phosphate buffer, 0.25 M potassium chloride, pH 6.2, 0.5 mL/min, the injection volume was 100 μg, and the detection wavelength was 280 nm.
非还原十二烷基磺酸钠毛细管电泳(CE-SDS(NR))：Capillary electrophoresis of sodium dodecyl sulfate (CE-SDS(NR)):
毛细管电泳仪(购自SCIEX公司，型号PA800PLUS)，毛细管卡盒SCIEX，货号U738；毛细管SCIEX，货号338451；Low pH SDS，SCIEX，货号C57805进行分析。25μL 4.0mg/mL样品溶液混合，加入Low pH SDS 70μL，200UM nem 5μL，离心后，70℃水浴10分钟，冷却。5KV进样20s，15KV分离40分钟。使用UV检测器，波长设定为214nm。Capillary electrophoresis was performed using a SCIEX capillary electrophoresis instrument (model PA800PLUS) using a SCIEX capillary cartridge (Cat. No. U738), SCIEX capillary tubes (Cat. No. 338451), and a SCIEX low-pH SDS (Cat. No. C57805). 25 μL of the 4.0 mg/mL sample solution was mixed, followed by the addition of 70 μL of low-pH SDS and 5 μL of 200 μM nem. After centrifugation, the sample was incubated in a 70°C water bath for 10 minutes and cooled. Injection was performed at 5 kV for 20 seconds, and separation was performed at 15 kV for 40 minutes. A UV detector was used, with a wavelength set to 214 nm.
全柱成像毛细管等电聚焦(icIEF)：Whole-column imaging capillary isoelectric focusing (icIEF):
实验条件设置：ProteinSimple Maurice毛细管等电聚焦仪(Protein Simple公司，美国)；两性电解溶液：3-10μL Pharmalyte 3-10；(购自GE公司，美国，货号：17045501)，8-10.5μL Pharmalyte 3-10，(购自GE公司，美国，货号：17045601)，0.5μL pI Marker 7.05(购自Protein Simple公司，美国，货号：102226)，0.5μL pI Marker 9.46(购自Protein Simple公司，美国，货号：102349)，适量1％甲基纤维素(购自Protein Simple公司，货号：101876，适量0.5％甲基纤维素(购自Protein Simple公司，货号：102505)，适量磺化二甲基乙基氨(NDSB)(购自Merck，D01955G)，适量牛磺酸(T)(购自Sigma，T0625-100G)。进样溶液由80μL上述两性电解溶液和20μL 1.0mg/mL的蛋白溶液混合而成。样品体系在1500V预聚焦1分钟，3000V聚焦10分钟进行检测。Experimental conditions: ProteinSimple Maurice capillary isoelectric focusing instrument (Protein Simple, USA); amphoteric electrolyte solution: 3-10 μL Pharmalyte 3-10 (purchased from GE, USA, catalog number: 17045501), 8-10.5 μL Pharmalyte 3-10 (purchased from GE, USA, catalog number: 17045601), 0.5 μL pI Marker 7.05 (purchased from Protein Simple, USA, catalog number: 102226), 0.5 μL pI Marker 9.46 (purchased from Protein Simple, USA, catalog number: 102226). in Simple Company, USA, Catalog No.: 102349), appropriate amount of 1% methylcellulose (purchased from Protein Simple Company, Catalog No.: 101876, appropriate amount of 0.5% methylcellulose (purchased from Protein Simple Company, Catalog No.: 102505), appropriate amount of sulfonated dimethylethylamine (NDSB) (purchased from Merck, D01955G), appropriate amount of taurine (T) (purchased from Sigma, T0625-100G). The injection solution was prepared by mixing 80 μL of the above-mentioned amphoteric electrolyte solution and 20 μL of 1.0 mg/mL protein solution. The sample system was pre-focused at 1500 V for 1 minute and focused at 3000 V for 10 minutes for detection.
结合活性检测：Binding activity assay:
采用ELISA方法检测ILT2和ILT4的结合活性。The binding activity of ILT2 and ILT4 was detected by ELISA.
表5零点样品结果
Table 5 Zero point sample results
表6 40℃避光7天样品结果
Table 6 Results of samples at 40℃ in the dark for 7 days
相比于表6，pH4～6聚集体相对较少且聚集增加相对较少，表明pH4～6为相对适宜抗ILT2/4抗体的缓冲pH。Compared with Table 6, the number of aggregates at pH 4-6 was relatively small and the increase in aggregation was relatively small, indicating that pH 4-6 is a relatively suitable buffer pH for anti-ILT2/4 antibodies.
表7 40℃光照7天样品结果
Table 7 Results of samples irradiated at 40°C for 7 days
如表7，对比表4，强光照射条件下，聚体相对于避光实验组进一步增加，表明强光照增加抗体聚集及沉淀。As shown in Table 7, compared with Table 4, under strong light irradiation conditions, the aggregates further increased compared with the light-proof experimental group, indicating that strong light irradiation increased antibody aggregation and precipitation.
表8冻融实验结果
Table 8 Freeze-thaw experiment results
冻融实验发现，如表8，冻融结果1中的样品均出现大量沉淀，后续补充实验，在冻融结果1中添加表面活性剂聚山梨酯20，发现添加聚山梨酯20后冻融样品沉淀明显减少，但是pH7至pH8样品沉淀添加聚山梨酯20后未明显减少，因此补充了冻融实验，添加聚山梨酯20后进行冻融。The freeze-thaw experiment found that, as shown in Table 8, a large amount of precipitation appeared in the samples in freeze-thaw result 1. In a subsequent supplementary experiment, the surfactant polysorbate 20 was added to the freeze-thaw result 1. It was found that the precipitation of the freeze-thaw samples was significantly reduced after the addition of polysorbate 20. However, the precipitation of the samples with pH 7 to pH 8 was not significantly reduced after the addition of polysorbate 20. Therefore, the freeze-thaw experiment was supplemented and freeze-thaw was performed after adding polysorbate 20.
表9为冻融补充实验结果
Table 9 shows the results of freeze-thaw supplementation experiments.
如表9，补充冻融实验样品均未见沉淀产生，证明处方中添加表面活性剂类辅料如聚山梨酯20、聚山梨酯80、泊洛沙姆188能够有效抑制蛋白沉淀。As shown in Table 9, no precipitation was observed in the samples of the supplementary freeze-thaw experiment, which proves that the addition of surfactant excipients such as polysorbate 20, polysorbate 80, and poloxamer 188 to the formulation can effectively inhibit protein precipitation.
因此pH4～6的处方，添加聚山梨酯20，添加蔗糖、海藻糖等糖醇类稳定剂，均能够有效提高抗ILT2/4抗体的稳定性。Therefore, a pH 4-6 formulation, the addition of polysorbate 20, and the addition of sugar alcohol stabilizers such as sucrose and trehalose can effectively improve the stability of anti-ILT2/4 antibodies.
实施例10辅料筛选Example 10 Excipient Screening
通过实验设计(Design of Experiments:DOE)进行辅料筛选，通过统计学方法同时研究pH、抗体浓度、聚山梨酯20含量、海藻糖含量对抗ILT2/4抗体稳定性的影响效果，具体研究条件见表10。Excipient screening was performed using Design of Experiments (DOE). Statistical methods were used to simultaneously investigate the effects of pH, antibody concentration, polysorbate 20 content, and trehalose content on the stability of anti-ILT2/4 antibodies. Specific research conditions are shown in Table 10.
表10 pH、抗体浓度、聚山梨酯20含量、海藻糖含量对抗ILT2/4抗体稳定性的影响
Table 10 Effects of pH, antibody concentration, polysorbate 20 content, and trehalose content on the stability of anti-ILT2/4 antibodies
DOE实验样品结果见表11。The results of the DOE experimental samples are shown in Table 11.
表11 DOE实验样品结果
注：结合活性分别为ILT2靶点及ILT4靶点。Table 11 DOE experimental sample results
Note: Binding activities are for ILT2 and ILT4 targets respectively.
冻融、振荡结果显示，抗ILT2/4抗体在上述实验研究参数范围内，稳定性较好，各质量参数均无明显变化。The freeze-thaw and oscillation results showed that the anti-ILT2/4 antibody had good stability within the above experimental research parameter range, and there was no significant change in various quality parameters.
40℃加速结果显示，不同处方聚体、CE-SDS(NR)小分子组分、icIEF酸性峰有不同程度的增加，通过DOE软件进行数据统计分析，结果显示聚体随蛋白浓度、pH的增加而增加(见图7)；CE-SDS(NR)小分子组分随蛋白浓度增加、pH的降低而增加(见图8)，二者受pH相反的影响，但总体而言，在研究范围内，加速变化程度均在可接受范围内，而icIEF酸性峰主要随储存温度和时间的增加而增加，加速试验结果显示该变化程度也在可接受范围内。The 40°C accelerated results showed that the aggregates, CE-SDS (NR) small molecule components, and icIEF acidic peaks of different formulations increased to varying degrees. Data statistical analysis using DOE software showed that the aggregates increased with increasing protein concentration and pH (see Figure 7); the CE-SDS (NR) small molecule components increased with increasing protein concentration and decreasing pH (see Figure 8). The two were affected in opposite ways by pH, but overall, within the research range, the degree of accelerated change was within an acceptable range. The icIEF acidic peak mainly increased with increasing storage temperature and time. The accelerated test results showed that the degree of change was also within an acceptable range.
即在上表的11个处方中，在所研究的处方的范围内，抗ILT2/4抗体均能保持较高的稳定性。That is, among the 11 prescriptions in the above table, within the range of the prescriptions studied, the anti-ILT2/4 antibodies were able to maintain a relatively high stability.
实施例11处方确认Example 11 Prescription Confirmation
通过对实施例10中心点处方，具体为表10中的编号2/7/11(为三个重复组)进行处方确认，进行冷冻、冻融、2-8℃、25℃、40℃稳定性试验。The central point prescription of Example 10, specifically No. 2/7/11 in Table 10 (three replicate groups), was confirmed by performing freezing, freeze-thaw, 2-8°C, 25°C, and 40°C stability tests.
结果如表12所示。The results are shown in Table 12.
表12稳定性试验
注：“/”代表未检测。Table 12 Stability test
Note: “/” means not tested.
上表结果显示，抗ILT2/4在该制剂处方中稳定性良好，能够长期保持稳定性。The results in the above table show that anti-ILT2/4 has good stability in this preparation and can maintain stability for a long time.
本发明用的抗体序列如表13所示。The antibody sequences used in the present invention are shown in Table 13.
表13抗体序列表
Table 13 Antibody sequence list
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410177579 | 2024-02-08 | ||
| CN202410177579.2 | 2024-02-08 |
| Publication Number | Publication Date |
|---|---|
| WO2025168070A1true WO2025168070A1 (en) | 2025-08-14 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2025/076275PendingWO2025168070A1 (en) | 2024-02-08 | 2025-02-07 | Anti-ilt2/4 monoclonal antibody preparation, kit containing same and use thereof |
| Country | Link |
|---|---|
| WO (1) | WO2025168070A1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019144052A1 (en)* | 2018-01-18 | 2019-07-25 | Adanate, Inc. | Anti-lilrb antibodies and uses thereof |
| WO2021222544A1 (en)* | 2020-05-01 | 2021-11-04 | Ngm Biopharmaceuticals, Inc. | Ilt-binding agents and methods of use thereof |
| WO2023170434A1 (en)* | 2022-03-11 | 2023-09-14 | Macomics Limited | Compositions and methods for modulation of macrophage activity |
| WO2023235703A1 (en)* | 2022-05-31 | 2023-12-07 | Ngm Biopharmaceuticals, Inc. | Therapeutic methods using ilt-binding agents |
| WO2024120526A1 (en)* | 2022-12-09 | 2024-06-13 | 诺纳生物（苏州）有限公司 | Anti-ilt4 antibody as well as preparation method therefor and use thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019144052A1 (en)* | 2018-01-18 | 2019-07-25 | Adanate, Inc. | Anti-lilrb antibodies and uses thereof |
| WO2021222544A1 (en)* | 2020-05-01 | 2021-11-04 | Ngm Biopharmaceuticals, Inc. | Ilt-binding agents and methods of use thereof |
| WO2023170434A1 (en)* | 2022-03-11 | 2023-09-14 | Macomics Limited | Compositions and methods for modulation of macrophage activity |
| WO2023235703A1 (en)* | 2022-05-31 | 2023-12-07 | Ngm Biopharmaceuticals, Inc. | Therapeutic methods using ilt-binding agents |
| WO2024120526A1 (en)* | 2022-12-09 | 2024-06-13 | 诺纳生物（苏州）有限公司 | Anti-ilt4 antibody as well as preparation method therefor and use thereof |
| Title |
|---|
| NAING, A. ET AL.: "First-in-human Study of NGM707, an ILT2/ILT4 Dual Antagonist Antibody in Advanced or Metastatic Solid Tumors: Preliminary Monotherapy Dose Escalation Data", IMMUNO-ONCOLOGY AND TECHNOLOGY, vol. 16, no. S1, 31 December 2022 (2022-12-31), XP093173258, DOI: 10.1016/j.iotech.2022.100284* |
| Publication | Publication Date | Title |
|---|---|---|
| CN110536903B (en) | Anti-OX40 antibody and use thereof | |
| AU2022201793A1 (en) | Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of use thereof | |
| KR20170102167A (en) | Anti-PD-L1 antibody | |
| KR20170070243A (en) | Anti-pd-1 antibodies | |
| JP2019532013A (en) | PD-L1-specific antibody and method of using the same | |
| JP2022529350A (en) | Antagonist anti-CD7 antibody | |
| CN111393529B (en) | anti-OX 40 antibodies that non-competitively bind to OX40L | |
| JP7624497B2 (en) | Human PD-L2 antibodies and methods of use thereof | |
| JP7514765B2 (en) | Bispecific antibodies against human PD-L1 and PD-L2 and methods of use thereof | |
| WO2021143826A1 (en) | Recombinant anti-programmed cell death protein 1 and anti-cluster of differentiation antigen 137 bispecific antibody preparation and use thereof | |
| WO2011108008A2 (en) | Antibody for targeted induction of apoptosis, cdc and adcc mediated killing of cancer cells, tbl-cln1 | |
| WO2020151761A1 (en) | Bispecific antibody binding to pd-l1 and ox40 | |
| TW201619198A (en) | Human anti-FGFR4 antibody | |
| KR20220148699A (en) | Anti-CNTN4 specific antibody and its use | |
| Yu et al. | A novel fully human anti-CD47 antibody as a potential therapy for human neoplasms with good safety | |
| CN110420316A (en) | Participate in the cellular factor of the cytotoxicity of staphylococcus aureus leukocidin: novel therapeutic target spot | |
| BR112020023265A2 (en) | use to prevent and treat myeloid-derived suppressor cell-related diseases | |
| TWI863317B (en) | Anti-CD40 antibody and anti-PD-L1×CD40 bispecific antibody and their applications | |
| WO2025168070A1 (en) | Anti-ilt2/4 monoclonal antibody preparation, kit containing same and use thereof | |
| WO2023109962A1 (en) | Antibody binding to human cd73, preparation method therefor, and use thereof | |
| CN119013309A (en) | Antigen binding proteins targeting PD-L1 and CD40, preparation and application thereof | |
| KR20220054600A (en) | IL-38-specific antibody | |
| EP3870610A1 (en) | Methods and compositions for the treatment of cancer and infectious diseases | |
| CN113842456B (en) | Anti-human 4-1BB monoclonal antibody preparation and application thereof | |
| CN117677396A (en) | IL-38 specific antibodies |
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | Ref document number:25751669 Country of ref document:EP Kind code of ref document:A1 |