Multispecific binding agents to PD-L1 and CD137 in combination with anti-PD-1 antibodies for treating cancer

Abstract

The present disclosure relates to combination therapies using binding agents that bind to human PD-L1 and human CD137, in combination with pembrolizumab, to reduce or prevent tumor progression or treat cancer.

Description

[Technical field]

[0001] The present invention relates to a combination therapy using a binding agent that binds to human PD-L1 and human CD137, in combination with pembrolizumab, to reduce or prevent tumor progression or treat cancer. [Background technology]

[0002] CD137 (4-1BB) is a member of the TNFR family and is involved in the regulation of CD8 + CD137 is a costimulatory molecule on CD4+ T cells, regulatory T cells (Treg), natural killer T cells (NK(T) cells), B cells, and neutrophils. On T cells, CD137 is not constitutively expressed and is induced upon activation of the T cell receptor (TCR), e.g., on tumor-infiltrating lymphocytes (TILs) (Gros et al., J. Clin Invest, 2014, 124(5):2246-59). Stimulation via its natural ligand 4-1BBL or agonistic antibodies results in signaling using TRAF-2 and TRAF-1 as adaptors. Initial signaling by CD137 involves K-63 polyubiquitination, which ultimately results in activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways. Signaling results in T cell costimulation, proliferation, increased cytokine production, maturation, and extended CD8+ T cell survival. Agonistic antibodies against CD137 have been shown to promote anti-tumor control by T cells in various preclinical models (Murillo et al., Clin Cancer Res, 2008, 14(21):6895-906). Antibodies that stimulate CD137 may induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art and include the human IgG4 antibody urelumab (AU2004279877) and the human IgG2 antibody utomilumab (Fisher et al., 2012, Cancer Immunol. Immunother., 61:1721-1733).

[0003] Programmed death-ligand 1 (PD-L1, PDL1, CD274, B7H1) is a 33 kDa type I single-pass transmembrane protein. Three isoforms of PD-L1 have been described based on alternative splicing. PD-L1 belongs to the immunoglobulin (Ig) superfamily and contains one Ig-like C2-type domain and one Ig-like V-type domain. Freshly isolated T and B cells express negligible amounts of PD-L1, whereas only CD14 expresses PD-L1 constitutively. + They are a small fraction of monocytes (approximately 16%). However, interferon gamma (IFNγ) is known to upregulate PD-L1 on tumor cells.

[0004] PD-L1 acts by 1) tolerizing tumor-reactive T cells by binding to its receptor, programmed cell death protein 1 (PD-1) (CD279), on activated T cells; and 2) PD-1 signaling through tumor cell-expressed PD-L1 mediates tumor cell-associated CD8 + PD-L1 impedes antitumor immunity by rendering them resistant to T cell / Fas ligand-mediated lysis; 3) by tolerizing T cells through reverse signaling via CD80 (B7.1) expressed by T cells; and 4) by promoting the development and maintenance of induced regulatory T cells. PD-L1 is expressed in many human cancers, including melanoma, ovarian, lung, and colon cancers (Latchman et al., 2004, Proc Natl Acad Sci USA, 101, 10691-6).

[0005] PD-L1 blocking antibodies have shown clinical activity in several cancers known to overexpress PD-L1, including melanoma and NSCLC. For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. Atezolizumab is currently in clinical trials as an immunotherapy for several indications, including various types of solid tumors (see, e.g., Rittmeyer et al., 2017, Lancet, 389:255-265), and has been approved for non-small cell lung cancer and bladder cancer indications. The PD-L1 antibody avelumab (Kaufman et al., Lancet Oncol., 2016, 17(10):1374-1385) has been approved by the FDA for the treatment of adult and pediatric patients aged 12 years and older with metastatic Merkel cell carcinoma and is currently in clinical trials in several cancer indications, including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, NSCLC, ovarian cancer, and kidney cancer. The PD-L1 antibody durvalumab has been approved for locally advanced or metastatic urinary tract cancer indications and is in clinical development in multiple solid tumors and hematological cancers (see, e.g., Massard et al., 2016, J Clin Oncol., 34(26):3119-25). Additional anti-PD-L1 antibodies are described, for example, in WO2004004771.

[0006] Horton et al. (J Immunother Cancer., 2015, Vol. 3 (Suppl. No. 2):O10) discloses the combination of an agonistic 4-1BB antibody with a neutralizing PD-L1 antibody. WO2019 / 025545 provides binding agents, e.g., bispecific antibodies that bind human PD-L1 and bind human CD137.

[0007] However, despite these advances in the art, there is a great need for improved therapies to prevent tumor progression or treat cancer. Summary of the Invention [Problem to be solved by the invention]

[0008] The inventors surprisingly found that a combination of (i) stimulation with a binding agent that binds human PD-L1 and binds human CD137, and (ii) an antibody that binds programmed death-1 (PD-1), amplifies the immune response. [Means for solving the problem]

[0009] Thus, in a first aspect, the disclosure provides a binding agent for use in a method of reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject a binding agent prior to, simultaneously with, or following administration of an antibody or antigen-binding fragment thereof that binds Programmed Death-1 (PD-1), wherein the binding agent comprises a first binding region that binds CD137, and a second binding region that binds PD-L1; a) a first binding region comprising a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 6, 7, and 8, respectively; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 16, 17, and 18, respectively; The binding agents provided herein are characterized in that the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, or the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively.

[0010] In a second aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (i) comprising a first binding region that binds to CD137 and a second binding region that binds to PD-L1; a) the first binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 6, 7, and 8, respectively; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 16, 17, and 18, respectively; Binders; and (ii) an antibody or antigen-binding fragment thereof that binds to PD-1, comprising a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 46, 47, and 48, respectively, or comprising a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 65, 66, and 67, respectively; A kit comprising:

[0011] In a third aspect, the present disclosure provides a kit of the second aspect for use in a method of reducing or preventing the progression of a tumor or treating cancer in a subject.

[0012] In a fourth aspect, the disclosure provides a method of reducing or preventing tumor progression or treating cancer in a subject, comprising administering to the subject prior to, simultaneously with, or following administration of an antibody or antigen-binding fragment thereof that binds PD-1, comprising a first binding region that binds CD137, and a second binding region that binds PD-L1; a) a first binding region comprising a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 6, 7, and 8, respectively; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 16, 17, and 18, respectively; The method includes administering a binding agent to a subject, wherein the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 46, 47, and 48, respectively, or the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 65, 66, and 67, respectively. A method is provided. [Brief description of the drawings]

[0013] [Figure 1] Figure 1 shows a schematic representation of the expected mode of action of CD137xPD-L1 bispecific antibody. (A) PD-L1 is expressed on antigen presenting cells (APCs) as well as on tumor cells. PD-L1 binding to T cells expressing the negative regulatory molecule PD-1 effectively neutralizes T cell activation signals, ultimately resulting in T cell inhibition. (B) Upon addition of CD137xPD-L1 bispecific antibody, the inhibitory PD-1:PD-L1 interaction is blocked via the PD-L1 specific arm, and at the same time, the bispecific antibody provides agonistic signaling to CD137 expressed on T cells via cell-cell interactions, resulting in strong T cell co-stimulation. [Diagram 2]Figure 1 shows IL-2 production induced by GEN1046 in combination with pembrolizumab in an MLR assay with LPS-matured mDCs and purified CD8+ T cells. Purified CD8+ T cells were co-cultured with allogeneic mDCs for 5 days in the presence of GEN1046 (0.001-30 μg / mL), pembrolizumab (0.01-100 μg / mL), alone or in combination, in the presence of control antibodies, or in the absence of any antibody (no Tx). IL-2 secretion was analyzed by Luminex. Data shown are the mean IL-2 ± SD of duplicate wells. Each individual graph represents one of three donor pairs. [Diagram 3] Figure 1 shows IFNγ production induced by GEN1046 in combination with pembrolizumab in a mixed lymphocyte reaction (MLR) with LPS-matured dendritic cells (mDCs) and purified CD8+ T cells. Purified CD8+ T cells were co-cultured with allogeneic mDCs for 5 days in the presence of GEN1046 (0.001-30 μg / mL), pembrolizumab (0.01-100 μg / mL), alone or in combination, in the presence of control antibodies, or in the absence of any antibody (no Tx). IFNγ secretion was analyzed by ELISA. Data shown are the mean IFNγ ± standard deviation (SD) of duplicate wells. Each individual graph represents one of three DC / T cell donor pairs. [Figure 4] Figure 1 shows TNFα production induced by GEN1046 in combination with pembrolizumab in an MLR assay with LPS-matured mDCs and purified CD8+ T cells. Purified CD8+ T cells were co-cultured with allogeneic mDCs in the presence of GEN1046 (0.001-30 μg / mL), pembrolizumab (0.01-100 μg / mL), alone or in combination, in the presence of control antibodies, or in the absence of any antibody (no Tx) for 5 days. TNFα secretion was analyzed by Luminex. Data shown are mean TNFα ± SD of duplicate wells. Each individual graph represents one of three donor pairs. [Diagram 5]Figure 1: MC38 syngeneic tumor model established by subcutaneous inoculation of 1x106 MC38 cells into C57BL / 6 mice. When tumors reached a mean volume of 64mm3, mice were randomized and treated with mbsIgG2a-PD-L1x4-1BB (5mg / kg), anti-mouse PD-1 antibody (anti-mPD-1; 10mg / kg), or PBS (all 2QWx3), alone or in combination. A: Data shown are median tumor volumes per treatment group (n=10) with data carried forward for animals that reached termination criteria. Growth curves were terminated when <50% of animals within a treatment group remained alive (PBS, mbsIgG2a-PD-L1x4-1BB, anti-mPD-1) or by day 35 (mbsIgG2a-PD-L1x4-1BB in combination with anti-mPD-1). Arrows indicate treatment days. B: Progression-free survival, defined as the percentage of mice with tumor volume less than 500 mm3, is shown as a Kaplan-Meier curve. Mantel-Cox analysis was used to compare survival rates between treatment groups at day 45 (Table Y). [Figure 6]Figure 1 shows the analysis of proliferation dose-response curves for GEN1046 (DuoBody-PD-L1x4-1BB) and the anti-PD-1 antibody pembrolizumab in an antigen-specific T cell assay with an active PD1 / PD-L1 axis. Carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells electroporated with claudin-6 specific T cell receptor (TCR) in vitro translated (IVT) RNA and PD-1 IVT RNA were incubated with immature dendritic cells electroporated with claudin-6 IVT RNA in the presence of (A) GEN1046 (at 3-fold serial dilutions from 1 to 0.00015 μg / mL) or (B) pembrolizumab (at 4-fold serial dilutions from 0.8 to 0.00005 μg / mL) for 5 days. CD8+ T cell proliferation was measured by flow cytometry. Data shown are expansion indices calculated as a function of antibody concentration using FlowJo software v10.7.1. Error bars (SD) indicate intra-experimental variation (n=3 replicates in (A); n=2 duplicates in (B) using cells from one representative donor). Curves were fitted with a four-parameter logarithmic fit, and EC50 values ​​and Hill coefficients (shown in Tables 1 and 2) were determined using GraphPad Prism software v9.0. [Figure 7]Figure 1 shows PD-1 release / PD-L1 mediated T cell inhibition and further costimulation of CD8+ T cell proliferation by GEN1046 in the absence or presence of anti-PD-1 antibody pembrolizumab. CFSE-labeled T cells electroporated with claudin-6 specific TCR in vitro translated (IVT) RNA and PD-1 IVT RNA were incubated with claudin-6 IVT RNA electroporated immature dendritic cells in the presence of 0.2 μg / mL, 0.0067 μg / mL, or 0.0022 μg / mL GEN1046 combined with a fixed concentration of 0.8 μg / mL pembrolizumab or isotype control antibody IgG1-ctrl for 5 days (n=2 technical replicates per condition using cells from n=3 individual donors). Baseline proliferation in the absence of GEN1046 was determined using media alone, IgG1-ctrl at 0.8 μg / mL alone, and pembrolizumab at 0.8 μg / mL alone. CD8+ T cell proliferation was measured by flow cytometry. Bar graphs represent the mean ± SD for the expansion index for each indicated condition, calculated using FlowJo software v10.7.1. The dashed line represents baseline proliferation in the presence of the anti-PD-1 antibody pembrolizumab. [Figure 8] Schematic representation of a first-in-human, open-label, dose-escalation study with expansion cohorts to assess the safety of GEN1046 in subjects with malignant solid tumors. [Figure 9] Waterfall plot showing progression-free survival in subjects previously treated with checkpoint inhibitors (grey line) and checkpoint inhibitor-naïve patients (black line). [Figure 10] Figure 1 compares time from last prior anti-PD-(L)1 in subjects across the CPI-experienced expansion cohort (GEN1046 monotherapy) with clinical response (PR) versus subjects with stable disease (SD) or progressive disease (PD). Wilcoxon test was used to compare response groups. PR vs. PD: p=0.0017; PR vs. SD: p=0.034. [Figure 11] FIG. 1 shows predicted partial response (PR) / complete response (CR) rates for GEN1046 administered as 100mg 1Q3W or 1Q6W in combination with pembrolizumab in an integrated quantitative systems pharmacology (QSP) model. [Figure 12] Characterization of the exhaustion phenotype of CD3+ T cells after two rounds of CD3 / CD28 stimulation. (A) In vitro exhausted or naive T cells were stimulated with CD3 / CD28 beads. IFNγ secretion was analyzed by ELISA. Data shown are the mean + standard deviation (SD) for duplicate wells from one representative donor pair. (B) Expression of TIM3, LAG3, PD-1, and 4-1BB in naive and in vitro exhausted CD3+ T cells was determined by flow cytometry. Data shown are median fluorescence intensity (ΔMFI) corrected for background fluorescence. (C) Expression of Ki67 in naive and in vitro exhausted CD3+ T cells was determined by flow cytometry. [Figure 13] Figure 1 shows IFNγ secretion induced by GEN1046 in combination with pembrolizumab in a mixed lymphocyte reaction (MLR) between mature dendritic cells (mDC) and in vitro depleted CD3+ T cells (Tex). Tex were co-cultured with allogeneic LPS-matured DC (DC:T cell ratio 1:4) in the presence of GEN1046 (0.001-30 μg / mL) or pembrolizumab (1 μg / mL) alone or in combination for 5 days. Co-cultures without antibody treatment (no antibody) or treated with bsIgG1-PD-L1×ctrl (30 μg / mL), bsIgG1-ctrl×4-1BB (30 μg / mL), IgG4 isotype control (1 μg / mL), or IgG1-ctrl-FEAL (30 μg / mL) were included as controls. IFNγ secretion was analyzed by ELISA. Data shown are the mean + standard deviation (SD) for duplicate wells from one representative donor pair out of four tested. [Figure 14] Figure 13 shows the highest single agent (HSA) synergy score for the combination of GEN1046 with pembrolizumab in the MLR with mDC and Tex. Tex was co-cultured with allogeneic LPS-matured DC (DC:T cell ratio 1:4) in the presence of GEN1046 (0.001-30 μg / mL) or pembrolizumab (1 μg / mL) alone or in combination for 5 days. Data shown are HSA synergy scores for one representative donor pair (same donor as shown in Figure 13) out of four donor pairs tested. A score of >10 indicates synergy in this model. [Figure 15] Figure 1: MC38 colon cancer model established by subcutaneous inoculation of 1x106 MC38 cells into C57BL / 6 mice. When tumors reached a mean volume of 60mm3, mice were randomized and treated with the indicated antibodies or combinations (all 2QWx3). A: Data shown are median tumor volumes per treatment group (n=10) with data carried forward for animals that reached termination criteria. Growth curves were terminated when <50% of animals within a treatment group remained alive (mIgG2a-ctrl-AAKR, mbsIgG2a-PD-L1x4-1BB, anti-mouse PD-1 antibody [anti-mPD-1]) or by day 60 (mbsIgG2a-PD-L1x4-1BB in combination with anti-mPD-1). Downward pointing triangles indicate days of treatment. B: Progression-free survival, defined as the percentage of mice with tumor volume less than 500 mm3, is shown as a Kaplan-Meier curve. Mantel-Cox analysis was used to compare survival rates between treatment groups at day 69 (Table 19). [Figure 16] Figure 1 shows (re)challenge of mice with complete tumor regression in treated and control groups with tumor-naive mice. Mice were (re)challenged with 1x106 MC38 tumor cells injected subcutaneously 121 days after the start of antibody treatment. Data shown are mean tumor volumes ± SEM. [Figure 17]Figure 1 shows quantitative IHC / ISH data for cellular immune and tumor markers expressed in tumor tissue excised from an MC38 colon cancer model. C57BL / 6 mice were inoculated with 1x106 MC38 cells. When tumors reached an average volume of 50-70mm3, mice were randomized and treated with mbsIgG2a-PD-L1x4-1BB, anti-mPD-1, or a combination thereof. Tumors were excised on day 7 (n=5 per treatment group) or day 14 (n=5 per treatment group) after the start of treatment. As a result of some of the excised tumor samples being too small to perform IHC analysis, 4-5 tumors were analyzed per treatment group. Excised tumor sections (4 μm) were stained by immunohistochemistry (IHC) using anti-CD3, anti-CD4, anti-CD8, or anti-PD-L1 antibodies, or stained for 4-1BB or PD-L2 by in situ hybridization (ISH). Data from IHC are presented as % marker positive cells out of total cells counted in the slide, as well as mean ± SEM per treatment group. Data from ISH are presented as RNAscope H score per slide, as well as mean ± SEM per treatment group. [Figure 18] Figure 1 shows expression of GzmB and Ki67 in CD8 T cell subsets derived from dissociated tumor tissue from an MC38 colon cancer model. C57BL / 6 mice were inoculated with 1x106 MC38 cells. When tumors reached an average volume of 50-70mm3, mice were randomized and treated with mbsIgG2a-PD-L1x4-1BB, anti-mPD-1, or a combination thereof. Tumors were excised 7 days after the start of treatment (n=5 per treatment group), dissociated into single cell suspensions, and analyzed by flow cytometry. Data shown are the percentage of GzmB+ cells (A) or Ki67+ cells (B) within the CD8+ T cell population for individual mice and mean ± SEM per treatment group. Statistical analysis was performed by Mann-Whitney with *p<0.05 and **p<0.01 to compare the percentage of GzmB+ cells or Ki67+ cells within the CD8+ T cell population between treatment groups. [Figure 19]Figure 1 shows cytokine levels in peripheral blood for C57BL / 6 mice bearing MC38 tumors treated with mbsIgG2a-PD-L1x4-1BB, anti-mPD-1 antibodies, or the non-binding control antibody IgG2a-ctrl-AAKR as single agents or in combination. Peripheral blood samples were collected at baseline (1 day before treatment [day -1]; dotted line) and 2 days after each treatment (days 2 and 5). Cytokine analysis was performed by ECLIA.

[0014] [Table 1] TIFF2024538755000003.tif248165TIFF2024538755000004.tif248165TIFF2024538755000005.tif248165TIFF20245387550 00006.tif248165TIFF2024538755000007.tif249165TIFF2024538755000008.tif250162TIFF2024538755000009.tif101162 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present disclosure is described in detail below, but it is understood that the present disclosure is not limited to the specific methods, protocols, and reagents described herein, as they may vary. It is also understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0016] In the following, the elements of the present disclosure are described in more detail. Although these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments are not to be construed as limiting the present disclosure to only the embodiments explicitly described. The present description is to be understood as endorsing and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application is considered to be disclosed by the description of this application, unless the context indicates otherwise. For example, in a preferred embodiment of the binding agent used herein, the first heavy chain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR]; in another preferred embodiment of the binding agent used herein, the second heavy chain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL]; in a further preferred embodiment of the binding agent used herein, the first heavy chain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR]; the second heavy chain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL].

[0017] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms (IUPAC Recommendations)", edited by HGW Leuenberger, B. Nagel, and H. Kolbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995).

[0018] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, which are described in the art (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin, 1990; Streitwieser / Heathcook, Organische Chemie, VCH, 1990; Beyer / Walter, Lehrbuch der Organischen Chemie, S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, Organische Chemie, VCH, 1995; March, Advanced Organic Chemistry, John Wiley & Sons, 1985; Roempp, Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular (See, for example, "Cloning: A Laboratory Manual," 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

[0019] Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. The use of any and all examples or illustrative language (e.g., "such as") provided herein is intended only to better illustrate the disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0020] The recitation of ranges of values ​​herein is intended to be used only as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated into this specification as if it were individually recited herein.

[0021] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, journal publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. No citation herein shall be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0022] definition The following provide definitions applicable to all aspects of this disclosure. Unless otherwise indicated, the following terms have the following meanings: Any undefined terms have their art-recognized meaning.

[0023] Throughout the following specification and claims, unless the context requires otherwise, the term "comprise" and variations thereof, such as "comprises" and "comprising" shall be understood to imply the inclusion of a stated member, integer, or step, or group of members, integers, or steps, but not the exclusion of any other member, integer, or step, or group of members, integers, or steps. The term "consisting essentially of" means the exclusion of other members, integers, or steps of any essential significance. The term "comprising" includes the term "consisting essentially of" and the term "consisting essentially of" includes the term "consisting of". Thus, at each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of". Similarly, at each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of".

[0024] The terms "a," "an," and "the," and similar references as used in the context of describing this disclosure (especially in the context of the claims), are intended to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0025] As used herein, "and / or" shall be understood as a specific disclosure of each of the two specified features or components, with or without the other features or components. For example, as used herein, "X and / or Y" shall be understood as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, exactly as if each were individually set forth.

[0026] In the context of the present disclosure, the term "about" denotes an interval of accuracy that a person skilled in the art would understand to still ascertain the technical effect of the feature in question. The term typically denotes a deviation from the indicated numerical value of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, for example, a deviation of ±0.01%. As would be appreciated by a person skilled in the art, such a specific deviation for a numerical value for a given technical effect would depend on the nature of the technical effect. For example, a natural technical effect or a biological technical effect may generally result in such a deviation that is greater than the deviation for a technical effect that is artificial or manipulated.

[0027] The term "binding agent" in the context of the present disclosure refers to any agent capable of binding to a desired antigen. In certain embodiments of the present disclosure, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also include synthetic, modified, or non-naturally occurring moieties, in particular non-peptide moieties. Such moieties may, for example, link a desired antigen-binding functional group or antigen-binding region, such as an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or an antigen-binding variable region.

[0028] As used herein, "immune checkpoint" refers to regulators of the immune system, particularly costimulatory and inhibitory signals that regulate the amplitude and quality of antigen recognition by the T cell receptor. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction of PD-1 with PD-L1 and / or PD-L2. In certain embodiments, the inhibitory signal is the interaction of CTLA-4 with CD80 or CD86, which displaces binding of CD28. In certain embodiments, the inhibitory signal is the interaction of LAG-3 with an MHC class II molecule. In certain embodiments, the inhibitory signal is the interaction of TIM-3 with one or more of its ligands, such as galectin 9, PtdSer, HMGB1, and CEACAM1. In certain embodiments, the inhibitory signal is the interaction of one or more KIRs with their ligands. In certain embodiments, the inhibitory signal is the interaction of TIGIT with one or more of its ligands, PVR, PVRL2, and PVRL3. In certain embodiments, the inhibitory signal is the interaction of CD94 / NKG2A with HLA-E. In certain embodiments, the inhibitory signal is the interaction of VISTA with its binding partner(s). In certain embodiments, the inhibitory signal is the interaction of one or more Siglecs with their ligands. In certain embodiments, the inhibitory signal is the interaction of GARP with one or more of its ligands. In certain embodiments, the inhibitory signal is the interaction of CD47 with SIRPα. In certain embodiments, the inhibitory signal is the interaction of PVRIG with PVRL2. In certain embodiments, the inhibitory signal is the interaction of CSF1R with CSF1. In certain embodiments, the inhibitory signal is the interaction of BTLA with HVEM. In certain embodiments, the inhibitory signal is the interaction of A2AR and / or A2BR with adenosine, which is mediated by parts of the adenosinergic pathway, such as CD39 and CD73.In certain embodiments, the inhibitory signal is the interaction of B7-H3 with its receptor and / or the interaction of B7-H4 with its receptor, hi certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX, or TDO.

[0029] As used herein, the terms "checkpoint inhibitor" (CPI) and "immune checkpoint (ICP) inhibitor" are used interchangeably. The terms refer to molecules that inhibit immune checkpoints, in particular to molecules that inhibit the inhibitory signals of immune checkpoints, such as binding agents, that fully or partially reduce, inhibit, interfere with, or negatively modulate one or more checkpoint proteins, or that fully or partially reduce, inhibit, interfere with, or negatively modulate the expression of one or more checkpoint proteins, such as binding agents. In one embodiment, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to one or more molecules that modulate the checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins, for example, at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor in accordance with the present disclosure may be used. The term "partially" as used herein means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in levels, e.g., levels of inhibition of a checkpoint protein.

[0030] In one embodiment, a checkpoint inhibitor can be any compound, e.g., any binding agent, that inhibits the inhibitory signals of immune checkpoints, where the inhibitory signals are the interaction of PD-1 with PD-L1 and / or PD-L2; the interaction of CTLA-4 with CD80 or CD86, which displaces the binding of CD28; the interaction of LAG-3 with MHC class II molecules; the interaction of TIM-3 with one or more of its ligands, e.g., galectin 9, PtdSer, HMGB1, and CEACAM1; the interaction of one or more KIRs with their ligands; the interaction of TIGIT with one or more of its ligands, PVR, PVRL2, and PVRL3; CD94 / NKG2 interaction of A with HLA-E; interaction of VISTA with its binding partner(s); interaction of one or more Siglecs with their ligands; interaction of GARP with one or more of its ligands; interaction of CD47 with SIRPα; interaction of PVRIG with PVRL2; interaction of CSF1R with CSF1; interaction of BTLA with HVEM; interaction of A2AR and / or A2BR with adenosine, which is mediated by parts of the adenosinergic pathway, such as CD39 and CD73; interaction of B7-H3 with its receptor, and / or interaction of B7-H4 with its receptor; inhibitory signals mediated by IDO, CD20, NOX, or TDO. In one embodiment, the checkpoint inhibitor is at least one checkpoint inhibitor selected from the group consisting of PD-1 inhibitor, PD-L1 inhibitor, PD-L2 inhibitor, CTLA-4 inhibitor, TIM-3 inhibitor, KIR inhibitor, LAG-3 inhibitor, TIGIT inhibitor, VISTA inhibitor, and GARP inhibitor. In one embodiment, the checkpoint inhibitor can be a blocking antibody, such as a PD-1 blocking antibody, a CTLA4 blocking antibody, a PD-L1 blocking antibody, a PD-L2 blocking antibody, a TIM-3 blocking antibody, a KIR blocking antibody, a LAG-3 blocking antibody, a TIGIT blocking antibody, a VISTA blocking antibody, or a GARP blocking antibody. Examples of PD-1 blocking antibodies include pembrolizumab, nivolumab, cemiplimab, and spartalizumab.Examples of CTLA4 blocking antibodies include ipilimumab and tremelimumab. Examples of PD-L1 blocking antibodies include atezolizumab, durvalumab, and avelumab.

[0031] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antigen receptors, such as antibodies or B-cell receptors (BCR). Immunoglobulins are characterized by structural domains, i.e. immunoglobulin domains with a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also commonly referred to as surface or membrane immunoglobulins, which are part of the BCR. Soluble immunoglobulins are commonly referred to as antibodies.

[0032] The structure of immunoglobulins has been well characterized. See, for example, "Fundamental Immunology," Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains that are linked via disulfide bonds. These chains are primarily divided into immunoglobulin domains or regions, e.g., V L or VL (variable light chain) domain / region, C L or CL (constant light chain) domain / region, V H or a VH (variable heavy chain) domain / region, and H or a CH (constant heavy chain) domain / region, H 1(CH1), C H 2(CH2), C H 3(CH3), and C HThe heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain is typically composed of VL and CL. The light chain constant region is typically composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or configuration of structurally defined loops), also referred to as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). Each VH and each VL is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, J. Mol. Biol., 196, 901-917 (1987)). Unless otherwise stated or refuted by context, CDR sequences herein are identified according to the IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research, 1999, 27:209-212; and Ehrenmann F., Kaas Q., and Lefranc M.-P., Nucleic Acids Res., 38, D301-307 (2010); see also the internet http address: www.imgt.org.). Unless stated otherwise or refuted by context, references to amino acid positions within constant regions in this disclosure follow EU numbering (Edelman et al., Proc Natl Acad Sci USA. May 1969, 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, NIH Publication No. 91-3242).

[0033] There are five types of immunoglobulin heavy chains in mammals, namely α, δ, ε, γ and μ heavy chains, which correspond to the different classes of antibodies, namely IgA, IgD, IgE, IgG and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini. In mammals, there are two types of light chains, namely lambda light chains and kappa light chains. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within the different isotypes of immunoglobulins, whereas the variable part is highly diverse and is responsible for antigen recognition.

[0034] As used herein, the terms "amino acid" and "amino acid residue" are used interchangeably and are not to be understood as limiting. An amino acid is an amine (-NH ) with a side chain (R group) specific to each amino acid. 2 Amino acids are organic compounds that contain a cyclic (-C) functional group and a carboxyl (-COOH) functional group. In the context of the present disclosure, amino acids may be classified based on structural and chemical characteristics. Thus, the classes of amino acids may be reflected in one or both of the following tables:

[0035] [Table 2]

[0036] [Table 3]

[0037] For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant, and / or an amino acid substitution variant. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformational, isoform, allelic variants, species variants, and species homologues, particularly variants that are naturally occurring. The term "variant" particularly includes fragments of an amino acid sequence.

[0038] Amino acid insertion variants include the insertion of a single amino acid or two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertions with appropriate screening of the resulting products are also possible.

[0039] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

[0040] Amino acid deletion mutants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein. Amino acid deletion mutants, including deletions at the N-terminus and / or C-terminus of the protein, are also referred to as N-terminal truncation mutants and / or C-terminal truncation mutants.

[0041] Amino acid substitution variants are characterized by at least one residue being removed in the sequence and another being inserted in its place. Substitution of one amino acid by another can be classified as conservative or non-conservative substitution. Preference is given to modifications in positions that are not conserved between homologous proteins or peptides in the amino acid sequence and / or replacement of an amino acid by another amino acid with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitution of a similarly charged or similarly uncharged amino acid. Conservative amino acid changes involve the substitution of one of a family of amino acids that are related in their side chains. In the context of the present disclosure, a "conservative substitution" is the substitution of one amino acid by another amino acid with similar structural and / or chemical characteristics, e.g., the substitution of one amino acid residue by another amino acid residue of the same class as defined in either of the two tables above: for example, leucine can be substituted with isoleucine, since both are branched, hydrophobic residues that are aliphatic. Similarly, aspartic acid may be substituted with glutamic acid, since both are small, negatively charged residues. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions are made within the following groups: Glycine, Alanine; · valine, isoleucine, leucine; · Aspartic acid, glutamic acid; · Asparagine, glutamine; ·Serine, Threonine; Lysine, arginine; and Phenylalanine, Tyrosine Includes substitutions within.

[0042] The term "amino acid corresponding to position" and similar expressions as used herein refer to the number of the amino acid position in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is an amino acid or segment that is aligned with the other amino acid or segment using a standard sequence alignment program, e.g., ALIGN, ClustalW, or similar sequence alignment program, typically with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain. It is believed that it is well known in the art how to align sequences or segments within a sequence and thereby determine the positions within a sequence that correspond to the amino acid positions according to the present disclosure.

[0043] The term "antibody" (Ab) in the context of the present disclosure refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of these, that has the ability to specifically bind to an antigen (particularly an epitope on an antigen) under typical physiological conditions, preferably with a half-life spanning a significant period of time, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, etc., or any other relevant functionally defined period of time (e.g., a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with binding of the antibody to the antigen, and / or a time sufficient for the antibody to recruit effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the foregoing. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and each VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3). The CDRs of the VL are referred to as LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3). The variable regions of the heavy and light chains contain binding domains that interact with an antigen.The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from amino terminus to carboxy terminus in the following order: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, e.g., C1q. An antibody can be an intact immunoglobulin from a natural source, an intact immunoglobulin from a recombinant source, or an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. An antibody can be, for example, a polyclonal antibody, a monoclonal antibody, an Fv, an Fab, and an F(ab). 2 In addition, antibodies may exist in a variety of forms, including single chain antibodies and humanized antibodies.

[0044] The heavy and light chain variable regions of an immunoglobulin molecule contain binding domains that interact with antigens. As used herein, the terms "binding region" and "antigen-binding region" are used interchangeably and refer to regions that interact with antigens, including both VH and VL regions. Antibodies as used herein include not only monospecific antibodies, but also multispecific antibodies that contain multiple different antigen-binding regions, for example, two or more, for example, three or more different antigen-binding regions.

[0045] As indicated above, unless stated otherwise or clearly refuted by the context, the term "antibody" in this specification includes fragments of antibodies that are antigen-binding fragments, i.e., that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) Fab' or Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or monovalent antibodies (Genmab) described in WO2007 / 059782; (ii) F(ab') , which are bivalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge regions. 2(iii) an Fd fragment, consisting essentially of the VH and CH1 domains; (iv) an Fv fragment, consisting essentially of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment, consisting essentially of a VH domain (Ward et al., Nature, 341, 544-546 (1989)), also called a domain antibody (Holt et al., Trends Biotechnol. November 2003, 21(11):484-90); (vi) a camelid or Nanobody molecule (Revets et al., Expert Opin Biol Ther. January 2005, 5(1):111-24); and (vii) an isolated complementarity determining region (CDR). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined using recombinant methods with a synthetic linker that renders the VL and VH into a single protein chain, where the VL and VH regions pair to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs), see, for example, Bird et al., Science, 242, 423-426 (1988); and Huston et al., PNAS USA, 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed within the scope of the term antibody, unless otherwise noted or clearly indicated by the context. Although such fragments are generally encompassed within the meaning of antibody, collectively, each independently represents a unique feature of the present disclosure and exhibits different biological properties and utilities. These and other antibody fragments useful in the context of the present disclosure, as well as bispecific formats of such fragments, are further discussed herein. Unless otherwise specified, the term antibody will also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric and humanized antibodies, as well as antibody fragments that retain the ability to specifically bind to an antigen (antigen-binding fragments) produced by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0046] The antibodies produced may possess any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgD, IgA (e.g., IgA1, IgA2), IgE, IgM, or IgY) encoded by heavy chain constant region genes. As used herein, when a particular isotype, e.g., IgG1, is referred to, the term "isotype" is not limited to a specific isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to this isotype, e.g., IgG1, than to other isotypes. Thus, for example, the IgG1 antibodies disclosed herein may be sequence variants of naturally occurring IgG1 antibodies that contain mutations in the constant region.

[0047] IgG1 antibodies can exist in multiple polymorphic variants, termed allotypes (Jefferis and Lefranc, 2009, mAb, vol. 1, no. 4, pp. 1-7), any of which are suitable for use in some of the embodiments herein. Common allotypic variants within the human population are those designated a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody can comprise a heavy chain Fc region that comprises a human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0048] The term "multispecific antibody" in the context of the present disclosure refers to an antibody having at least two different antigen-binding regions defined by different antibody sequences. In some embodiments, the different antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding regions bind to different target antigens. In one embodiment, the multispecific antibody is a "bispecific antibody" or "bs". The multispecific antibody, e.g., bispecific antibody, can be an antibody of any format, including any of the bispecific or multispecific antibody formats described herein below.

[0049] The term "full length" when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of a particular isotype that are normally found for that isotype in nature, e.g., the VH domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, VL domain, and CL domain for an IgG1 antibody.

[0050] The term "human antibody" as used herein is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant regions. The human antibodies disclosed herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., insertions or deletions that are mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, e.g., a mouse, have been grafted onto human framework sequences.

[0051] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, e.g., from a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a term generally used for the modification of different types of antibodies, and the process for antibody engineering is well known to those skilled in the art. In particular, chimeric antibodies can be produced by using standard DNA methods as described in Sambrook et al., 1989, "Molecular Cloning: A laboratory Manual", New York: Cold Spring Harbor Laboratory Press, Chapter 15. Thus, chimeric antibodies can be genetically engineered or enzymatically engineered recombinant antibodies. Since the production of chimeric antibodies is within the knowledge of those skilled in the art, the production of chimeric antibodies can be performed by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic applications in humans are developed to reduce the immunogenicity of the antibody that is expected for non-human antibodies, e.g., rodent antibodies. A chimeric monoclonal antibody typically contains a non-human (e.g., mouse or rabbit) variable region specific for an antigen of interest, and may contain human antibody heavy and light chain constant domains. The term "variable region" or "variable domain" as used in the context of a chimeric antibody refers to the regions that contain the CDRs and framework regions of both the heavy and light immunoglobulin chains, as described below.

[0052] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs), which together form the antigen-binding site, into the homologous human acceptor framework regions (FRs) (see WO92 / 22653 and EP0629240). To fully reconstitute the binding affinity and specificity of the parent antibody, replacement (backmutation) of the human framework regions with framework residues from the parent antibody (i.e., the non-human antibody) may be required. Structural homology modeling can help identify amino acid residues within the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain primarily human framework regions, including non-human CDR sequences, optionally one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications, not necessarily back mutations, can be applied to obtain a humanized antibody with favorable characteristics, e.g., affinity and biochemical properties.

[0053] As used herein, a protein "derived from" another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical or similar to one or more amino acid sequences in the other protein or in the parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, etc. derived from another antibody, binding arm, antigen-binding region, constant region, or parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to those of the other antibody, binding arm, antigen-binding region, or constant region, or of the parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, the amino acid sequences of the VH CDRs and VL CDRs, and / or one or more or all of the framework regions, VH regions, VL regions, CL regions, hinge regions, or CH regions. For example, a humanized antibody may be described herein as "derived from" a non-human parent antibody, meaning that at least the VL and VH CDR sequences are identical or similar to the VH and VL CDR sequences of said non-human parent antibody. A chimeric antibody may be described herein as "derived from" a non-human parent antibody, meaning that typically the VH and VL sequences may be identical or similar to the VH and VL sequences of said non-human parent antibody. Another example is a binding arm or antigen-binding region that may be described herein as "derived from" a particular parent antibody, meaning that said binding arm or antigen-binding region typically comprises VH and / or VL CDRs, or VH and / or VL sequences that are identical or similar to the binding arm or antigen-binding region of said parent antibody. However, as described elsewhere herein, amino acid modifications, e.g., mutations, may be made within the CDRs, within the constant regions, or elsewhere within the antibody, binding arms, antigen-binding regions, etc., to introduce desired characteristics.When used in the context of one or more sequences derived from a first or parent protein, a "similar" amino acid sequence preferably has at least about 50%, e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, 98%, or 99% sequence identity.

[0054] Non-human antibodies can be generated in a number of different species, including mice, rabbits, chickens, guinea pigs, llamas, and goats.

[0055] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody techniques, such as the standard somatic cell hybridization method according to Kohler and Milstein, Nature, 256:495 (1975). Other techniques for preparing monoclonal antibodies can be employed, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of antibody genes, and such methods are well known to those skilled in the art.

[0056] The production of hybridomas in such non-human species is a well-established procedure. Immunization protocols and methods for isolating spleen cells of immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0057] As used herein, unless otherwise indicated by context, the term "Fab arm" or "arm" refers to one heavy-light chain pair and is used interchangeably herein with "half molecule."

[0058] The term "binding arm comprising an antigen-binding region" refers to an antibody molecule or antibody fragment comprising an antigen-binding region. Thus, a binding arm can comprise, for example, six VH and VL CDR sequences, a VH and VL sequence, a Fab or Fab' fragment, or a Fab arm.

[0059] As used herein, unless otherwise indicated by context, the term "Fc region" refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the term "Fc region" as used herein refers to a region including at least a hinge region, a CH2 domain, and a CH3 domain in the N-terminal to C-terminal orientation of an antibody. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0060] In the context of the present disclosure, the term "induces Fc-mediated effector function to a lesser extent", as used in relation to antibodies, including multispecific antibodies, means that the antibody induces an Fc-mediated effector function, in particular selected from the list of binding to IgG Fc receptors (Fc gamma R, FcγR), binding to C1q, ADCC or CDC, to a lesser extent compared to a human IgG1 antibody that (i) comprises in particular the same CDR sequences, including the same first and second antigen-binding regions as said antibody, and (ii) two heavy chains comprising the hinge, CH2 and CH3 regions of human IgG1.

[0061] Fc-mediated effector function can be measured by binding to FcγR, binding to C1q, or induction of Fc-mediated cross-linking through FcγR.

[0062] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering set forth in Kabat (Kabat, EA et al., "Sequences of proteins of immunological interest", 5th ed.: US Department of Health and Human Services, NIH Publication No. 91-3242, pp. 662, 680, 689 (1991)). However, the hinge region can also be of any of the other subtypes described herein.

[0063] The term "CH1 region" or "CH1 domain" as used herein refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the EU numbering set forth in Kabat (ibid.). However, the CH1 region can also be of any of the other subtypes described herein.

[0064] The term "CH2 region" or "CH2 domain" as used herein refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering set forth in Kabat (ibid.). However, the CH2 region can also be of any of the other subtypes described herein.

[0065] The term "CH3 region" or "CH3 domain" as used herein refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering set forth in Kabat (ibid.). However, the CH3 region can also be of any of the other subtypes described herein.

[0066] In the context of the present disclosure, the term "monovalent antibody" means that the antibody molecule is capable of binding to a single antigen molecule and is therefore not capable of cross-linking antigens.

[0067] A "CD137 antibody" or "anti-CD137 antibody" is an antibody as described above that specifically binds to the antigen CD137.

[0068] A "CD137xPD-L1 antibody" or "anti-CD137xPD-L1 antibody" is a bispecific antibody that contains two different antigen-binding domains, one of which specifically binds to the antigen CD137, and the other of which specifically binds to the antigen PD-L1.

[0069] As used herein, the term "biosimilar" (e.g., an approved reference drug / biologic) refers to a biologic that is similar to the reference drug based on data from: (a) analytical studies that support that the biologic is highly similar to the reference drug, despite minor differences in clinically inactive ingredients; (b) animal studies (including evaluation of toxicity); and / or (c) one or more clinical studies (including evaluation of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to support safety, purity, and potency under one or more appropriate conditions of use for which the reference drug is approved and intended for use and for which approval is sought (e.g., approval that there are no clinically significant differences between the biologic and the reference drug in terms of drug safety, purity, and potency). In some embodiments, the biosimilar biologic and the reference drug utilize one or more of the same mechanisms of action for one or more conditions of use indicated, recommended, or suggested in the proposed labeling, but only to the extent that one or more mechanisms of action are known for the reference drug. In some embodiments, one or more conditions of use indicated, recommended, or suggested in the labeling proposed for the biopharmaceutical have already been approved for the reference pharmaceutical product. In some embodiments, the route of administration, dosage form, and / or strength of the biopharmaceutical is the same as the route of administration, dosage form, and / or strength of the reference pharmaceutical product. A biosimilar can be, for example, a currently known antibody that has the same primary amino acid sequence as a commercially available antibody, but may be produced in a different cell type or by a different production, purification, or formulation method.

[0070] The terms "binding to" or "capable of binding to" as used herein in the context of antibody binding to a given antigen or epitope typically refer to binding to an antigen or epitope that is greater than or equal to about 10% of the total antigen or epitope as determined using biolayer interferometry (BLI) or surface plasmon resonance (SPR) technology, e.g., in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10-10 M or less, or about 10 -11 M or less K D An antibody will bind with an affinity corresponding to its K for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D K that is 1 / 10 or less, for example, 1 / 100 or less, for example, 1 / 1,000 or less, for example, 1 / 10,000 or less, for example, 1 / 100,000 or less D The amount of higher affinity is the antibody's K D As a result, the K D is very low (i.e., the antibody is highly specific), the degree to which the affinity for the antigen is lower than the affinity for a nonspecific antigen can be at least 10,000-fold.

[0071] As used herein, "k d ”(seconds -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called the value.

[0072] As used herein, "K D The term "" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0073] Two antibodies have the "same specificity" if they bind to the same antigen and to the same epitope. Whether a test antibody recognizes the same epitope as a particular antigen-binding antibody, i.e., whether the antibody binds to the same epitope, can be examined by different methods well known to those skilled in the art.

[0074] Competition between antibodies can be detected by cross-blocking assay. For example, competitive ELISA assay can be used as cross-blocking assay. For example, target antigen can be coated on the well of a microtiter plate, and antigen-binding antibody and candidate competing test antibody can be added. The amount of antigen-binding antibody binding to the antigen in the well indirectly correlates with the binding ability of the candidate competing test antibody that competes with it for binding to the same epitope. Specifically, the greater the affinity of the candidate competing test antibody for the same epitope, the smaller the amount of antigen-binding antibody that binds to the well coated with the antigen. The amount of antigen-binding antibody that binds to the well can be measured by labeling the antibody with a detection labeling substance or a measurement labeling substance.

[0075] An antibody that competes with another antibody, e.g., an antibody comprising a heavy chain variable region and a light chain variable region described herein, for binding to an antigen, or has the specificity for an antigen of another antibody, e.g., an antibody comprising a heavy chain variable region and a light chain variable region described herein, may be a variant of the heavy chain variable region and / or light chain variable region described herein, e.g., an antibody comprising modifications and / or a certain degree of identity within the CDRs, as described herein.

[0076] As used herein, an "isolated multispecific antibody" is intended to refer to a multispecific antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds CD137 and PD-L1 is substantially free of monospecific antibodies that specifically bind to CD137 or PD-L1).

[0077] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0078] As used herein, the term "heterodimeric interaction between a first CH3 region and a second CH3 region" refers to the interaction between a first CH3 region and a second CH3 region within a first CH3 / second CH3 heterodimeric antibody.

[0079] As used herein, the term "homodimeric interaction between a first CH3 region and a second CH3 region" refers to an interaction between a first CH3 region and another first CH3 region in a first CH3 / first CH3 homodimeric antibody, and an interaction between a second CH3 region and another second CH3 region in a second CH3 / second CH3 homodimeric antibody.

[0080] As used herein, the term "homodimeric antibody" refers to an antibody that comprises two first Fab arms or half molecules, where the amino acid sequences of said Fab arms or half molecules are the same.

[0081] As used herein, the term "heterodimeric antibody" refers to an antibody comprising a first Fab arm or half molecule and a second Fab arm or half molecule, where the amino acid sequences of said first Fab arm or half molecule are different from the amino acid sequences of the second Fab arm or half molecule, in particular the CH3 region, or antigen binding region, or CH3 region and antigen binding region of said first Fab arm / half molecule are different from the CH3 region, or antigen binding region, or CH3 region and antigen binding region of the second Fab arm / half molecule.

[0082] The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, for example, a cysteine ​​residue in the hinge region of an antibody, is more likely to be reduced than oxidized.

[0083] The present disclosure also describes multispecific antibodies, e.g., bispecific antibodies, that comprise functional variants of the VL region, VH region, or one or more CDRs of the example bispecific antibodies. A functional variant of a VL, VH, or CDR used in the context of a bispecific antibody still allows each antigen-binding region of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or specificity / selectivity of the parent bispecific antibody, and in some cases such bispecific antibodies may be associated with greater affinity, selectivity, and / or specificity than the parent bispecific antibody.

[0084] Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences when considering the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm by E. Meyers and W. Miller, Comput. Appl. Biosci, 4, 11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a gap length penalty of 12, a gap penalty of 4, and a PAM120 weighted residue table. In addition, the percent identity between two amino acid sequences can also be determined using the algorithm by Needleman and Wunsch, J. Mol. Biol., 48, 444-453 (1970).

[0085] In the context of the present disclosure, unless otherwise indicated, the following notation is used to describe mutations: i) substitution of an amino acid at a given position is written, for example, K409R, meaning substitution of lysine at position 409 of the protein with arginine; ii) for specific variants, a specific three-letter or one-letter code is used, with Xaa and X being codes to indicate any amino acid residue. Thus, substitution of lysine at position 409 with arginine is written as K409R, and substitution of lysine at position 409 with any amino acid residue is written as K409X. Deletion of lysine at position 409 is written as K409R. * It is pointed out by.

[0086] Exemplary variants include variants that differ from the VH and / or VL and / or CDRs of a parent sequence primarily by conservative substitutions; for example, at least 12, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions in the variant are conservative amino acid residue substitutions.

[0087] In the context of the present disclosure, conservative substitutions may be defined by substitutions within the classes of amino acids defined in Tables 2 and 3.

[0088] The term "CD137" as used herein refers to CD137(4-1BB), also referred to as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is a receptor for the ligand TNFSF9 / 4-1BBL. CD137(4-1BB) is thought to be involved in T cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDw137, T cell antigen 4-1BB homolog, and T cell antigen ILA. In one embodiment, CD137(4-1BB) is human CD137(4-1BB) having UniProt accession number: Q07011. The sequence of human CD137 is also shown in SEQ ID NO:37. Amino acids 1 to 23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; whereas amino acids 24 to 186 of SEQ ID NO: 37 correspond to the extracellular domain of human CD137; the remainder of the protein, i.e., amino acids 187 to 213 and 214 to 255 of SEQ ID NO: 37, are the transmembrane and cytoplasmic domains, respectively.

[0089] "Programmed Death-1 (PD-1)" receptor refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 (also known as CD279) is expressed primarily on T cells that have already been activated in vivo and binds to two ligands, PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs that share at least one common epitope with hPD-1. The sequence of human PD-1 is also shown in SEQ ID NO: 39. "Programmed Death Ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other ligand is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1.

[0090] As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, such as PD-L1 from macaque monkeys (cynomolgus monkeys), African elephant, wild boar, and mouse (see, for example, Genbank Accession Nos. NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), and analogs that share at least one shared epitope with hPD-L1. The sequence of human PD-L1 is also shown in SEQ ID NO:40, in which amino acids 1-18 are predicted to be a signal peptide. The term "PD-L2" as used herein includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, as well as analogs that share at least one common epitope with hPD-L2. Ligands for PD-1 (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation. Cancer cells that express PD-L1 and / or PD-L2 can switch off T cells that express PD-1, resulting in suppression of anti-cancer immune responses. Interaction of PD-1 with its ligands results in a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. Immunosuppression is reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is also blocked.

[0091] The term "dysfunctional" as used herein refers to immune cells in a state of reduced immune responsiveness to antigenic stimulation. Dysfunctionality includes non-responsiveness to antigen recognition and impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, production of cytokines (e.g., IL-2), and / or killing of target cells.

[0092] The term "anergy" as used herein refers to a state of non-responsiveness to antigenic stimulation resulting from defective or insufficient signals delivered via the T cell receptor (TCR). T cell anergy can also occur upon stimulation with an antigen in the absence of costimulation, resulting in cells being refractory to subsequent activation by antigen even in the context of costimulation. The non-responsive state can often be abrogated by the presence of IL-2. Anergic T cells do not undergo clonal expansion and do not acquire effector function / do not undergo clonal expansion or acquire effector function.

[0093] The term "exhaustion" as used herein refers to immune cell exhaustion, e.g., T cell exhaustion as a state of T cell dysfunction resulting from persistent TCR signaling, which occurs in many chronic infections and cancers. "Exhaustion" is distinct from anergy in that it does not result from incomplete or defective signaling, but from persistent signaling. Exhaustion is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that is significantly different from that of functional effector or memory T cells. Exhaustion prevents optimal control of disease (e.g., infections and tumors). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) and cell-intrinsic negative regulatory pathways (e.g., inhibitory immune checkpoint pathways, as described herein).

[0094] "Enhancing T cell function" means inducing, directing, or stimulating T cells to have a sustained or amplified biological function, or regenerating or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include increased secretion of gamma interferon from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor elimination) compared to such levels before the intervention. In some embodiments, the level of enhancement is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or more. Methods for measuring this enhancement are known to those of skill in the art.

[0095] As used herein, the term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" refers to a nucleic acid molecule, e.g., DNA or RNA, that fully or partially attenuates, inhibits, interferes with, or negatively modulates one or more PD-1 proteins. Inhibitory nucleic acid molecules include, but are not limited to, oligonucleotides, siRNAs, shRNAs, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

[0096] The term "oligonucleotide" as used herein refers to a nucleic acid molecule capable of reducing protein expression, in particular the expression of a PD-1 protein, e.g., a PD-1 protein as described herein. An oligonucleotide is typically a short DNA or RNA molecule comprising 2-50 nucleotides. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide that is a PD-1 inhibitor may be an antisense oligonucleotide.

[0097] Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, particularly the nucleic acid sequence of the PD-1 protein (or a fragment thereof). Antisense RNA is typically used to bind to mRNA, e.g., the mRNA encoding the PD-1 protein, thereby preventing the translation of said mRNA into protein. Antisense DNA is typically used to target a specific complementary (coding or non-coding) RNA. Upon binding, such DNA / RNA hybrids can be degraded by the enzyme RNase H. Additionally, morpholino antisense oligonucleotides can also be used for gene knockdown in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed a B7-H4-specific morpholino that specifically blocked expression of B7-H4 in macrophages, resulting in increased T cell proliferation and reduced tumor volume in mice with tumor-associated antigen (TAA)-specific T cells.

[0098] As used herein, the terms "siRNA" or "small interfering RNA" or "small inhibitory RNA" are used interchangeably and refer to double-stranded RNA molecules, typically 20-25 base pairs in length, that interfere with the expression of a specific gene, e.g., the gene encoding the PD-1 protein, by virtue of a complementary nucleotide sequence. In one embodiment, the siRNA interferes with mRNA, thereby blocking translation, e.g., translation of the PD-1 protein. Transfection of exogenous siRNA can be used for gene knockdown, but the effect may be only a transient effect, especially in rapidly dividing cells. Stable transfection may be achieved, for example, by modification of the RNA or by using an expression vector. Modifications and vectors useful for stable transfection of cells with siRNA are known in the art. siRNA sequences may also be modified to introduce a short loop between the two strands, resulting in a "small hairpin RNA" or "shRNA". The shRNA may be processed by Dicer into functional siRNA. shRNAs have a relatively low rate of degradation and turnover. Therefore, the PD-1 inhibitor can be an shRNA.

[0099] The term "aptamer" as used herein refers to a single-stranded nucleic acid molecule, e.g., DNA or RNA, typically 25-70 nucleotides in length, capable of binding to a target molecule, e.g., a polypeptide. In one embodiment, an aptamer binds to a PD-1 protein, e.g., a PD-1 protein described herein. For example, an aptamer according to the present disclosure may specifically bind to a PD-1 protein or polypeptide, or a molecule in a signaling pathway that modulates expression of a PD-1 protein or polypeptide. The generation and therapeutic use of aptamers is well known in the art (see, e.g., US 5,475,096).

[0100] As used herein, the term "small molecule inhibitor" or "small molecule" is used interchangeably and refers to a low molecular weight organic compound, usually up to 1000 Daltons, that completely or partially reduces, inhibits, interferes with, or negatively modulates one or more of the above-mentioned PD-1 proteins.Such small molecule inhibitors are usually synthesized by organic chemical reactions, but can also be isolated from natural sources, such as plants, fungi, and microorganisms.The low molecular weight allows small molecule inhibitors to diffuse rapidly across cell membranes.For example, various A2AR antagonists known in the art are organic compounds with molecular weights below 500 Daltons.

[0101] The term "cell-based therapy" refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells, or stem cells) expressing a PD-1 inhibitor into a subject for the purpose of treating a disease or disorder (e.g., a cancerous disease).

[0102] The term "oncolytic virus" as used herein refers to a virus that can selectively replicate in cancerous or hyperproliferative cells in vitro or in vivo and slow their growth or induce their death, while having no or minimal effect on normal cells. Oncolytic viruses for delivery of PD-1 inhibitors include an expression cassette that can encode an inhibitory nucleic acid molecule, such as a PD-1 inhibitor that is an siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or a soluble PD-1 protein or fusion. The oncolytic virus is preferably replication competent, and the expression cassette is under the control of a viral promoter, such as a synthetic early / late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picornaviruses, e.g., Seneca Valley virus; SVV-001), coxsackieviruses, parvoviruses, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza viruses), measles viruses, reoviruses, Sindbis viruses, vaccinia viruses (including Copenhagen strains, Western Reserve strains, and Wyeth strains) as exemplarily described in WO2017 / 209053, and adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF). Methods for making recombinant oncolytic viruses containing soluble forms of PD-1 inhibitors and their use are disclosed in WO2018 / 022831, which is incorporated by reference in its entirety. Oncolytic viruses can be used as attenuated viruses.

[0103] As used herein, a "treatment cycle" is defined as the time within the additive effect of individual doses of a binding agent due to the pharmacodynamics of the binding agent, or in other words, the time after the administered binding agent has essentially been cleared from the subject's systemic body. Administration of multiple small doses within a small time window, e.g., within a few hours, e.g., within 2-24 hours, e.g., within 2-12 hours, or on the same day, can be equivalent to a single administration of a high dose.

[0104] In the present context, the terms "treatment", "treating" or "therapeutic intervention" refer to the management and care of a subject with the aim of combating a condition, e.g., a disease or disorder. These terms are intended to include the full spectrum of treatments for a given condition suffered by a subject, e.g., the administration of therapeutically effective compounds to alleviate symptoms or complications, delay the progression of a disease, disorder or condition, relieve or alleviate symptoms and complications, and / or cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is understood as the management and care of an individual with the aim of combating a disease, condition or disorder, and prevents the onset of symptoms or complications. In one embodiment, "treatment" refers to the administration of an effective amount of a therapeutically active binding agent, e.g., a therapeutically active antibody of the present disclosure, with the aim of alleviating, ameliorating, arresting or eradicating (curing) a symptom or disease state.

[0105] Response to treatment with the binding agents of the present disclosure, as well as resistance to, non-response to, and / or future recurrence, may be determined according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST criteria v1.1). The RECIST criteria are shown in the table below (LD: maximum width).

[0106] [Table 4]

[0107] "Best overall response" is the best response recorded from the start of treatment until disease progression / relapse (the minimum measurement recorded since treatment began shall be used as the reference for PD). Subjects with CR or PR are considered to be objective responders. Subjects with CR, PR, or SD are considered to be in disease control. Subjects with NE are counted as non-responders. "Best overall response" is the best response recorded from the start of treatment until disease progression / relapse (the minimum measurement recorded since treatment began shall be used as the reference for PD). Subjects with CR, PR, or SD are considered to be in disease control. Subjects with NE are counted as non-responders.

[0108] "Duration of response (DOR)" applies only to subjects with a confirmed best overall response of CR or PR and is defined as the time from the time of first documentation of an objective tumor response (CR or PR) to the date of first PD or death from the underlying cancer.

[0109] "Progression-free survival (PFS)" is defined as the number of days from day 1 in cycle 1 to first recorded progression or death from any cause.

[0110] "Overall survival (OS)" is defined as the number of days from day 1 to death from any cause in cycle 1. If the subject is not known to have died, OS will be censored at the last date the subject was known to be alive (prior to the cutoff date).

[0111] In the context of this disclosure, the term "treatment regimen" refers to a structured treatment plan designed to improve and maintain health.

[0112] The term "effective amount" or "therapeutically effective amount" refers to an effective amount, at a dosage required for a period of time necessary to achieve a desired therapeutic result. The therapeutically effective amount of a binding agent, for example, an antibody, such as a multispecific antibody or a monoclonal antibody, can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding agent to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the binding agent or fragment thereof are outweighed by the therapeutically beneficial effects. Should an initial dose result in an insufficient response in the patient, a higher dose (or an effective higher dose achieved by a different, more localized route of administration) can be used. Should a dose result in undesirable side effects in the patient, a lower dose (or an effective lower dose achieved by a different, more localized route of administration) can be used.

[0113] As used herein, the term "cancer" includes diseases characterized by dysregulation of cell proliferation, growth, differentiation, adhesion, and / or migration. "Cancer cells" refers to abnormal cells that proliferate by rapid, uncontrolled cell proliferation and continue to proliferate even after the stimuli that triggered the new proliferation have ceased.

[0114] The term "cancer" according to the present disclosure also includes cancer metastasis. "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that depends on the dissociation of malignant cells from the primary tumor, infiltration into the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then infiltration into the target organ after being carried by the blood. Finally, the growth of new tumors at the target site, i.e., secondary or metastatic tumors, depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, since tumor cells or tumor components are still present and may develop metastatic potential. In one embodiment, the term "metastasis" according to the present disclosure relates to "distant metastasis", which refers to metastasis far from the primary tumor and regional lymph node system.

[0115] As used herein, for example, the terms "reduce," "inhibit," "interfere with," and "negatively modulate" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete inhibition, or essentially complete inhibition, i.e., a reduction to zero, or a reduction to essentially zero.

[0116] In one embodiment, for example, the terms "increase" or "enhance" refer to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0117] As used herein, "physiological pH" refers to a pH of 7.5 or about 7.5.

[0118] As used in this disclosure, "wt. %" refers to weight percent, which is a concentration unit measuring the amount of a substance in grams (g) expressed as a percentage of the total weight of the entire composition in grams (g).

[0119] The term "TPS" or "tumor proportion score" refers to the percentage of tumor cells that express PD-L1 on the cell membrane. TPS includes the percentage of neoplastic cells that express PD-L1 at any strength (weak, moderate, or strong), which can typically be determined using an immunohistochemical assay using a diagnostic anti-human PD-L1 mAb, such as antibody 20C3 and antibody 22C3 described in WO2014 / 100079. If membrane staining is present, cells are considered to express PD-L1, including cells with partial membrane staining.

[0120] The term "freezing" refers to the solidification of a liquid, usually with the removal of heat.

[0121] The term "freeze-drying" or "freeze-drying" refers to freeze-drying a material by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, e.g., below 10 Pa, below 5 Pa, or below 1 Pa) to allow the frozen medium in the material to sublime directly from the solid phase to the gas phase. Thus, the terms "freeze-drying" and "freeze-drying" are used interchangeably herein.

[0122] In the context of this disclosure, the term "recombinant" means "created through genetic engineering." In one embodiment, in the context of this disclosure, a "recombinant subject" is not a naturally occurring subject.

[0123] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "naturally found" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0124] In accordance with the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance that contains about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The term "protein" refers to larger peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are typically used synonymously herein.

[0125] A "therapeutic protein" when administered to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or disease state. In one embodiment, a therapeutic protein has curative or palliative properties and may be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides, and also refers to therapeutically active fragments thereof. The term "therapeutic protein" may also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.

[0126] The term "portion" refers to a small portion. With respect to a particular structure, e.g., an amino acid sequence or a protein, the term "portion" can refer to a contiguous or non-contiguous portion of said structure.

[0127] As used herein, the terms "portion" and "fragment" are used interchangeably and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or a protein, refers to a continuous element of said structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion of the composition that is between 0.1% and 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0128] A "fragment" when referring to an amino acid sequence (peptide or protein) relates to a part of the amino acid sequence, i.e. a sequence that represents an amino acid sequence truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 3'-end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 5'-end of the open reading frame, insofar as the truncated open reading frame contains the initiation codon used to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues derived from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids derived from the amino acid sequence.

[0129] According to the present disclosure, a portion or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective binding to nucleic acids. For example, a pharmacologically active fragment of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which it is derived. A portion or fragment of a peptide or protein preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30, or up to 55 consecutive amino acids of the peptide or protein.

[0130] As used herein, the term "mutant (variant)" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a naturally occurring amino acid sequence or a wild-type (WT) amino acid sequence, or may be a modified form of the wild-type amino acid sequence. Preferably, the mutant amino acid sequence has at least one amino acid modification, for example, 1 to about 20 amino acid modifications, compared to the parent amino acid sequence, and preferably has 1 to about 10 or 1 to about 5 amino acid modifications, compared to the parent amino acid sequence.

[0131] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variants. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally altered.

[0132] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, for consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be done by tools known in the art, for example, using best sequence alignment, using Align, with standard settings, preferably EMBOSS::Needle, using matrix: Blosum62, Gap Open: 10.0, Gap Extend: 0.5.

[0133] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of identical nucleotides between the sequences.

[0134] The terms "% identical" and "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. Said percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences being compared. The comparison of two sequences is usually carried out by comparing the sequences over segments or "comparison regions" in order to identify local regions of corresponding sequences after optimal alignment. Optimal alignment for comparison may be performed manually, with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math., 2, 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol., 48, 443, with the aid of the search for similarity algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA, 88, 2444, or with the aid of computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available from the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include (i) an Expect Threshold set to 10; (ii) a Word Size set to 28; (iii) Max matches in query range set to 0; (iv) Match / Mismatch Scores set to 1,-2; (v) Gap Costs set to Linear; and (vi) use of a filter for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include (i) an Expect Threshold set to 10; (ii) a Word Size set to 3; (iii) Max matches in query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence:11, Extension:1; and (vi) a conditional composite score matrix correction.

[0135] The percentage of identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence) and multiplying the result by 100.

[0136] In some embodiments, the degree of similarity or identity is given over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is given over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acid residues, in some embodiments, contiguous amino acid residues. In some embodiments, the degree of similarity or identity is given over the entire length of the reference sequence.

[0137] Homologous amino acid sequences, according to the present disclosure, exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% identity of the amino acid residues, preferably at least 95%, at least 98% or at least 99% identity.

[0138] The amino acid sequence variants described herein can be readily prepared by one skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptide variants and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, by solid phase synthesis and similar methods.

[0139] In one embodiment, the fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties that are identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein refers in particular to a variant molecule or variant sequence that comprises an amino acid sequence that has been altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, but is still capable of performing one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect or change the characteristics of the molecule or sequence. In different embodiments, the functionality of the functional fragment or functional variant may be reduced, but still significantly present, for example the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however in other embodiments the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0140] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a named amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be altered such that their sequence varies from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0141] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or isolated protein may exist in a substantially purified form or may exist in a non-native environment, e.g., in a host cell. In a preferred embodiment, the binding agent used in the present disclosure is in a substantially purified form.

[0142] The term "genetic modification" or simply "modification" includes the transfection of a nucleic acid into a cell. The term "transfection" relates to the introduction of a nucleic acid, particularly RNA, into a cell. For the purposes of this disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell, where the cell may be present in a subject, e.g., a patient. Thus, in accordance with the present disclosure, the cells for transfection of a nucleic acid as described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and / or organism of a patient. In accordance with the present disclosure, the transfection may be a transient or stable transfection. For some applications of transfection, it is sufficient that the genetic material to be transfected is only transiently expressed. RNA can be transfected into a cell to transiently express its encoded protein. Since the nucleic acid introduced in the transfection process is not usually integrated into the nuclear genome, the foreign nucleic acid will be diluted or degraded through mitosis. Cells that allow for episomal amplification of nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually persists in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection. In general, the nucleic acid encoding the antigen is transiently transfected into the cell. RNA can be transfected into the cell to transiently express its encoded protein.

[0143] According to the present disclosure, an analog of a peptide or protein is a modified form of said peptide or protein from which it is derived, and has at least one functional property of said peptide or protein. For example, a pharmacologically active analog of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which it is derived. Such modifications include any chemical modification, including one or more substitutions, deletions, and / or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids, and / or proteins or peptides. In one embodiment, an "analog" of a protein or peptide includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of a protecting / blocking group, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also extends to all functional chemical equivalents of said proteins and peptides.

[0144] As used herein, "activation" or "stimulation" refers to the state of an immune effector cell, e.g., a T cell, that is sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the induction of signal transduction pathways, induction of cytokine production, and detectable effector functions. The term "activated immune effector cell" refers, inter alia, to an immune effector cell that is undergoing cell division.

[0145] The term "priming" refers to the process by which an immune effector cell, e.g., a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, e.g., an effector T cell.

[0146] The term "clonal expansion" or "expansion" refers to a process in which a specific entity is multiplied. In the context of the present disclosure, the term "expansion" is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize said antigen are amplified. Preferably, clonal expansion results in differentiation of immune effector cells.

[0147] An "antigen" according to the present disclosure is intended to be any substance that induces an immune response and / or any substance against which an immune response or immune mechanism, e.g. a cellular response, is directed. It also includes the situation in which an antigen is processed into an antigenic peptide and an immune response or immune mechanism is directed against one or more antigenic peptides, particularly when presented in the context of an MHC molecule. In particular, an "antigen" relates to any substance, preferably a peptide or protein, that reacts specifically with an antibody or a T lymphocyte (T cell). According to the present disclosure, the term "antigen" includes any molecule that includes at least one epitope, e.g. a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that induces an immune response, optionally after processing, and is preferably specific for the antigen (including the cell that expresses the antigen). In one embodiment, the antigen is a disease-associated antigen, e.g. a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0148] The term "epitope" refers to an antigenic determinant within a molecule, e.g., an antigen, i.e., a portion or fragment thereof within a molecule that is recognized by the immune system, e.g., an antibody, T cell, or B cell, particularly when presented in the context of an MHC molecule. In one embodiment, "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of surface molecular groupings, e.g., amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents, whereas binding to the latter is not lost. Epitopes may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked or covered by the specific antigen-binding peptide (in other words, amino acid residues within the footprint of the specific antigen-binding peptide).

[0149] An epitope of a protein preferably comprises a contiguous or discontinuous portion of said protein and is preferably between about 5 and about 100 amino acids in length, preferably between about 5 and about 50 amino acids in length, more preferably between about 8 and about 0 amino acids in length, and most preferably between about 10 and about 25 amino acids in length, for example, an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that an epitope in the context of the present disclosure is a T cell epitope.

[0150] As used herein, the term "optionally" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and the description includes cases where said event, circumstance, or condition occurs as well as cases where it does not occur.

[0151] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining together of two or more elements or components or domains.

[0152] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition affecting an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. A disease may be a disease caused by an agent originally derived from an external source, e.g., an infectious disease, or a disease caused by an internal malfunction, e.g., an autoimmune disease. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death to the affected individual, or that causes similar problems to individuals who come into contact with the affected individual. In this broader sense, "disease" may include injury, physical dysfunction, disorder, syndrome, infection, isolated symptoms, deviant behavior, and atypical variations in structure and function, although in other contexts and for other purposes, "disease" may also be considered a distinct classification. Illnesses usually not only affect an individual physically, but also emotionally, as suffering from and living with many illnesses can change one's outlook on life and can alter one's personality.

[0153] The term "therapeutic treatment" relates to any treatment that improves the health status of an individual, extends (increases) / improves or extends (increases) the lifespan of an individual. Said treatment may / may eliminate the disease in an individual, may halt or slow the onset of the disease in an individual, may inhibit or slow the onset of the disease in an individual, may reduce the frequency or severity of symptoms in an individual, and may reduce recurrence in an individual who currently has or has previously had the disease.

[0154] The term "prophylactic treatment" or "preventive treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. In this specification, the terms "prophylactic treatment" or "preventive treatment" are used interchangeably. Similarly, the term "method of preventing" in the context of disease progression, e.g. tumor or cancer progression, relates to any method intended to prevent the disease from progressing in an individual.

[0155] As used herein, the terms "individual" and "subject" are used interchangeably. The terms "individual" and "subject" refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate), or any other non-mammalian, including bird (chicken), fish, or any other animal species, that may be affected by or susceptible to a disease or disorder (e.g., cancer). Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, geriatrics, children, and newborns. In some embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0156] The term "patient" refers to an individual or subject for treatment, in particular an affected individual or subject.

[0157] Aspects and embodiments of the present disclosure In a first aspect, the disclosure provides a method of reducing or preventing tumor progression or treating cancer in a subject, comprising administering to the subject a binding agent prior to, concurrently with, or following administration of an antibody or antigen-binding fragment thereof that binds Programmed Death-1 (PD-1), wherein the binding agent comprises a first binding region that binds CD137 and a second binding region that binds PD-L1; a) a first binding region comprising a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 6, 7, and 8, respectively; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 16, 17, and 18, respectively; The antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 46, 47, and 48, respectively, or the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 65, 66, and 67, respectively. A binding agent for use in the method is provided.

[0158] Binding agents that bind to CD137 and PD-L1 In one embodiment, the CD137 is human CD137, particularly human CD137 comprising the sequence set forth in SEQ ID NO: 38. In one embodiment, the PD-L1 is human PD-L1, particularly human PD-L1 comprising the sequence set forth in SEQ ID NO: 40. In one embodiment, the CD137 is human CD137 and the PD-L1 is human PD-L1. In one embodiment, the CD137 is human CD137 comprising the sequence set forth in SEQ ID NO: 38 and the PD-L1 is human PD-L1 comprising the sequence set forth in SEQ ID NO: 40.

[0159] In one embodiment of the binding agent according to the first aspect, the first binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 9, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or 10.

[0160] In a further embodiment of the binding agent according to the first aspect, the second binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:11, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:15.

[0161] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD137, comprising a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 9, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or 10; b) The second binding domain that binds to human PD-L1 comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:11, and a light chain variable domain (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:15.

[0162] In one embodiment of the binding agent according to the first aspect, the first binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 or 9, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 5 or 10.

[0163] In a further embodiment of the binding agent according to the first aspect, the second binding domain that binds to human PD-L1 comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO:15.

[0164] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD137, comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 or 9, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 5 or 10; b) The second binding domain that binds to human PD-L1 comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO:15.

[0165] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD137, comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; b) The second binding domain that binds to human PD-L1 comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO:15.

[0166] The binding agent may in particular be an antibody, such as a multispecific antibody, such as a bispecific antibody. The binding agent may also be in the format of a full length antibody or an antibody fragment.

[0167] It is further preferred that the binding agent is a human or humanized antibody.

[0168] Each variable region can include three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0169] The complementarity determining regions (CDRs) and framework regions (FRs) may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0170] In one embodiment of the first aspect, the binder is i) a polypeptide comprising the first heavy chain variable region (VH) and a first heavy chain constant region (CH); and ii) A polypeptide comprising the second heavy chain variable region (VH) and a second heavy chain constant region (CH).

[0171] In one embodiment of the first aspect, the binder is i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL), and ii) A polypeptide comprising said second light chain variable region (VL) and further comprising a second light chain constant region (CL).

[0172] In one embodiment of the first aspect, the binding agent is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising: i) a polypeptide comprising the first heavy chain variable region (VH), and the first heavy chain constant region (CH), and ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). and the second binding arm comprises iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH); and iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). The antibody comprises:

[0173] In one embodiment of the first aspect, the binding agent comprises i) a first heavy chain and a light chain comprising the antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a light chain comprising the antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region.

[0174] Each of the first heavy chain constant region and the second heavy chain constant region (CH) can include one or more of a constant heavy chain 1 (CH1) region, a hinge region, a constant heavy chain 2 (CH2) region, and a constant heavy chain 3 (CH3) region, preferably at least the hinge region, the CH2 region, and the CH3 region.

[0175] Each of the first and second heavy chain constant regions (CH) may comprise a CH3 region, where the two CH3 regions comprise asymmetric mutations. Asymmetric mutations mean that the sequences of the first and second CH3 regions contain amino acid substitutions at positions that are not identical. For example, one of the first and second CH3 regions contains a mutation at a position corresponding to position 405 in the human IgG1 heavy chain according to EU numbering, and the other of the first and second CH3 regions contains a mutation at a position corresponding to position 409 in the human IgG1 heavy chain according to EU numbering.

[0176] In the first heavy chain constant region (CH), at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering is substituted, and in the second heavy chain constant region (CH), at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering is substituted. In certain embodiments, the first heavy chain and the second heavy chain are not substituted at the same positions (i.e., the first heavy chain and the second heavy chain contain asymmetric mutations).

[0177] In one embodiment of the binding agent according to the first aspect, (i) the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said first heavy chain and the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said second heavy chain.

[0178] In one embodiment of the first aspect, the binding agent induces Fc-mediated effector function to a lesser extent than another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs), comprising the hinge, CH2, and CH3 regions of human IgG1.

[0179] In one particular embodiment of the binding agent according to the first aspect, said first and second heavy chain constant regions (CH) are modified to induce Fc-mediated effector function to a lesser extent than an antibody that is identical except that the antibody comprises the unmodified first and second heavy chain constant regions (CH). In particular, each or both of said unmodified first and second heavy chain constant regions (CH) may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 19 or 25.

[0180] Fc-mediated effector function may be determined by measuring binding of a binding agent to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking to Fcγ receptors. In particular, Fc-mediated effector function may be determined by measuring binding of a binding agent to C1q.

[0181] The first heavy chain constant region and the second heavy chain constant region of the binding agent may be modified such that binding of C1q to the antibody is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, where C1q binding is preferably determined by ELISA.

[0182] In one embodiment of the binding agent according to the first aspect, in at least one of said first and second heavy chain constant regions (CH), one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

[0183] In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering may be F and E, respectively, in said first and second heavy chains.

[0184] In particular, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering may be F, E, and A, respectively, in said first heavy chain constant region and second heavy chain constant region (HC).

[0185] In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F and E, respectively, in which (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0186] In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, respectively, wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0187] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain comprises: a) the sequence shown in SEQ ID NO: 19 or SEQ ID NO: 25 [IgG1-FC]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence is selected from the group consisting of:

[0188] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain or the second heavy chain, e.g. the second heavy chain, comprises: a) the sequence shown in SEQ ID NO: 20 or SEQ ID NO: 26 [IgG1-F405L]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 9 substitutions, e.g. at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0189] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain or the second heavy chain, e.g. the first heavy chain, comprises: a) the sequence shown in SEQ ID NO: 21 or 27 [IgG1-F409R]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2, or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0190] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain comprises: a) the sequence shown in SEQ ID NO: 22 or SEQ ID NO: 28 [IgG1-Fc_FEA]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 7 substitutions, e.g. at most 6 substitutions, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0191] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain, e.g. the second heavy chain, comprises: a) the sequence shown in SEQ ID NO: 24 or SEQ ID NO: 30 [IgG1-Fc_FEAL]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 6 substitutions, e.g. at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0192] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain, e.g. the first heavy chain, comprises: a) the sequence shown in SEQ ID NO: 23 or SEQ ID NO: 29 [IgG1-Fc_FEAR]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 6 substitutions, e.g. at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0193] In one embodiment of the first aspect, the binding agent comprises a kappa (κ) light chain constant region.

[0194] In one embodiment of the first aspect, the binding agent comprises a lambda (λ) light chain constant region.

[0195] In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0196] In one embodiment of the binding agent according to the first aspect, the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0197] In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region, or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0198] In one embodiment of the binding agent according to the first aspect, the kappa (κ) light chain is a) the sequence shown in SEQ ID NO:35; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2, or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence is selected from the group consisting of:

[0199] In one embodiment of the binding agent according to the first aspect, the lambda (λ) light chain comprises a) the sequence shown in SEQ ID NO:36; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2, or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence is selected from the group consisting of:

[0200] The binding agent (particularly an antibody) according to the first aspect is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In particular, the binding agent may be a full length IgG1 antibody. In a preferred embodiment of the first aspect, the binding agent (particularly an antibody) is of the IgG1m(f) allotype.

[0201] In a preferred embodiment of the binder according to the first aspect, the binder comprises i) a first heavy and light chain capable of binding to CD137, wherein the first heavy chain comprises the antigen-binding region comprising the sequence set forth in SEQ ID NO: 31 and the first light chain comprises the sequence set forth in SEQ ID NO: 32; ii) comprises a second heavy chain and a light chain which is capable of binding to PD-L1, wherein the second heavy chain comprises the antigen-binding region comprising the sequence set forth in SEQ ID NO: 33 and the second light chain comprises the antigen-binding region comprising the sequence set forth in SEQ ID NO: 34.

[0202] The binding agent for use according to the first aspect may in particular be akasunlimab or a biosimilar thereof.

[0203] In a preferred embodiment herein, the amount of binding agent administered in each dose and / or each treatment cycle is: a) about 0.3 to 5 mg per kg of body weight or about 25 to 400 mg in total; and / or b) Approximately 2.1 x 10 per kg of body weight -9 ~3.4×10 -8 moles or total of about 1.7 x 10 -7 ~2.7×10 -6 Mole It is.

[0204] According to these embodiments, doses specified in mg / kg can be converted to flat doses based on the median body weight of the subjects to whom the binding agent is administered, which is 80 kg, and vice versa.

[0205] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.3 to 4.0 mg per kg of body weight or about 25 to 320 mg in total; and / or Approximately 2.1 x 10 per kg of body weight -9 ~2.7×10 -8 moles or total of about 1.7 x 10 -7 ~2.2×10 -6 Mole It is possible.

[0206] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.38-4.0 mg per kg of body weight or about 30-320 mg in total; and / or Approximately 2.6 x 10 per kg of body weight -9 ~2.7×10 -8 moles or total of about 2.4 x 10 -7 ~2.2×10 -6 Mole It is possible.

[0207] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be About 0.5 to 3.3 mg per kg of body weight or about 40 to 260 mg in total; and / or Approximately 3.4 x 10 per kg of body weight -9 ~2.2×10 -8 moles or total of about 2.7 x 10 -7 ~1.8×10 -6 Mole It is possible.

[0208] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.6-2.5 mg per kg of body weight or about 50-200 mg in total; and / or Approximately 4.3 x 10 per kg of body weight -9 ~1.7×10 -8 moles or total approx. 3.4 x 10 -7 ~1.4×10 -6 Mole It is possible.

[0209] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.8-1.8 mg per kg of body weight or about 60-140 mg in total; and / or Approximately 5.1 x 10 per kg of body weight -9 ~1.2×10 -8 moles or total of about 4.1 x 10 -7 ~9.5×10 -7 Mole It is possible.

[0210] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.9-1.8 mg per kg of body weight or about 70-140 mg in total; and / or Approximately 6.0 x 10 per kg of body weight -9 ~1.2×10 -8 moles or total of about 4.8 x 10 -7 ~9.5×10 -7 Mole It is possible.

[0211] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 1-1.5 mg per kg of body weight or about 80-120 mg in total; and / or Approximately 6.8 x 10 per kg of body weight -9 ~1.0×10 -8 moles or total of about 5.5 x 10 -7 ~8.2×10 -7 Mole It is possible.

[0212] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 1.1 to 1.4 mg per kg of body weight or about 90 to 110 mg in total; and / or Approximately 7.7 x 10 per kg of body weight -9 ~9.4×10 -9 moles or total of about 6.1 x 10 -7 ~7.5×10 -7 Mole It is possible.

[0213] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 1.2-1.3 mg / kg body weight or about 95-105 mg total; and / or Approximately 6.8 x 10 per kg of body weight -9 ~8.9×10 -9 moles or total of about 6.5 x 10 -7 ~7.2×10 -7 Mole It is possible.

[0214] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.8-1.5 mg per kg of body weight or about 65-120 mg in total; and / or Approximately 5.5 x 10 per kg of body weight -9 ~1.0×10 -8 moles or total of about 4.4 x 10 -7 ~8.2×10 -7 Mole It is possible.

[0215] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.9-1.3 mg per kg of body weight or about 70-100 mg in total; and / or Approximately 6.0 x 10 per kg of body weight -9 ~8.5×10 -9 moles or total of about 4.8 x 10 -7 ~6.8×10 -7 Mole It is possible.

[0216] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be about 0.9-1.1 mg / kg body weight or about 75-90 mg total; and / or Approximately 6.4 x 10 per kg of body weight -9 ~7.7×10 -9 moles or total of about 5.1 x 10 -7 ~6.1×10 -7 Mole It is possible.

[0217] Furthermore, the amount of binding agent administered in each administration and / or each treatment cycle may, inter alia, be 0.3 to 4.0 mg per kg of body weight or a total of 25 to 320 mg; and / or 2.1 x 10 per kg of body weight -9 ~2.7×10 -8 moles or total 1.7 x 10 -7~2.2×10 -6 Mole It is possible.

[0218] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.38-4.0 mg per kg of body weight or 30-320 mg in total; and / or 2.6 x 10 per kg of body weight -9 ~2.7×10 -8 moles or total 2.4 x 10 -7 ~2.2×10 -6 Mole It is possible.

[0219] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.5-3.3 mg / kg body weight or a total of 40-260 mg; and / or 3.4 x 10 per kg of body weight -9 ~2.2×10 -8 moles or total 2.7 x 10 -7 ~1.8×10 -6 Mole It is possible.

[0220] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.6 to 2.5 mg per kg of body weight or a total of 50 to 200 mg; and / or 4.3 x 10 per kg of body weight -9 ~1.7×10 -8 moles or total 3.4 x 10 -7 ~1.4×10 -6 Mole It is possible.

[0221] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.8 to 1.8 mg per kg of body weight or a total of 60 to 140 mg; and / or 5.1 x 10 per kg of body weight -9 ~1.2×10 -8 moles or total 4.1 x 10-7 ~9.5×10 -7 Mole It is possible.

[0222] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.9-1.8 mg / kg body weight or a total of 70-140 mg; and / or 6.0 x 10 per kg of body weight -9 ~1.2×10 -8 moles or total 4.8 x 10 -7 ~9.5×10 -7 Mole It is possible.

[0223] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 1-1.5 mg / kg body weight or a total of 80-120 mg; and / or 6.8 x 10 per kg of body weight -9 ~1.0×10 -8 moles or total 5.5 x 10 -7 ~8.2×10 -7 Mole It is possible.

[0224] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 1.1 to 1.4 mg per kg of body weight or a total of 90 to 110 mg; and / or 7.7 x 10 per kg of body weight -9 ~9.4×10 -9 moles or total 6.1 x 10 -7 ~7.5×10 -7 Mole It is possible.

[0225] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 1.2-1.3 mg / kg body weight or a total of 95-105 mg; and / or 6.8 x 10 per kg of body weight -9 ~8.9×10 -9moles or total 6.5 x 10 -7 ~7.2×10 -7 Mole It is possible.

[0226] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.8-1.5 mg per kg of body weight or a total of 65-120 mg; and / or 5.5 x 10 per kg of body weight -9 ~1.0×10 -8 moles or total 4.4 x 10 -7 ~8.2×10 -7 Mole It is possible.

[0227] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.9-1.3 mg / kg body weight or a total of 70-100 mg; and / or 6.0 x 10 per kg of body weight -9 ~8.5×10 -9 moles or total 4.8 x 10 -7 ~6.8×10 -7 Mole It is possible.

[0228] The amount of binding agent administered in each administration and / or each treatment cycle may, in particular, be 0.9-1.1 mg / kg body weight or a total of 75-90 mg; and / or 6.4 x 10 per kg of body weight -9 ~7.7×10 -9 moles or total 5.1 x 10 -7 ~6.1×10 -7 Mole It is possible.

[0229] The amount of binding agent administered in each dose and / or each treatment cycle is a) about 1.1 mg / kg body weight or about 80 mg total; and / or b) Approximately 6.8 x 10 per kg of body weight -9moles or total of about 5.5 x 10 -7 Mole It is possible.

[0230] The amount of binding agent administered in each dose and / or each treatment cycle is a) 1.1 mg / kg body weight or a total of 80 mg; and / or b) 6.8 x 10 per kg of body weight -9 moles or total 5.5 x 10 -7 Mole It is possible.

[0231] As used herein, the amount of binding agent administered in each dose and / or each treatment cycle is a) about 1.25 mg per kg of body weight or about 100 mg total; and / or b) Approximately 8.5 x 10 per kg of body weight -9 moles or total of about 6.8 x 10 -7 Mole It is preferable that:

[0232] The amount of binding agent administered in each dose and / or each treatment cycle is a) 1.25 mg per kg of body weight or a total of 100 mg; and / or b) 8.5 x 10 per kg of body weight -9 moles or total 6.8 x 10 -7 Mole It is also preferred that

[0233] The binding agent may be administered in any manner and by any route known in the art, in preferred embodiments, the binding agent is administered systemically, e.g., parenterally, particularly intravenously.

[0234] The binding agent may be administered in the form of any suitable pharmaceutical composition as described herein, hi a preferred embodiment, the binding agent is administered in the form of an injection.

[0235] The binding agent for use according to the invention may be administered by intravenous (IV) infusion, e.g., by intravenous infusion for a minimum of 30 minutes, e.g., for a minimum of 60 minutes, e.g., by intravenous infusion for 30 to 120 minutes. Preferably, the binding agent for use according to the invention is administered by intravenous (IV) infusion for 30 minutes.

[0236] The binding agent may be administered prior to, simultaneously with, or after administration of the PD-1 inhibitor.

[0237] In one embodiment, the binding agent is administered prior to administration of the PD-1 inhibitor. For example, the interval between administration of the binding agent and administration of the PD-1 inhibitor is at least about 10 minutes, e.g., at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, and up to about 14 days (up to about 2 weeks), e.g., , up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days (up to about 1 week), up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day (up to about 24 hours), up to about 18 hours, up to about 12 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2.5 hours, or up to about 2 hours.

[0238] In one embodiment, the binding agent is administered after administration of the PD-1 inhibitor. For example, the interval between administration of the PD-1 inhibitor and administration of the binding agent is at least about 10 minutes, e.g., at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, and up to about 14 days (up to about 2 weeks), e.g., , up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days (up to about 1 week), up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day (up to about 24 hours), up to about 18 hours, up to about 12 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2.5 hours, or up to about 2 hours.

[0239] In one embodiment, the binding agent is administered simultaneously with the PD-1 inhibitor. For example, the binding agent and the PD-1 inhibitor can be administered using a composition that includes both drugs. Alternatively, the binding agent can be administered to one limb of the subject and the PD-1 inhibitor can be administered to another limb of the subject.

[0240] Antibodies that bind to PD-1 An antibody or antigen-binding fragment thereof that binds to PD-1 preferably comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity, e.g., at least 90% sequence identity, 95% sequence identity, 98% sequence identity, or 99% sequence identity, to the amino acid sequence of SEQ ID NO:49, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity, e.g., at least 90% sequence identity, 95% sequence identity, 98% sequence identity, or 99% sequence identity, to the amino acid sequence of SEQ ID NO:50.

[0241] In the most preferred embodiments herein, the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain variable region that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:49, and a light chain variable region that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:50.

[0242] An antibody or antigen-binding fragment thereof that binds to PD-1 may comprise a heavy chain that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:51, and a light chain that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:52.

[0243] Antibodies that bind PD-1 for use in accordance with the present invention preferably prevent inhibitory signaling associated with PD-1.Antibodies that bind PD-1 preferably disrupt or inhibit inhibitory signaling associated with PD-1.

[0244] Inhibition or blockage of PD-1 signaling, as described herein, results in the prevention or reversal of immune suppression and the establishment or enhancement of T cell immunity against cancer cells. In one embodiment, inhibition of PD-1 signaling, as described herein, reduces or inhibits immune system dysfunction. In one embodiment, inhibition of PD-1 signaling, as described herein, reduces the dysfunction of dysfunctional immune cells. In one embodiment, inhibition of PD-1 signaling, as described herein, reduces the dysfunction of dysfunctional T cells.

[0245] In one embodiment, the PD-1 inhibitor prevents the interaction of PD-1 with PD-L1, hi another embodiment, the PD-1 inhibitor prevents the interaction of PD-1 with PD-L2.

[0246] In particular, the antibody or antigen-binding fragment thereof that binds to PD-1 is a chimeric antibody, a humanized antibody, or a human antibody.

[0247] In a preferred embodiment, the antibody that binds to PD-1 is an isolated antibody.

[0248] Without being bound by theory, it is believed that the combination of a binding agent comprising a first binding domain that binds CD137, as defined above, and a second binding domain that binds PD-L1, as defined above, together with an antibody that binds PD-1, as defined above, increases the response rate and improves the duration of response in subjects receiving the combination therapy, since the combination therapy results in a complete blockade of the PD-1 pathway with a concomitant conditional activation of 4-1BB. The PD-1 blocking antibody blocks the interaction with both PD-L1 and PD-L2. Furthermore, it is believed that the combination therapy with an antibody that binds PD-1 increases the amount of PD-L1 available for binding by the binding agent.

[0249] The PD-1 inhibitor may in particular be pembrolizumab or a biosimilar thereof.

[0250] In a further embodiment, the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) that comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 49, and a light chain variable region (VL) that comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 50. The PD-1 inhibitor may in particular be an antibody comprising a heavy chain that comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 51, and a light chain that comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 52.

[0251] The anti-PD-1 antibodies of the present disclosure are preferably monoclonal antibodies, and can be multispecific antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, and PD-1-binding fragments of any of the above. In some embodiments, the anti-PD-1 antibodies described herein specifically bind to PD-1 (e.g., human PD-1). The immunoglobulin molecules of the present disclosure can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0252] Antigen-binding fragments (e.g., human antigen-binding fragments) described herein include Fab, Fab', and F(ab') 2 , Fd, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), and V L Domain or V H The antigen-binding fragments include, but are not limited to, fragments comprising the variable region(s), alone or in combination with all or a portion of the following: hinge region, CH1 domain, CH2 domain, CH3 domain, and CL domain. The present disclosure also includes antigen-binding fragments comprising any combination of the variable region(s) with the hinge region, CH1 domain, CH2 domain, CH3 domain, and CL domain. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken anti-PD-1 antibody or antigen-binding fragment thereof.

[0253] In some embodiments, the numbering of amino acid residues within the CDR sequences of the anti-PD-1 antibodies, or antigen-binding fragments thereof, provided herein is according to the IMGT numbering scheme as described in Lefranc, MP et al., Dev. Comp. Immunol., 2003, 27, 55-77.

[0254] Antibodies or antigen-binding fragments thereof that bind PD-1 also include derivatives and constructs that have been modified, i.e., modified by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from binding to PD-1. For example, and without limitation, derivatives of anti-PD-1 antibodies include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivatives or constructs may contain one or more non-classical amino acids.

[0255] Preferably, the antibody or antigen-binding fragment thereof that binds PD-1 is administered in a suitable amount. The amount of the antibody or antigen-binding fragment thereof that binds PD-1 administered in each dose and / or each treatment cycle may be in a range such that more than 5%, preferably more than 10%, more preferably more than 15%, even more preferably more than 20%, even more preferably more than 25%, even more preferably more than 30%, even more preferably more than 35%, even more preferably more than 40%, even more preferably more than 45%, and most preferably more than 50% of the PD-1 inhibitors bind to PD-1.

[0256] In certain embodiments, the antibody that binds PD-1 is pembrolizumab or a biosimilar thereof, and for example, the amount of PD-1 inhibitor administered in each dose and / or each treatment cycle is about 10 to about 1000 mg total, for example, about 100 to about 600 mg total, for example, about 150 to about 600 mg total, about 150 to about 500 mg total, about 175 to about 500 mg total, about 175 to about 450 mg total, about 200 to about 450 mg total, or, for example, about 200 to about 400 mg total.

[0257] In certain embodiments, the antibody that binds PD-1 is pembrolizumab or a biosimilar thereof, and for example, the amount of PD-1 inhibitor administered in each dose and / or each treatment cycle is between 10 and 1000 mg total, such as between 100 and 600 mg total, for example between 150 and 600 mg total, between 150 and 500 mg total, between 175 and 500 mg total, between 175 and 450 mg total, between 200 and 450 mg total, or, for example, between 200 and 400 mg total.

[0258] In certain embodiments, the antibody that binds PD-1 is pembrolizumab or a biosimilar thereof, e.g., the amount of the antibody that binds PD-1 administered in each dose and / or each treatment cycle is about 100-600 mg in total; and / or Total approx. 6.84 x 10 -7 ~4.11×10 -7 Mole It is.

[0259] In certain embodiments, the antibody that binds PD-1 is pembrolizumab or a biosimilar thereof, e.g., the amount of the antibody that binds PD-1 administered in each dose and / or each treatment cycle is about 100-400 mg in total; and / or about 6.84 x 10 -7 ~2.73×10 -6 moles, e.g., a total of 100 to 400 mg; and / or a total of 6.84 x 10 -7 ~2.73×10 -6 It is a mole.

[0260] In certain embodiments, the antibody that binds PD-1 is pembrolizumab or a biosimilar thereof, e.g., the amount of the antibody that binds PD-1 administered in each dose and / or each treatment cycle is about 200-400 mg in total; and / or about 6.84 x 10 -7 ~2.73×10 -6 moles, e.g., a total of 200 to 400 mg; and / or a total of 6.84 x 10 -7 ~2.73×10 -6 It is a mole.

[0261] In certain embodiments, for example, the amount of antibody or antigen-binding fragment thereof that binds PD-1 administered in each dose and / or each treatment cycle is about 200 mg or about 1.37×10 -6 moles, e.g., 200 mg total or 1.37 x 10 -6 It is a mole.

[0262] In certain embodiments, the antibody that binds PD-1 is pembrolizumab or a biosimilar thereof, e.g., the amount of the antibody that binds PD-1 or an antigen-binding fragment thereof administered in each dose and / or each treatment cycle is about 200 mg total or about 1.37×10 -6 moles, e.g., 200 mg total or 1.37 x 10 -6 It is a mole.

[0263] In certain embodiments, for example, the amount of antibodies or antigen-binding fragments thereof that bind PD-1 administered in each dose and / or each treatment cycle is about 400 mg total or about 2.73×10 -6 moles, e.g., 400 mg total or 2.73 x 10 total -6 It is a mole.

[0264] In certain embodiments, the antibody that binds PD-1 is pembrolizumab or a biosimilar thereof, e.g., the amount of the antibody that binds PD-1 or an antigen-binding fragment thereof administered in each dose and / or each treatment cycle is about 400 mg total or about 2.73×10 total. -6 moles, e.g., 400 mg total or 2.73 x 10 total -6 It is a mole.

[0265] The antibody or antigen-binding fragment thereof that binds PD-1 may be administered in any manner and by any route known in the art. The manner and route of administration will depend on the type of antibody used. In a preferred embodiment, the antibody or antigen-binding fragment thereof that binds PD-1 is administered systemically, e.g., parenterally, particularly intravenously.

[0266] The antibody or antigen-binding fragment thereof that binds PD-1 may be administered in the form of any suitable pharmaceutical composition as described herein, hi a preferred embodiment, the antibody or antigen-binding fragment thereof that binds PD-1 is administered in the form of an infusion, e.g., an intravenous infusion.

[0267] Subjects and Tumors or Cancers to be Treated The subjects treated in accordance with the present disclosure are preferably human subjects.

[0268] In a preferred embodiment, the tumor or cancer to be treated is a solid tumor or solid cancer. The tumor or cancer may be a metastatic tumor or metastatic cancer.

[0269] Preferably, the tumor or cancer may be selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, urinary tract cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancer, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma. More preferably, the tumor or cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, pancreatic cancer, and head and neck cancer.

[0270] In certain embodiments, the tumor or cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urinary tract cancer (bladder cancer, ureteral cancer, urethral cancer, or renal pelvis cancer), endometrial cancer (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g., oral cavity cancer, laryngeal cancer, or pharyngeal cancer), and cervical cancer.

[0271] Preferably, the tumor is a PD-L1 positive tumor. In certain embodiments, PD-L1 is preferably expressed in > 1% of cancer or tumor cells. PD-L1 expression can be determined using techniques known to those skilled in the art, and can be assessed, for example, by immunohistochemistry (IHC).

[0272] The tumor or cancer may in particular be lung cancer. The lung cancer may be non-small cell lung cancer (NSCLC), e.g., squamous NSCLC or non-squamous NSCLC. Lung cancer is the second most common malignancy with an estimated age-standardized incidence of 22.4 cases per 100,000 people and is the leading cause of cancer death in both men and women (Kantar, 2021). In 2020, approximately 2,206,771 new lung cancer cases and 1,796,144 deaths are estimated worldwide (GLOBOCAN, 2020). NSCLC accounts for 85%-90% of all cases, with a 5-year survival rate across all stages of the disease of approximately 18%, and metastatic disease in only 3.5% (Jemal et al., 2011) (Kantar, 2021; SEER, 2018). In the 1L setting, treatment typically consists of platinum-based chemotherapy combined with immunotherapy or targeted therapy depending on the molecular / biomarker analysis and histology of the tumor (NCCN, 2021d). More recently, the advent of PD-1 inhibitors and programmed death-ligand 1 (PD-L1) inhibitors has improved outcomes for patients without driving mutations (approximately 62% of the non-squamous population and 77% of the squamous population (Kantar, 2021)). Additional treatment alternatives are needed for patients whose tumors do not harbor certain oncogenic mutations or express biomarkers for checkpoint inhibitor (CPI) options. Novel combinations with complementary approaches to enhance response may further address unmet needs in this population. For patients in the second-line setting, SOC is limited to platinum-based chemotherapy, CPI monotherapy, or docetaxel with or without ramucirumab depending on the prior treatment administered. For patients in the third-line (3L) setting, chemotherapy monotherapy is the standard of care. Novel therapies are needed to limit toxicity and potentially enhance efficacy in this population (NCCN, 2021d).

[0273] In one embodiment where the tumor or cancer is lung cancer, the tumor or cancer is non-small cell lung cancer (NSCLC), e.g., squamous NSCLC or non-squamous NSCLC. The tumor or cancer may in particular be a metastatic cancer, e.g., metastatic NSCLC.

[0274] In one embodiment, where the tumor or cancer is lung cancer, particularly NSCLC, the tumor or cancer does not have an epidermal growth factor receptor (EGFR) sensitizing mutation and / or an anaplastic lymphoma karyogram (ALK) translocation / ROS1 rearrangement. An EGFR sensitizing mutation is one that is amenable to treatment with an approved tyrosine kinase inhibitor (TKI).

[0275] In one embodiment, where the tumor or cancer is lung cancer, particularly NSCLC, the tumor or cancer comprises cancer cells and PD-L1 is expressed in > 1% of the cancer cells. Such expression may be determined by any means and methods known to those skilled in the art, such as, for example, by a local SOC test (preferably an FDA approved test) or by immunohistochemistry (IHC) determined in a central laboratory.

[0276] In one embodiment, where the tumor or cancer is lung cancer, particularly NSCLC, the tumor or cancer comprises cancer cells and PD-L1 is expressed in between 1% and 49% of the cancer cells. Such expression may be determined by any means and methods known to those of skill in the art, such as, for example, by a local SOC test (preferably an FDA approved test) or by immunohistochemistry (IHC) determined in a central laboratory.

[0277] In one embodiment, where the tumor or cancer is lung cancer, particularly NSCLC, the tumor or cancer comprises cancer cells and PD-L1 is expressed in >50% of the cancer cells. Such expression may be determined by any means and methods known to those skilled in the art, such as, for example, by a local SOC test (preferably an FDA approved test) or by immunohistochemistry (IHC) determined in a central laboratory.

[0278] In one embodiment, the subject has not had prior systemic treatment for metastatic disease, i.e., the subject has not had any systemic treatment for metastatic disease prior to receiving treatment according to the present invention. In accordance with this embodiment, the tumor or cancer is preferably lung cancer, e.g., NSCLC.

[0279] In one embodiment, the subject has not had prior treatment with a checkpoint inhibitor / immune checkpoint (ICP) inhibitor, i.e., prior to treatment according to the first aspect, the subject has not had treatment with an ICP inhibitor. In a further embodiment, the subject has not had prior treatment with a PD-1 inhibitor or PD-L1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody. In these embodiments, the tumor or cancer is preferably lung cancer, e.g., NSCLC.

[0280] In further embodiments, the subject has not been previously treated with a 4-1BB (CD137) targeting agent, an anti-tumor vaccine, or an autologous cell immunotherapy. In one embodiment, the subject has not been previously treated with an anti-4-1BB (CD137) antibody. In these embodiments, the tumor or cancer is preferably a lung cancer, e.g., NSCLC.

[0281] In other embodiments, the tumor or cancer has relapsed, is refractory / has relapsed, or is refractory following treatment, e.g., systemic treatment with a checkpoint inhibitor.

[0282] The subject may have received at least one prior systemic selective therapy, e.g., a systemic therapy including a PD-1 inhibitor or PD-L1 inhibitor, e.g., an anti-PD-1 antibody or anti-PD-L1 antibody. The cancer or tumor has relapsed, is refractory / has relapsed or is refractory, or the subject has progressed following treatment with a PD-1 inhibitor or PD-L1 inhibitor, e.g., an anti-PD-1 antibody or anti-PD-L1 antibody, a PD-1 inhibitor or PD-L1 inhibitor, particularly administered as a monotherapy or as part of a combination therapy.

[0283] In a particular embodiment, treatment according to the invention is administered to a subject who has received a previous treatment, for example a subject whose last previous treatment was with a PD1 inhibitor or a PD-L1 inhibitor, e.g. an anti-PD-1 antibody or an anti-PD-L1 antibody, a PD-1 inhibitor or a PD-L1 inhibitor, administered as a monotherapy or as part of a combination therapy, as defined above. The last previous treatment may be a treatment with a PD1 inhibitor or a PD-L1 inhibitor as defined above.

[0284] Preferably, treatment according to the invention is administered to a subject when the subject's time since progression at the time of last treatment with a PD1 inhibitor or PD-L1 inhibitor, e.g. an anti-PD-1 antibody or anti-PD-L1 antibody, PD-1 inhibitor or PD-L1 inhibitor, is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0285] By analogy, it may be preferable to administer a treatment according to the invention to a subject where the time since the last administration of a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, as part of the last previous treatment, is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0286] In a further embodiment, the cancer or tumor is recurrent or refractory, or the subject is i) treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody followed by platinum-based chemotherapy; or ii) Platinum-based chemotherapy followed by treatment with anti-PD-1 or anti-PD-L1 antibodies It is ongoing during or after the period.

[0287] Also in these embodiments, the tumor or cancer is preferably a lung cancer, e.g., NSCLC.

[0288] The subject to be treated according to the present invention may in particular be a subject who has not been previously treated with a taxane chemotherapeutic agent; e.g., docetaxel or paclitaxel, e.g., a taxane chemotherapeutic agent, e.g., docetaxel, for NSCLC.

[0289] Treatment regimen The binding agent and the PD-1 inhibitor may be administered in any suitable manner, for example, intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, intralymph node, or intratumorally.

[0290] In one embodiment of the first aspect, the binding agent as defined above is administered to the subject by systemic administration. Preferably, the binding agent is administered to the subject by intravenous injection or infusion. In one embodiment, the binding agent is administered in at least one treatment cycle.

[0291] In one embodiment, the antibody or antigen-binding fragment thereof that binds PD-1 is administered to the subject, particularly by systemic administration. Preferably, the antibody or antigen-binding fragment thereof that binds PD-1 is administered to the subject by intravenous injection or infusion. In one embodiment, the antibody or antigen-binding fragment thereof that binds PD-1 is administered in at least one treatment cycle.

[0292] In one embodiment, the binding agent as defined above and the antibody or antigen-binding fragment thereof that binds PD-1 are administered to the subject by systemic administration, in particular. Preferably, the binding agent and the antibody or antigen-binding fragment thereof that binds PD-1 are administered to the subject by intravenous injection or infusion. In one embodiment, the binding agent and the antibody or antigen-binding fragment thereof that binds PD-1 are administered in at least one treatment cycle.

[0293] In one embodiment, each treatment cycle is about 2 weeks (14 days), 3 weeks (21 days), or 4 weeks (28 days), 5 weeks (35 days), or 6 weeks (48 days). In a preferred embodiment, each treatment cycle is 3 weeks (21 days). In another preferred embodiment, each treatment cycle is 6 weeks (48 days).

[0294] In certain embodiments, one dose of the binding agent defined above and one dose of the antibody or antigen-binding fragment thereof that binds PD-1 are administered or infused every 2 weeks (1Q2W), every 3 weeks (1Q3W), or every 4 weeks (1Q4W), every 5 weeks (1Q5W), preferably every 3 weeks (1Q3W). In other embodiments, one dose of the binding agent defined above and one dose of the antibody or antigen-binding fragment thereof that binds PD-1 are administered every 6 weeks (1Q6W). The amounts of the binding agent and the amount of the antibody or antigen-binding fragment thereof that binds PD-1 are preferably as defined above.

[0295] In some embodiments, the or each dose is administered or infused on day 1 of each treatment cycle. For example, one dose of the binding agent defined above and one dose of the antibody or antigen-binding fragment thereof that binds to PD-1 may be administered on day 1 of each treatment cycle.

[0296] In some embodiments, a 100 mg dose of the binding agent defined above and a 200 mg dose of the antibody or antigen-binding fragment thereof that binds to PD-1 are administered every three weeks (1Q3W).

[0297] In another embodiment, a 100 mg dose of the binding agent defined above and a 400 mg dose of the antibody or antigen-binding fragment thereof that binds to PD-1 are administered every six weeks (1Q6W).

[0298] In a specific embodiment, a 100 mg dose of the acasunlimab or biosimilar binding agent and a 200 mg dose of the pembrolizumab or biosimilar antibody or antigen-binding fragment thereof that binds PD-1 are administered every three weeks (1Q3W), e.g., on day 1 of each three-week treatment cycle.

[0299] In a particular embodiment, the tumor or cancer is NSCLC; a 100 mg dose of the binding agent that is akasunlimab or a biosimilar thereof, and a 200 mg dose of the antibody or antigen-binding fragment thereof that binds PD-1 that is pembrolizumab or a biosimilar thereof, are administered every three weeks (1Q3W), e.g., on day 1 of each three-week treatment cycle.

[0300] In other embodiments, a 100 mg dose of acasunlimab or a biosimilar binding agent and a 400 mg dose of pembrolizumab or a biosimilar antibody or antigen-binding fragment thereof that binds PD-1 are administered every six weeks (1Q6W), e.g., on day 1 of every 6-week treatment cycle.

[0301] In yet other embodiments, the tumor or cancer is NSCLC; in this case, a 100 mg dose of the binding agent that is acasunlimab or a biosimilar thereof, and a 400 mg dose of the antibody that binds PD-1 or antigen-binding fragment thereof that is pembrolizumab are administered every six weeks (1Q6W), e.g., on day 1 of every six-week treatment cycle.

[0302] The antibody or antigen-binding fragment thereof that binds PD-1 may be administered first, followed by the binding agent. Alternatively, the binding agent is administered first, followed by the antibody or antigen-binding fragment thereof that binds PD-1.

[0303] Each dose may be administered or infused over a minimum of 30 minutes, such as a minimum of 60 minutes, a minimum of 90 minutes, a minimum of 120 minutes, or a minimum of 240 minutes.

[0304] The binding agent may in particular be administered using an intravenous (IV) infusion over a period of 30 minutes, such as a minimum of 40 minutes, a minimum of 50 minutes, or such as a minimum of 60 minutes.

[0305] The antibody or antigen-binding fragment thereof that binds to PD-1 may be administered as an intravenous infusion over a period of 30 minutes, e.g., a minimum of 40 minutes, a minimum of 50 minutes, or e.g., a minimum of 60 minutes.

[0306] The binding agent as defined above and the antibody or antigen-binding fragment thereof that binds to PD-1 may be administered simultaneously. In an alternative preferred embodiment, the binding agent and the PD-1 inhibitor are administered separately.

[0307] The binding agent and the antibody or antigen-binding fragment thereof that binds PD-1 as defined above may be administered in any suitable form (e.g., naked form as such). However, the binding agent and the PD-1 inhibitor are preferably administered in the form of any suitable pharmaceutical composition as described herein. In one embodiment, at least the binding agent and the antibody or antigen-binding fragment thereof that binds PD-1 are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent and one pharmaceutical composition for the antibody or antigen-binding fragment thereof that binds PD-1), preferably the binding agent and the antibody or antigen-binding fragment thereof that binds PD-1 are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent and one pharmaceutical composition for the antibody or antigen-binding fragment thereof that binds PD-1).

[0308] The composition or pharmaceutical composition may be formulated with carriers, excipients, and / or diluents, as well as any other ingredients suitable for pharmaceutical compositions, including known adjuvants, according to conventional techniques, e.g., those disclosed in Remington, "The Science and Practice of Pharmacy", 19th Edition, Gennaro Edition, Mack Publishing Co., Easton, PA, 1995. The pharma- ceutically acceptable carriers or diluents, as well as any known adjuvants and excipients, shall be suitable for the binding agent and / or antibody or antigen-binding fragment that binds PD-1 and the selected mode of administration. Suitability of carriers and other ingredients of the pharmaceutical composition is determined based on lack of significant negative effect on the desired biological properties of the selected compound or pharmaceutical composition (e.g., less than substantially affecting binding to the antigen [10% or less relative inhibition, 5% or less relative inhibition, etc.]).

[0309] Compositions, in particular pharmaceutical compositions with binding agents as defined above, with an antibody or antigen-binding fragment thereof that binds PD-1, may contain diluents, bulking agents, salts, buffers, detergents (e.g. non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g. sugars or non-protein containing amino acids), preservatives, solubilizers, and / or other materials suitable for incorporation into a pharmaceutical composition.

[0310] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in "Remington's Pharmaceutical Sciences", Mack Publishing Co. (ed. A. R Gennaro, 1985).

[0311] The pharmaceutical carrier, excipient, or diluent can be selected in view of the intended route of administration and standard pharmaceutical practice.

[0312] Pharmaceutically acceptable carriers include any suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents, and all such agents, that are physiologically compatible with the active compounds, particularly the binding agents defined above, and antibodies or antigen-binding fragments thereof that bind PD-1.

[0313] Examples of suitable aqueous and non-aqueous carriers that may be incorporated in the (pharmaceutical) compositions include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical art.

[0314] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the (pharmaceutical) composition is contemplated.

[0315] The term "excipient" as used herein refers to a substance that may be present in the (pharmaceutical) composition of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0316] The term "diluent" refers to dilution and / or thinning agents. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension, and / or mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0317] The (pharmaceutical) compositions may also include pharma- ceutically acceptable antioxidants, such as: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0318] The (pharmaceutical) compositions may also include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0319] The (pharmaceutical) composition may also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, humectants, emulsifiers, dispersing agents, and preservatives or buffers that may enhance the shelf life or effectiveness of the composition. The compositions used herein may be prepared with carriers that protect the compound against rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable polymers, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with wax or other materials known in the art. Methods for preparing such formulations are known to those skilled in the art, see, for example, "Sustained and Controlled Release Drug Delivery Systems", edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0320] "Pharmaceutically acceptable salts" include, for example, acid addition salts, which may be formed, for example, by using pharma- ceutically acceptable acids, such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carboxylic acid, or phosphoric acid. Further suitable pharma- ceutically acceptable salts include alkali metal salts (e.g., sodium salts or potassium salts); alkaline earth metal salts (e.g., calcium salts or magnesium salts); ammonium salts (NH 4 +); and salts formed with appropriate organic ligands (e.g., quaternary ammonium and amine cations formed with counter anions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates, and aryl sulfonates).Illustrative examples of pharma- ceutically acceptable salts include acetate, adipate, alginate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphor, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edta ... Disylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycosylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isobutyrate Salt, isothioate, hydroxynaphthoate, iodide, isobutyrate, isothioate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate Salts include, but are not limited to, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, suberate, succinate, tannate, tartrate, theoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al., "Pharmaceutical Salts," J. Pharm.Sci., 66, pp. 1-19 (1977)). Pharmaceutically unacceptable salts may also be used to prepare pharma-ceutically acceptable salts and are incorporated into the present disclosure.

[0321] In one embodiment, the binding agent and PD-1 inhibitor used herein can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutical active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Other active or therapeutic compounds can also be incorporated into the composition.

[0322] Pharmaceutical compositions for injections must typically be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier may be an aqueous or non-aqueous solvent, or a dispersion medium, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, e.g., olive oil, and injectable organic esters, e.g., ethyl oleate. Proper fluidity may be maintained, for example, by using a coating, e.g., lecithin, to maintain the required particle size in the case of dispersions, and by using surfactants. In many cases, it will be preferable to include isotonic agents, e.g., sugars, polyalcohols, e.g., glycerol, mannitol, sorbitol, or sodium chloride, in the composition. Delayed absorption of the injectable compositions may be brought about by incorporating into the composition agents that delay absorption, e.g., monostearate salts and gelatin. Sterile injectable solution can be prepared by incorporating active compound in a suitable solvent in the required amount, for example, with one or a combination of the above-listed ingredients as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile medium that contains a basic dispersion medium and other required ingredients, for example, from the above-listed ingredients.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and lyophilization (freeze drying), which produces a powder of active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0323] Sterile injectable solution can be prepared by incorporating active compound in the required amount in a suitable solvent with one or combination of the above-listed ingredients as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile medium that contains a basic dispersion medium and other required ingredients from the above-listed other ingredients.In the case of sterile powder for preparing sterile injectable solution, examples of preparation methods are vacuum drying and freeze-drying (freeze-drying), which produces a powder of active ingredient plus any other desired ingredients from the solution that has already been sterile-filtered.

[0324] In certain embodiments, the binder for use according to the invention is formulated in a composition or formulation comprising histidine, sucrose, and polysorbate 80, and having a pH of about 5 to about 6, e.g., 5 to 6. In particular, the binder for use according to the invention can be a binder in a composition or formulation comprising about 20 mM histidine, about 250 mM sucrose, about 0.02% polysorbate 80, and having a pH of about 5.5, e.g., a binder in a composition or formulation comprising 20 mM histidine, 250 mM sucrose, 0.02% polysorbate 80, and having a pH of 5.5. In certain embodiments, the formulation can comprise about 10 to about 30 mg of binder per mL, e.g., 10 to 30 mg of binder per mL, in particular about 20 mg of binder per mL, e.g., 20 mg of binder per mL.

[0325] The binding agent for use according to the invention may be provided in a composition as defined above, which may then be diluted in 0.9% NaCl (saline) prior to administration.

[0326] In a second aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (i) comprising a first binding region that binds to CD137 and a second binding region that binds to PD-L1; a) the first binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 6, 7, and 8, respectively; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 16, 17, and 18, respectively; Binders; and (ii) an antibody or antigen-binding fragment thereof that inhibits PD-1 activity and binds to PD-1, wherein the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 46, 47, and 48, respectively, or the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 65, 66, and 67, respectively; A kit comprising:

[0327] The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, and the antibody or antigen-binding fragment thereof that binds to PD-1) also apply to the kit of the second aspect. In one embodiment, the kit comprises at least two containers, one of which contains the binding agent (per se or in the form of a (pharmaceutical) composition) and the second container contains the antibody or antigen-binding fragment thereof that binds to PD-1 (per se or in the form of a (pharmaceutical) composition).

[0328] In a third aspect, the present disclosure provides a kit of the second aspect for use in a method of reducing or preventing tumor progression or treating cancer in a subject. The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agents, PD-1 inhibitors, treatment regimens, specific tumors / cancers, and subjects) and / or the second aspect also apply to the kit for use of the third aspect.

[0329] In a fourth aspect, the disclosure provides a method of reducing or preventing tumor progression or treating cancer in a subject, comprising administering to the subject a binding agent prior to, concurrently with, or following administration of an antibody or antigen-binding fragment thereof that binds PD-1, wherein the binding agent comprises a first binding region that binds CD137 and a second binding region that binds PD-L1; a) a first binding region comprising a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 6, 7, and 8, respectively; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 16, 17, and 18, respectively; The antibody inhibits PD-1 activity and comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 46, 47, and 48, respectively, or the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 65, 66, and 67, respectively. A method is provided.

[0330] Embodiments disclosed herein with respect to the first aspect (particularly with respect to binding agents, PD-1 inhibitors, treatment regimens, specific tumors / cancers, and subjects) also apply to the method of the fourth aspect.

[0331] In a further aspect, the disclosure provides a method of reducing or preventing tumor progression or treating cancer in a subject, comprising administering to the subject a PD-1 inhibitor prior to, concurrently with, or following administration of an antibody or antigen-binding fragment thereof that binds PD-1; the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 46, 47, and 48, respectively; or the antibody comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 65, 66, and 67, respectively; the binding agent comprises a first binding region that binds CD137 and a second binding region that binds PD-L1; a) the first binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 6, 7, and 8, respectively; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 16, 17, and 18, respectively; The present invention provides an antibody or antigen-binding fragment thereof that binds to PD-1 for use in the method.

[0332] The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, the PD-1 inhibitor, the optional one or more further therapeutic agents, the treatment regimen, the specific tumor / cancer, and the subject) also apply to the PD-1 inhibitor for use in this further aspect.

[0333] Citation of the documents and works referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the contents of these documents.

[0334] This description (including the following examples) is presented to enable one of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be accorded the scope consistent with the appended claims.

[0335] Items of this disclosure 1. A binding agent for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject the binding agent prior to, concurrently with, or following administration of an antibody that binds programmed death-1 (PD-1), or an antigen-binding fragment thereof; where the binding agent comprises a first binding domain that binds CD137 and a second binding domain that binds PD-L1; (a) said first binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; And (b) said second antigen-binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18; and the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; or the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively; The binding substance.

[0336] 2. The binding agent for use according to item 1, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:49, and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:50.

[0337] 3. The binding agent for use of any one of the preceding items, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:49, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:50.

[0338] 4. The binding agent for use of any one of the preceding items, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:51, and a light chain comprising the amino acid sequence of SEQ ID NO:52.

[0339] 5. The binding agent for use according to any one of the preceding items, wherein the antibody that binds to PD-1 is pembrolizumab or a biosimilar thereof.

[0340] 6. The binding agent for use according to any one of the preceding items, wherein the PD-L1 is human PD-L1, particularly human PD-L1 comprising the sequence set forth in SEQ ID NO:40, and / or the CD137 is human CD137, particularly human CD137 comprising the sequence set forth in SEQ ID NO:38.

[0341] 7. A binding agent for use according to any one of the preceding items, wherein the first binding region of the binding agent comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 9, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or 10.

[0342] 8. A binding agent for use according to any one of the preceding items, wherein the second binding region of the binding agent comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 25-100% sequence identity to SEQ ID NO:11, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:15.

[0343] 9. A binding agent for use according to any one of the preceding items, wherein the first binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 or 9, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 5 or 10.

[0344] 10. A binding agent for use according to any one of the preceding items, wherein the second binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:15.

[0345] 11. The following (a) and (b) are true: (a) the first binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and (b) the second binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 11, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 15; A binding agent for use according to any one of the preceding items.

[0346] 12. A binding agent for use according to any one of the preceding items, wherein the binding agent is a multispecific antibody, such as a bispecific antibody.

[0347] 13. A binding agent for use according to any one of the preceding items, wherein said binding agent is in the format of a full length antibody or an antibody fragment.

[0348] 14. A binding agent for use according to any one of the preceding items, wherein each variable region comprises three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0349] 15. A binding agent for use according to item 13, wherein the complementarity determining regions and the framework regions are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0350] 16. The binding substance, (i) a polypeptide comprising, consisting of, or consisting essentially of said first heavy chain variable region (VH) and first heavy chain constant region (CH), and (ii) a polypeptide comprising, consisting of, or consisting essentially of said second heavy chain variable region (VH) and second heavy chain constant region (CH); 2. A binding agent for use according to any one of the preceding items, comprising:

[0351] 17. The binding substance, (i) a polypeptide comprising the first light chain variable region (VL) and further comprising a first light chain constant region (CL); and (ii) a polypeptide comprising the second light chain variable region (VL) and further comprising a second light chain constant region (CL). 2. A binding agent for use according to any one of the preceding items, comprising:

[0352] 18. The binding agent is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm is: (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH); and (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL) and the second binding arm comprises (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH); and (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). 2. A binding agent for use according to any one of the preceding items, comprising:

[0353] 19. The binding substance, (i) a first heavy chain and a light chain comprising an antigen-binding region capable of binding to CD137; and (ii) a second heavy chain and a light chain comprising an antigen-binding region capable of binding to PD-L1. 2. A binding agent for use according to any one of the preceding items, comprising:

[0354] 20. The binding substance, (i) a first heavy chain and a light chain comprising said antigen-binding region capable of binding to CD137, wherein said first heavy chain comprises a first heavy chain constant region and said first light chain comprises a first light chain constant region; and (ii) a second heavy chain and a light chain comprising said antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region, and the second light chain comprises a second light chain constant region; 2. A binding agent for use according to any one of the preceding items, comprising:

[0355] 21. The binding agent for use according to any one of items 16 to 20, wherein each of the first and second heavy chain constant regions (CH) comprises one or more of the constant heavy chain 1 (CH) region, the hinge region, the constant heavy chain 2 (CH2) region and the constant heavy chain 3 (CH3) region, preferably at least the hinge region, the CH2 region and the CH3 region.

[0356] 22. The binding agent for use according to any one of items 16 to 21, wherein each of the first and second heavy chain constant regions (CH) comprises a CH3 region, and wherein these two CH3 regions comprise an asymmetric mutation.

[0357] 23. A binding agent for use according to any one of items 16 to 21, wherein in the first heavy chain constant region (CH) at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering is substituted, and in the second heavy chain constant region (CH) at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering is substituted, and wherein the first and the second heavy chains are not substituted at the same position.

[0358] 24. A binding agent for use according to item 23, which is either (i) or (ii), namely (i) the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said first heavy chain and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said second heavy chain.

[0359] 25. A binding agent for use according to any one of the preceding items, wherein said binding agent induces Fc-mediated effector functions to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CH) comprising human IgG1 hinge, CH2 and CH3 regions.

[0360] 26. The binding agent for use according to item 25, wherein the first and second heavy chain constant regions (CH) have been modified so as to induce Fc-mediated effector function to a lesser extent compared to an identical antibody except that the antibody comprises unmodified first and second heavy chain constant regions (CH).

[0361] 27. The binding agent for use according to item 26, wherein each of the unmodified first and second heavy chain constant regions (CH) comprises an amino acid sequence as set forth in SEQ ID NO: 19 or 25.

[0362] 28. The binding agent for use according to item 26 or 27, wherein the Fc-mediated effector function is measured by binding to Fcγ receptors, binding to C1q, or induction of Fe-mediated cross-linking of Fcγ receptors.

[0363] 29. The binding agent for use according to item 28, wherein the Fc-mediated effector function is measured by binding to C1q.

[0364] 30. The binding agent for use according to any one of items 25 to 29, wherein the first and second heavy chain constant regions are modified such that binding of C1q to the antibody is reduced compared to a wild type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, and wherein C1q binding is preferably determined by ELISA.

[0365] 31. A binding agent for use according to any one of the preceding items, wherein in at least one of the first and second heavy chain constant regions (CH), one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

[0366] 32. A binding agent for use according to item 31, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E in the first and second heavy chains, respectively.

[0367] 33. A binding agent for use according to item 31 or 32, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in the first and second heavy chain constant regions (HC).

[0368] 34. A binding agent for use according to any one of items 31 to 33, wherein the positions in both the first and the second heavy chain constant regions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, and wherein (i) the position in the first heavy chain constant region corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L and the position in the second heavy chain corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, or (ii) the position in the first heavy chain constant region corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R and the position in the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L.

[0369] 35. The binding agent for use according to any one of items 31 to 34, wherein the positions in both the first and the second heavy chain constant regions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, and wherein (i) the position in the first heavy chain constant region corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L and the position in the second heavy chain constant region corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, or (ii) the position in the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R and the position in the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L.

[0370] 36. The constant region of the first and / or second heavy chain comprises: (a) the sequence set forth in SEQ ID NO: 19 or 25 [IgG1-FC]; (b) a subsequence of the sequence (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having up to 10 substitutions, such as up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in (a) or (b). 36. The binding agent for use according to any one of items 16 to 35, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

[0371] 37. The constant region of the first or second heavy chain, e.g., the constant region of the second heavy chain, (a) the sequence set forth in SEQ ID NO: 20 or 26 [IgG1-F405L]; (b) a subsequence of the sequence of (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having up to 9 substitutions, such as up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in (a) or (b). 37. The binding agent for use according to any one of items 16 to 36, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

[0372] 38. The constant region of the first or second heavy chain, e.g., the constant region of the first heavy chain, (a) the sequence set forth in SEQ ID NO: 21 or 27 [IgG1-K409R]; (b) a subsequence of the sequence of (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having up to 10 substitutions, such as up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in (a) or (b). 37. The binding agent for use according to any one of items 16 to 36, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

[0373] 39. The constant region of the first and / or second heavy chain comprises: (a) the sequence set forth in SEQ ID NO: 22 or 28 [IgG1-Fc_FEA]; (b) a subsequence of the sequence of (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having up to seven substitutions, such as up to six substitutions, up to five, up to four, up to three, up to two substitutions or up to one substitution, compared to the amino acid sequence defined in (a) or (b). 16. The binding agent for use according to any one of items 16 to 15, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

[0374] 40. The constant region of the first and / or second heavy chain, e.g. the constant region of the second heavy chain, (a) the sequence set forth in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL]; (b) a subsequence of the sequence of (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having at most 6 substitutions, such as at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). 40. The binding agent for use according to any one of items 16 to 39, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

[0375] 41. The constant region of the first and / or second heavy chain, e.g. the constant region of the first heavy chain, (a) the sequence set forth in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR]; (b) a subsequence of the sequence of (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having at most six substitutions, such as at most five substitutions, at most four, at most three, at most two substitutions or at most one substitution, compared to the amino acid sequence defined in (a) or (b). 41. The binding agent for use according to any one of items 16 to 40, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

[0376] 42. A binding agent for use according to any one of the preceding items, wherein the binding agent comprises a kappa (κ) light chain constant region.

[0377] 43. A binding agent for use according to any one of the preceding items, wherein the binding agent comprises a lambda (λ) light chain constant region.

[0378] 44. A binding agent for use according to any one of the preceding items, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0379] 45. A binding agent for use according to any one of the preceding items, wherein the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0380] 46. ​​The binding agent for use according to any one of the preceding items, wherein the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region, or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0381] 47. The kappa (κ) light chain is (a) the sequence set forth in SEQ ID NO: 35; (b) a subsequence of the sequence of (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having up to 10 substitutions, such as up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in (a) or (b). 47. The binding substance for use according to any one of items 42 to 46, comprising an amino acid sequence selected from the group consisting of:

[0382] 48. The lambda (λ) light chain is (a) the sequence set forth in SEQ ID NO: 36; (b) a subsequence of the sequence of (a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted from the N-terminus or C-terminus of the sequence defined in (a), and (c) a sequence having up to 10 substitutions, such as up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in (a) or (b). 48. The binding substance for use according to any one of items 43 to 47, comprising an amino acid sequence selected from the group consisting of:

[0383] 49. A binding agent for use according to any one of the preceding items, wherein the binding agent is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0384] 50. A binding agent for use according to any one of the preceding items, wherein the binding agent is a full-length IgG1 antibody.

[0385] 51. A binding agent for use according to any one of the preceding items, wherein the binding agent is an antibody of the IgG1m(f) allotype.

[0386] 52. The binding substance, (i) a first heavy chain and a light chain comprising an antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises the sequence set forth in SEQ ID NO:31 and the first light chain comprises the sequence set forth in SEQ ID NO:32; (ii) a second heavy chain and a light chain comprising an antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a sequence set forth in SEQ ID NO: 33, and the second light chain comprises a sequence set forth in SEQ ID NO: 34. 2. A binding agent for use according to any one of the preceding items, comprising:

[0387] 53. The binding agent for use according to any one of the preceding items, wherein the binding agent is akasunlimab or a biosimilar thereof.

[0388] 54. The binding substance for use according to any one of the preceding items, wherein the binding substance is in the form of a composition or formulation comprising histidine, sucrose and polysorbate-80, and has a pH of 5 to 6.

[0389] 55. The binding material for use according to any one of the preceding items, wherein the binding material is in the form of a composition or formulation comprising about 20 mM histidine, about 250 mM sucrose, about 0.02% polysorbate-80, and having a pH of about 5.5.

[0390] 56. The binding substance for use according to any one of the preceding items, wherein the binding substance is in the form of a composition or formulation comprising 10 to 30 mg of binding substance / mL, for example 20 mg of binding substance / mL.

[0391] 57. The binding substance for use according to any one of the preceding items, wherein the binding substance is in the form of a composition as defined in any one of items 54 to 56 and is diluted with 0.9% NaCl (physiological saline) before administration.

[0392] 58. The binding agent for use according to any one of the preceding items, wherein the subject is a human subject.

[0393] 59. The binding agent for use according to any one of the preceding items, wherein the tumor or cancer is a solid tumor or solid cancer.

[0394] 60. The binding agent for use according to any one of the preceding items, wherein the tumor is a PD-L1 positive tumor.

[0395] 61. The binding agent for use according to any one of the preceding items, wherein the tumor or cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, renal cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic cancer, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma and mesothelioma.

[0396] 62. The binding agent for use according to any one of the preceding items, wherein the tumor or cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urothelial carcinoma (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial carcinoma (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)) and squamous cell carcinoma of the head and neck (SCCHN) (e.g., cancer of the oral cavity, pharynx, or larynx).

[0397] 63. The binding agent for use according to item 61 or 62, wherein the tumor or cancer is a lung cancer, in particular a non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

[0398] 64. The binding agent for use according to any one of items 61 to 63, wherein the tumor or cancer is metastatic, such as metastatic NSCLC.

[0399] 65. The binding agent for use according to items 61 to 64, wherein the lung cancer, in particular NSCLC, does not have an epidermal growth factor receptor (EGFR) sensitizing mutation and / or an anaplastic lymphoma (ALK) translocation / ROS1 rearrangement.

[0400] 66. The binding agent for use according to any one of items 61 to 65, wherein the lung cancer, in particular NSCLC, comprises cancer cells and PD-L1 is expressed in 1% or more of the cancer or tumor cells, e.g. as assessed by immunohistochemistry (IHC).

[0401] 67. The binding agent for use according to item 66, wherein the lung cancer, in particular NSCLC, comprises cancer cells and PD-L1 is expressed in 1% to 49% of the cancer or tumor cells, e.g. as assessed by immunohistochemistry (IHC).

[0402] 68. The binding agent for use according to item 66, wherein the lung cancer, in particular NSCLC, comprises cancer cells and PD-L1 is expressed in more than 50% of the cancer or tumor cells, e.g. as assessed by immunohistochemistry (IHC).

[0403] 69. A binding agent for use according to the preceding paragraph, wherein the subject has not received prior systemic treatment for metastatic disease.

[0404] 70. The binding agent for use according to any one of the preceding items, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g. a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0405] 71. The binding agent for use according to any one of the preceding items, wherein the subject has not been pretreated with a 4-1BB (CD137) targeting agent, such as an anti-4-1BB (CD137) antibody, with an anti-tumor vaccine, or with autologous cellular immunotherapy.

[0406] 72. The binding agent for use according to any one of items 1 to 68, wherein the tumor or cancer has relapsed and / or is refractory after treatment, such as systemic treatment with a checkpoint inhibitor.

[0407] 73. The binding agent for use according to any one of items 1 to 68 and 72, wherein the subject has received at least one line of prior systemic therapy, e.g. a systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0408] 74. The binding agent for use according to any one of items 1 to 68, 72 and 73, wherein the cancer or tumour has recurred and / or is refractory or the subject has progressed following treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein the PD-1 inhibitor or PD-L1 inhibitor is administered as monotherapy or as part of a combination therapy.

[0409] 75. The binding agent for use according to any one of items 1 to 68 and 72 to 74, wherein the last pretreatment is with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

[0410] 76. The binding agent for use according to any one of items 1 to 68 and 72 to 74, wherein the time from progression during the last treatment with a PD-1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, or a PD-L1 inhibitor, is within 8 months, such as within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, or for example within 2 weeks.

[0411] 77. The binding agent for use according to any one of items 1 to 68 and 72 to 74, wherein the time since the last dose of a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, as part of the last conditioning treatment, is within 8 months, such as within 7 months, within 6 months, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 3 weeks, or for example within 2 weeks.

[0412] 78. The following (i) or (ii), i.e. (i) treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody followed by platinum-based chemotherapy; or (ii) Treatment with anti-PD-1 or anti-PD-L1 antibodies following platinum-based chemotherapy 75. The binding agent for use according to any one of items 1 to 68 and 72 to 74, wherein the cancer or tumor recurs and / or is refractory or the subject progresses during or after the treatment.

[0413] 79. The binding agent for use according to any one of the preceding items, wherein the subject has not undergone prior treatment with a taxane chemotherapeutic agent, such as docetaxel, e.g. prior treatment of NSCLC with a taxane chemotherapeutic agent, such as docetaxel.

[0414] 80. The binding agent for use according to any one of the preceding items, wherein the binding agent and the antibody that binds PD-1, or antigen-binding fragment thereof, are administered in at least one treatment cycle, each treatment cycle being 3 weeks (21 days) or 6 weeks (42 days).

[0415] 81. The binding agent for use according to any one of the preceding items, wherein one dose of the binding agent and one dose of the antibody that binds to PD-1, or antigen-binding fragment thereof, are administered every three weeks (1Q3W).

[0416] 82. The binding agent for use according to any one of the preceding items, wherein one dose of the binding agent and one dose of the antibody that binds to PD-1, or antigen-binding fragment thereof, are administered every six weeks (1Q6W).

[0417] 83. The binding agent for use according to any one of the preceding items, wherein one dose of the binding agent and one dose of the antibody that binds PD-1, or antigen-binding fragment thereof, are administered on day 1 of each treatment cycle.

[0418] 84. The binding agent for use according to any one of the preceding items, wherein the amount of said binding agent administered in each dose and / or each treatment cycle is 100 mg.

[0419] 85. The binding agent for use according to any one of the preceding items, wherein the amount of the antibody that binds PD-1, or an antigen-binding fragment thereof, administered in each dose and / or each treatment cycle is 200 mg.

[0420] 86. The binding agent for use according to any one of the preceding items, wherein the amount of the antibody that binds PD-1, or an antigen-binding fragment thereof, administered in each dose and / or each treatment cycle is 400 mg.

[0421] 87. The binding agent for use according to any one of the preceding items, wherein a 100 mg dose of said binding agent and a 200 mg dose of said antibody that binds to PD-1, or antigen-binding fragment thereof, are administered every three weeks (1Q3W).

[0422] 88. The binding agent for use according to any one of the preceding items, wherein a 100 mg dose of the binding agent and a 400 mg dose of the antibody that binds to PD-1, or antigen-binding fragment thereof, are administered every six weeks (1Q6W).

[0423] 89. The binding agent for use according to any one of the preceding items, wherein the tumor or cancer is NSCLC, and wherein a 100 mg dose of the binding agent which is acasunlimab or a biosimilar thereof, and a 200 mg dose of the antibody that binds to PD-1 which is pembolizumab are administered every three weeks (1Q3W), e.g., on day 1 of each three-week treatment cycle.

[0424] 90. The binding agent for use according to any one of items 1 to 88, wherein the tumor or cancer is NSCLC, and wherein a 100 mg dose of the binding agent which is acasunlimab or a biosimilar thereof, and a 400 mg dose of the antibody that binds to PD-1 which is pembolizumab are administered every 6 weeks (1Q6W), e.g. on day 1 of a treatment cycle every 6 weeks.

[0425] 91. A binding agent for use according to any one of the preceding items, wherein the antibody that binds to PD-1, or an antigen-binding fragment thereof, is administered first, followed by administration of the binding agent.

[0426] 92. The binding agent for use according to any one of the preceding items, wherein the binding agent is administered by intravenous (IV) infusion over a period of at least 30 minutes, such as at least 60 minutes.

[0427] 93. The binding agent for use according to any one of the preceding items, wherein the binding agent is administered by intravenous (IV) infusion over 30 minutes.

[0428] 94. The binding agent for use according to any one of the preceding items, wherein the PD-1 inhibitor is administered as an intravenous infusion over 30 minutes.

[0429] 95. Subparagraphs (i) and (ii) below, i.e. (i) a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1; (a) said first binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; (b) said second antigen-binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; A binding agent comprising (ii) an antibody, or antigen-binding fragment thereof, that binds to PD-1, wherein the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; or the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively; Kit including:

[0430] 96. The kit according to item 95, wherein the binding agent and / or the antibody that binds to PD-1, or an antigen-binding fragment thereof, is as defined in any one of items 1 to 94.

[0431] 97. The kit according to item 95 or 96, wherein the binding agent and the antibody that binds PD-1, or antigen-binding fragment thereof, are for systemic administration, in particular for injection or infusion, such as intravenous injection or infusion.

[0432] 98. The kit according to any one of items 95 to 97, for use in a method for reducing or preventing the progression of a tumor or treating cancer in a subject.

[0433] 99. The kit for use according to item 98, wherein the tumor or cancer and / or the subject and / or the method are as defined in any one of items 1 to 94.

[0434] 100. A method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject a binding agent prior to, concurrently with, or following administration of an antibody that binds PD-1, or an antigen-binding fragment thereof; wherein the binding agent comprises a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1; (a) said first binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; And (b) said second antigen-binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; and wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; or the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively; The above method.

[0435] 101. The method according to item 100, wherein the tumor or cancer and / or the subject and / or the method and / or the binding agent and / or the PD-1 inhibitor are as defined in any one of items 1 to 94.

[0436] Further aspects of the disclosure are described herein. (Example)

[0437] [Example 1] Purified CD8 + Cytokine secretion in co-culture of T cells and allogeneic mature dendritic cells (mDCs) method Monocytes and T cells from healthy donors CD14 + Monocytes and purified CD8 + T cells were obtained from Precision Medicine or BioIVT. Allogeneic donor pairs were used for allogeneic mixed lymphocyte reactions (MLR assays).

[0438] Differentiation of monocytes into immature dendritic cells Human CD14 + Monocytes were obtained from healthy donors (see above). To differentiate into immature dendritic cells (iDCs), 1–1.5 × 10 monocytes were cultured in T25 culture flasks (Falcon, Cat. No. 353108). 6Cells / mL were cultured in Roswell Park Memorial Institute (RPMI) 1640 complete medium (ATCC modification formula; ThermoFisher, Cat. No. A1049101) supplemented with 10% heat inactivated fetal bovine serum (FBS; Gibco, Cat. No. 16140071), 100 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF; BioLegend, Cat. No. 766106), and 300 ng / mL interleukin-4 (IL-4; BioLegend, Cat. No. 766206) at 37°C for 6 days. The medium was replaced with fresh medium containing the supplements once during these 6 days.

[0439] The maturity of iDC To mature iDCs, cells were harvested by collecting non-adherent cells, counted, and cultured at 1–1.5 × 10 in RPMI 1640 complete medium supplemented with 10% FBS, 100 ng / mL GM-CSF, 300 ng / mL IL-4, and 1× lipopolysaccharide (LPS; ThermoFisher, catalog no. 00-4976-93). 6 cells / mL and incubated at 37° C. for 24 hours prior to initiation of the MLR assay.

[0440] Mixed lymphocyte reaction (MLR) The day before the start of the MLR assay, purified CD8 + T cells were thawed. Cells were cultured at 1 × 10 in complete RPMI 1640 medium supplemented with 10% FBS and 10 ng / mL IL-2 (BioLegend, Cat. No. 589106). 6 The cells were resuspended at 100 cells / mL and incubated overnight at 37°C.

[0441] The next day, LPS-matured dendritic cells (mDCs, see "Maturation of iDCs") and purified allogeneic CD8 + T cells were harvested and diluted to 4 × 10 5 cells / mL and 4 x 10 6The cells were resuspended in AIM-V medium (ThermoFisher, cat. no. 12055091) at 100 cells / mL.

[0442] For co-culture, 20,000 mDCs were cultured with 200,000 purified allogeneic CD8 + DCs were incubated with T cells (DC:T cell ratio 1:10) in AIM-V medium in the presence of GEN1046 (0.001-30 μg / mL) alone or in combination with research grade pembrolizumab (0.1-30 μg / mL or 0.1-100 μg / mL), bsIgG1-PD-L1×ctrl (30 μg / mL), bsIgG1-ctrl×4-1BB (30 μg / mL), isotype control antibody IgG4 (100 μg / mL), or IgG1-ctrl-FEAL (30 μg / mL; Table 5) at 37°C. After 5 days, plates were centrifuged at 500×g for 5 min and supernatants were carefully transferred from each well to a new 96-well round-bottom plate.

[0443] Supernatants collected from the MLR assays were analyzed for interferon (IFN)γ levels by enzyme-linked immunosorbent assay (ELISA) using the Alpha Lisa IFNγ kit (Perkin Elmer, catalog no. AL217) on an Envision instrument according to the manufacturer's instructions. TNFα and IL-2 were measured on a Luminex FLEXMAP 3D instrument as part of the Milliplex MAP- Human Cytokine / TH17 Panel (Millipore Sigma, catalog no. SPR1526).

[0444] [Table 5]

[0445] result GEN1046 significantly increased the number of purified CD8 +Co-cultures of T cells and allogeneic mDCs induced secretion of IL-2 (see Figure 2). In contrast, pembrolizumab induced only a modest increase in IL-2 (<50 pg / mL) compared to the isotype control IgG4 antibody. Simultaneous exposure to GEN1046 and pembrolizumab induced a robust increase in IL-2 compared to either GEN1046 or pembrolizumab alone, with a maximum IL-2 concentration approximately 2-3 fold higher than GEN1046 alone.

[0446] In addition, GEN1046 significantly increased the number of purified CD8 + Co-cultures of T cells and allogeneic mDCs induced IFNγ secretion (see FIG. 3). Similarly, pembrolizumab enhanced IFNγ secretion in all three donor pairs, albeit to a more limited extent compared to GEN1046. Simultaneous exposure to GEN1046 and pembrolizumab induced a further increase in IFNγ, especially at low doses of GEN1046 (<1 μg / mL), compared to either GEN1046 or pembrolizumab alone.

[0447] Furthermore, GEN1046 significantly reduced the number of purified CD8 + GEN1046 induced the secretion of TNFα in co-cultures of T cells and allogeneic mDCs (see FIG. 4). In contrast, pembrolizumab only induced limited amounts of TNFα compared to GEN1046. Co-exposure to GEN1046 and pembrolizumab induced a slight increase in TNFα for all pembrolizumab concentrations tested compared to GEN1046 alone, especially at 0.1 μg / mL GEN1046, indicating a left shift in efficacy.

[0448] These results suggest that the combination of GEN1046 and pembrolizumab enhances mDC / CD8 +In a T cell MLR assay, it was shown to enhance the secretion of IFNγ, IL-2, and TNFα compared to each antibody alone. Enhancement of IFNγ was mainly observed at low concentrations of GEN1046, but the combination of GEN1046 and pembrolizumab showed enhancement of IL-2 at various concentrations.

[0449] [Example 2] Tumor growth of MC38 mouse colon cancer method MC38 mouse colon carcinoma cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C and 5% CO. 2 MC38 cells were harvested from logarithmic growth phase cell cultures and quantified.

[0450] MC38 cells (1 × 10 tumor cells in 100 mL PBS) 6 ) were injected subcutaneously into the right lower flank of female C57BL / 6 mice (obtained from Vital River Laboratories Research Models and Services; 6-8 weeks old at the start of the experiment).

[0451] Tumor growth was assessed three times a week using a caliper. Tumor volume (mm 3 ) is calculated from the caliper measurement ([length] x [width] 2 ) / 2, where length is the longest dimension of the tumor and width is the longest dimension of the tumor perpendicular to the length).

[0452] Treatment was performed on patients with a median tumor volume of 64 mm 3 Mice were treated with 100 mg / kg of 10 ... 3). On the day of dosing, mice were intraperitoneally injected with mbsIgG2a-PD-L1x4-1BB (5 mg / kg; injection volume 10 μL / g body weight; twice weekly for 3 weeks [2QW×3]), anti-mouse PD-1 antibody (anti-mPD-1; 10 mg / kg; injection volume 10 μL / g body weight; 2QW×3; clone RMP1-14; Leinco Technologies, catalog no. P372), a combination of mbsIgG2a-PD-L1x4-1BB (5 mg / kg) and anti-mPD-1 (10 mg / kg) (two separate injections [mbsIgG2a-PD-L1x4-1BB followed by anti-mPD-1 20 min later], injection volume 10 μL / g body weight; 2QW×3), or PBS at an injection volume of 10 μL / g body weight (Table 6).

[0453] Mice were monitored daily for clinical signs of disease. Body weights were measured three times a week after randomization. Individual mice were cultured until tumor volumes reached 1500 mm3. 3 Experiments were terminated when the total number of mice exceeded 100 or when animals reached a welfare endpoint (e.g., when mice showed more than 20% weight loss, tumors showed ulceration [>75%], severe clinical signs were observed, and / or tumor growth inhibited physical activity of the mice).

[0454] [Table 6]

[0455] result Rapid tumor growth was observed in MC38 tumor-bearing mice treated with PBS (Figure 5A). Tumor growth delay was observed in mice treated with anti-mPD-1 (10 mg / kg) or mbsIgG2a-PD-L1x4-1BB (5 mg / kg), with a more pronounced tumor growth delay induced by mbsIgG2a-PD-L1x4-1BB (Figure 5A). In mice treated with mbsIgG2a-PD-L1x4-1BB (5 mg / kg) in combination with anti-mPD-1 (10 mg / kg; both 2QW×3), complete tumor regression was observed in 6 / 10 mice at day 21 after the start of treatment, whereas no complete tumor regression was observed with either agent alone in this model (Figure 5A). Kaplan-Meier analysis showed that the combination of mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 significantly reduced tumor volume below 500 mm compared with PBS (p<0.001) and either antibody alone (p≦0.001). 3 The combination induced a significant increase in progression-free survival, defined as a smaller proportion of mice with gliomas ...

[0456] These results provide a rationale for evaluating the combination of GEN1046 with anti-PD-1 antibodies to further amplify antitumor immune responses in cancer patients, leading to durable and profound clinical responses and improved survival.

[0457] [Table 7]

[0458] [Example 3] Antigen-specific CD8 to measure the proliferative dose response of GEN1046 and the anti-PD-1 antibody pembrolizumab in an antigen-specific T cell assay with an activated PD1 / PD-L1 system + T cell proliferation assay To measure the induction of T cell proliferation by DuoBody-PD-L1x4-1BB or pembrolizumab, an antigen-specific T cell proliferation assay with an activated PD1 / PD-L1 system was performed.

[0459] HLA-A2+ peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs by magnetic-activated cell sorting (MACS) using anti-CD14 MicroBeads (Miltenyi; catalog number 130-050-201) according to the manufacturer's instructions. Peripheral blood lymphocytes (PBLs, CD14 negative fraction) were cryopreserved for later isolation of T cells. For differentiation into immature DCs (iDCs), monocytes 1 × 10 6 Cells / mL were cultured for 5 days in RPMI GlutaMAX (Life technologies GmbH, Cat. No. 61870-044) containing 5% human AB serum (Sigma-Aldrich Chemie GmbH, Cat. No. H4522-100ML), sodium pyruvate (Life technologies GmbH, Cat. No. 11360-039), non-essential amino acids (Life technologies GmbH, Cat. No. 11140-035), 100 IU / mL penicillin-streptomycin (Life technologies GmbH, Cat. No. 15140-122), 1000 IU / mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi, Cat. No. 130-093-868) and 1000 IU / mL interleukin-4 (IL-4; Miltenyi, Cat. No. 130-093-924). Half of the medium was replaced with fresh medium once during these 5 days. iDCs were harvested by collecting non-adherent cells, and adherent cells were detached by incubation with PBS containing 2 mM EDTA for 10 min at 37° C. After washing, iDCs were cryopreserved in RPMI GlutaMAX containing 10% v / v DMSO (AppliChem GmbH, Cat. No. A3672,0050) + 50% v / v human AB serum for future antigen-specific T cell assays.

[0460] antigen-specific CD8 +Frozen PBL and iDC from the same donor were thawed the day before starting the T cell proliferation assay. + T cells were isolated from PBLs by the MACS method using anti-CD8 MicroBeads (Miltenyi, catalog no. 130-045-201) according to the manufacturer's instructions. Approximately 10–15 × 10 T cells were isolated in 250 μL of X-Vivo15 (Biozym Scientific GmbH, catalog no. 881026) in a 4 mm electroporation cuvette (VWR International GmbH, catalog no. 732-0023) using a BTX ECM® 830 Electroporation System device (BTX; 500 V, 1x3 ms pulse). 6 CD8 + T cells were electroporated with 10 μg of in vitro translated (IVT)-RNA encoding the α-chain and β-chain of claudin 6-specific mouse TCR (HLA-A2 restricted; described in WO 2015150327 A1), and 10 μg of IVT-RNA encoding PD-1. Immediately after electroporation, cells were transferred to fresh IMDM medium (Life Technologies GmbH, Cat. No. 12440-061) supplemented with 5% human AB serum and incubated at 37°C, 5% CO 2 T cells were labeled with 1.6 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, catalog no. C34564) in PBS according to the manufacturer's instructions and incubated overnight in IMDM medium supplemented with 5% human AB serum.

[0461] Using the electroporation system described above (300 V, 1x12 ms pulse), in 250 µL of X-Vivo15 medium, 1 µg (GEN1046 dose response) or 3 µg (pembrolizumab dose response) of IVT-RNA encoding full-length claudin-6 were injected into up to 5 × 10 6 Thawed iDCs were electroporated and incubated overnight in IMDM medium supplemented with 5% human AB serum.

[0462] The next day, cells were harvested. The cell surface expression of claudin-6 and PD-L1 on DCs, and TCR and PD-1 on T cells were confirmed by flow cytometry. DCs were stained with Alexa647-labeled CLDN6-specific antibody (not commercially available; produced in-house) and anti-human CD274 antibody (PD-L1, eBioscienes, Cat. No. 12-5983), and T cells were stained with anti-mouse TCR β chain antibody (Becton Dickinson GmbH, Cat. No. 553174) and anti-human CD279 antibody (PD-1, eBioscience, Cat. No. 17-2799). Electroporated DCs were incubated at a ratio of 1:10 with electroporated CFSE-labeled T cells in the presence of GEN1046 (3-fold serial dilutions from 1 to 0.00015 μg / mL) or in the presence of clinical grade pembrolizumab (4-fold serial dilutions from 0.8 to 0.00005 μg / mL; Keytruda, Phoenix Apotheke, PZN 10749897) in IMDM GlutaMAX supplemented with 5% human AB serum in 96-well round-bottom plates. Flow cytometric analysis of T cell proliferation based on CFSE dilution was performed after 5 days on a BD FACSCanto™ II or BD FACSCelesta™ flow cytometer (Becton Dickinson GmbH). Acquired data were analyzed using FlowJo software version 10.7.1. Proliferation index values ​​(determining fold proliferation of the entire culture) for each treatment condition were calculated and plotted as a function of GEN1046 or pembrolizumab concentration. Dose-response curves were generated and EC values ​​were calculated using a four-parameter logarithmic fit in GraphPad Prism version 9 (GraphPad Software, Inc.). 20 , E.C. 50 , E.C. 90 , and Hill-Slope values ​​were calculated.

[0463] The dose response of GEN1046 was analyzed in three-fold serial dilutions from 1 to 0.00015 μg / mL (Figure 6A), with EC 20 , E.C. 50, E.C. 90 The mean EC of the four donors tested is shown in Table 8. 50 At 0.0064 μg / mL, a strong proliferation-inducing effect was observed.

[0464] The dose response of pembrolizumab was analyzed in four-fold serial dilutions from 0.8 to 0.00005 μg / mL (Figure 6B), with EC 20 , E.C. 50 , E.C. 90 The mean EC of the four donors tested is shown in Table 9. 50 At 0.0149 μg / mL, a strong proliferation-inducing effect was observed.

[0465] [Table 8]

[0466] [Table 9]

[0467] [Example 4] GEN1046 reverses PD-1 / PD-L1-associated T cell suppression and releases additional CD8 + Costimulation of T cell proliferation. To measure the T cell proliferation induction by the combination of GEN1046 with the anti-PD-1 antibody pembrolizumab or IgG1-ctrl antibody, an antigen-specific T cell proliferation assay with an active PD1 / PD-L1 system was performed (the general assay setup is the same as in Example 1). That is, DCs electroporated with claudin-6-IVT-RNA were incubated with CFSE-labeled T cells electroporated with claudin-6-specific TCR- and PD1-IVT-RNA (ratio 1:10) in IMDM GlutaMAX supplemented with 5% human AB serum in a 96-well round-bottom plate in combination with a fixed concentration of pembrolizumab or isotype control antibody IgG1-ctrl in the presence of GEN1046. Three different concentrations of GEN1046 were tested, which correspond to the optimal, 50% and suboptimal effective concentrations determined in previous experiments (0.2 μg / mL >EC90; 0.0067 μg / mL ≒EC50; 0.0022 μg / mL ≒EC20, see Example 1, Table 1). Pembrolizumab and IgG1-ctrl antibodies were tested at a concentration of 0.8 μg / mL, which is well above the EC90 value of pembrolizumab (see Example 1, Table 2). Baseline proliferation was measured using only medium and 0.8 μg / mL IgG1-ctrl. Pembrolizumab (0.8 μg / mL) was used as an additional checkpoint inhibition control. Flow cytometric analysis of T cell proliferation based on CFSE dilution was performed after 5 days using a BD FACSCanto® II or BD FACSCelesta® flow cytometer (Becton Dickinson GmbH). Acquired data were analyzed using FlowJo software version 10.7.1. Proliferation index values ​​for each treatment condition were calculated and plotted using GraphPad Prism version 9 (GraphPad Software, Inc.).

[0468] Incubation of CD8+ T cells expressing PD-1 and claudin-6 specific TCR with DCs expressing PD-L1 and cognate antigen resulted in minimal proliferation induction with proliferation indices only slightly above 1 for media only and IgG1-ctrl treated cultures for all three donors tested (see Figure 7). Relief of PD-1:PD-L1 mediated inhibition by adding pembrolizumab to the co-culture environment resulted in a mild increase in the proliferation index as shown by the dashed line in the graph. Following addition of GEN1046, a dose-dependent as well as more pronounced increase in T cell proliferation was observed, with the highest concentration tested resulting in the highest proliferation induction compared to the medium and low concentration single compound treatment conditions. Notably, the lowest concentration of 0.0022 μg / mL GEN1046 (without pembrolizumab) resulted in proliferation index values ​​comparable to or lower than those recorded in the pembrolizumab-only control, indicating a suboptimal PD-1:PD-L1 checkpoint inhibitor. In stark contrast, the T cell proliferation induction of GEN1046 with pembrolizumab was always superior to DuoBody-PD-L1x4-1BB without pembrolizumab, regardless of the GEN1046 concentration tested. The difference in proliferation index between the pembrolizumab and non-pembrolizumab conditions was particularly large at medium and low concentrations of GEN1046. Notably, at suboptimal GEN1046 concentrations (0.0022 μg / mL ≒ EC20), the addition of pembrolizumab significantly increased the proliferation of CD8 + It induced T cell proliferation, and a significantly higher proliferation index was observed compared to the pembrolizumab-only control.

[0469] [Example 6] A First-in-Human, Open-Label, Dose-Escalation Study with Expansion Cohort to Evaluate the Safety of GEN1046 in Subjects with Malignant Solid Tumors This study is an open-label, multicenter, Phase 1 / 2a safety study of GEN1046 (DuoBody® PD-L1×4 1BB). The study consists of two parts: a first-in-human (FIH) dose-escalation study (Phase 1) and an expansion study (Phase 2a). The dose-escalation study evaluated GEN1046 in subjects with solid malignancies to determine the maximum tolerated dose (MTD) or maximum dose and / or recommended Phase 2 dose (RP2D).

[0470] Additionally, the expansion study will evaluate the safety, tolerability, PK, and antitumor activity of selected doses in expansion cohorts of selected solid tumors: non-small cell lung cancer (NSCLC) (PD-1 / L1 pre-treated and PD-1 / L1 naive), urothelial carcinoma (UC), endometrial carcinoma (EC), triple-negative breast cancer (TNBC) (subjects previously treated with PD-1 / L1 inhibitors and subjects not previously treated with PD-1 / L1 inhibitors), and squamous cell carcinoma of the head and neck (SCCHN).

[0471] [Table 10]

[0472] A schematic of the study design is shown in Figure 8. Further disclosure of the dose escalation study and expansion cohorts, as well as preliminary results of the dose escalation study, are described in International Patent Application WO 2021 / 156326.

[0473] Expansion cohorts (EC) A and B: Previously untreated NSCLC for metastatic disease: GEN1046 in combination with pembrolizumab Expansion cohorts ECA and ECB will evaluate 100mg GEN1046 in combination with pembrolizumab on two different dosing schedules: The ECA will test a GEN1046 dosing regimen of 100mg 1Q3W with a pembrolizumab dosing regimen of 200mg 1Q3W. Based on PK / pharmacodynamic modeling, this GEN1046 regimen is expected to result in peak trimer formation and sustained 4-1BB activation, which in combination with pembrolizumab may allow optimal co-target / pathway engagement and improved antitumor efficacy.

[0474] The ECB will evaluate a GEN1046 dosing regimen of 100 mg 1Q6W alongside a pembrolizumab dosing regimen of 400 mg 1Q6W. Based on PK / pharmacodynamic modeling, this GEN1046 regimen is expected to result in sustained activation of 4-1BB over a 3-week dosing cycle versus intermittent / transient activation of 4-1BB over a 6-week dosing cycle. Transient activation of 4-1BB is expected to allow resetting of T cell responses and reduce long-term interferon signaling (Weber, EW, et al. (2021), Science 372 (6537)), but it is unclear whether sustained 4-1BB activation may result in tumor-infiltrating CD8 + It prevents T cell exhaustion and, in combination with pembrolizumab, can improve depth of response and duration of response (DoR).

[0475] Pembrolizumab dosing regimens of 200 mg Q3W and 400 mg Q6W are approved as first-line and second-line SOC treatments, respectively, for NSCLC.

[0476] Discontinuation of treatment Treatment continues until the subject meets one of the discontinuation criteria (see below).

[0477] Selection Criteria Expansion Cohorts A and B Subjects with metastatic NSCLC who have not received prior systemic treatment regimens for metastatic disease. Subjects must not have received prior treatment with a PD-1 / L1 inhibitor. Subjects must not have had radiographic disease progression on or since their previous treatment, except for subjects with newly diagnosed disease.

[0478] b. Subjects with NSCLC of any histology will be enrolled. Subjects with histologically or cytologically diagnosed non-squamous NSCLC must not have EGFR sensitizing mutations and / or ALK translocations / ROS1 rearrangements. EGFR sensitizing mutations are those that are treatable with approved TKIs.

[0479] c. Subjects must have PD-L1 expression results available from a central laboratory prior to Cycle 1 Day 1 (C1D1) from a fresh tumor sample obtained by core needle or excision biopsy, or from tumor tissue resected at the time of diagnosis of metastatic disease.

[0480] d. Tumors demonstrate PD-L1 expression on ≥ 1% of tumor cells (TPS ≥ 1%) as assessed by immunohistochemistry (IHC) determined by a central laboratory.

[0481] Dose Escalation and Expansion 3. Subjects must be male or female, aged 18 years or older.

[0482] 4. Prior to any study-related evaluation or procedure, subjects must sign an Informed Consent Form (ICF) indicating that they understand the purpose of the study and the procedures involved, and that they are willing to participate in the study.

[0483] 5. Subjects must have disease assessable according to RECIST 1.1.

[0484] 6. Subjects must be Eastern Cooperative Oncology Group (ECOG) 0-1.

[0485] 7. Subjects must have the following organ and bone marrow function: a. Bone marrow / blood function: Absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L; hemoglobin ≥ 9.0 g / dL; platelet count ≥ 100 × 10 9 / L b. Liver function: - Total bilirubin ≦ upper limit of normal (ULN) - ALT ≦1.5×ULN - AST ≤ 1.5 × ULN - Albumin ≥ 30g / L c. Coagulation ability: - Prothrombin time (PT) / International normalized ratio (INR) ≦ 1.5 - Activated partial thromboplastin time (aPTT) ≤ 1.5 x ULN (without anticoagulation) - Subjects receiving anticoagulant therapy must have PT and aPTT within the therapeutic range for the intended use of the anticoagulant. d. Renal function: Glomerular filtration rate (GFR) ≥ 45 mL / min / 1.73 m 2 - For example, the MDRD (Modification of Diet in Renal Disease) simplified formula: GFR = 186 × (SCr -1.154 )×(age -0.203 ) by (where SCr is the serum creatinine level expressed in mg / dL; multiply this by 0.742 if the subject is female; multiply this by 1.212 if the subject is African American (Levey et al., 1999)).

[0486] 12. A) In the dose escalation part, all subjects must provide a tumor tissue sample (formalin-fixed paraffin-embedded block / slide), preferably from advanced stage disease, from archival tissue or fresh biopsy taken on or before Day 1 of Cycle 1. B) In the expansion part, all subjects must provide a defined fresh biopsy specimen (formalin-fixed paraffin-embedded [FFPE] block / slide) containing tumor tissue obtained after failure / discontinuation of the most recent prior treatment (bronchoscopy-guided biopsies, fine needle aspirates, cell blocks, cell pellets, clots, bone marrow, and cytology specimens are not acceptable).

[0487] Exclusion criteria Potential subjects meeting any of the following criteria will be excluded from participating in the study:

[0488] 1. Subject has uncontrolled comorbidities, including but not limited to: Ongoing or active infection requiring intravenous treatment with anti-infective medication administered within 2 weeks prior to the first dose. b. Symptomatic congestive heart failure (grade III or IV as classified by the New York Heart Association), unstable angina, or cardiac arrhythmia. c. Uncontrolled hypertension, defined as a systolic blood pressure of 160 mmHg or greater and / or a diastolic blood pressure of 100 mmHg or greater despite optimal medical management. d. Ongoing or recent (within 1 year) evidence of significant autoimmune disease requiring treatment with systemic immunosuppressive therapy that may indicate risk for immune-related adverse events (irAEs). e. Subjects with a history of Grade ≥ 3 irAEs leading to treatment discontinuation from prior immunotherapy should be excluded. Subjects with < Grade 3 irAEs leading to treatment discontinuation should be discussed with the sponsor. f. Exclude subjects with a history of myositis, Guillain-Barré syndrome, or myasthenia gravis, regardless of stage. g. History of chronic liver disease or evidence of cirrhosis. h. History of or current pneumonia requiring steroids. i. History of allogeneic organ transplant (excluding corneal transplant), autologous or allogeneic bone marrow transplant, or stem cell rescue within 3 months prior to the first dose of GEN1046. j. Severe non-healing wounds, skin ulcers (any degree), or broken bones.

[0489] 2. All subjects must undergo a brain computed tomography (CT) scan or magnetic resonance imaging (MRI) to document any new or existing central nervous system (CNS) lesions. Any history of cerebral arteriovenous malformations, cerebral aneurysms, spinal cord compression (due to disease), carcinomatous meningitis, or stroke will be excluded. a. Transient ischemic attack occurring ≥1 month prior to screening. b. Subjects with newly identified or known unstable or symptomatic CNS metastases will be excluded. Subjects with previously treated brain metastases may be enrolled provided they have radiographically stable disease (i.e., no evidence of progression) for at least 28 days on repeat imaging (note that repeat imaging must be performed during study screening). Subjects must be clinically stable and not have received short-term corticosteroid treatment or steroid tapering therapy, or stereotactic or whole-brain radiation therapy within 14 days prior to C1D1. Long-term steroid treatment is permitted as long as the dose has been stable for 14 days prior to C1D1 (≤10 mg prednisone daily or equivalent).

[0490] 3. Prior treatment Radiotherapy: Radiotherapy within 14 days prior to the first dose of GEN1046. Palliative radiotherapy is permitted. b. Anticancer therapy prior to GEN1046 administration (within 28 days or at least 5 drug half-lives, whichever is shorter), unless otherwise noted below. Exceptions are permitted for bisphosphonates (e.g., pamidronate, zoledronic acid, etc.) and denosumab. c. Subject has received an investigational medicinal product (including an investigational vaccine) within 28 days prior to the first scheduled dose of GEN1046, has used an invasive investigational medical device, or is currently enrolled in an interventional trial. NOTE: Subjects in the follow-up phase of the interventional study may participate if they have not received any investigational drug within 28 days of the first dose of GEN1046. d. History of treatment with live attenuated vaccines within 3 weeks prior to initiating GEN1046 administration. e. Chronic systemic administration of immunosuppressive corticosteroids, i.e., greater than 10 mg prednisone per day or a cumulative dose of prednisone greater than 150 mg within 14 days prior to the first dose of GEN 1046. Replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment and is permitted. f. Having received granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) supplementation 4 weeks prior to the first dose of GEN1046 or being long-term transfusion dependent. g. History of Grade 3 or greater allergic reaction to monoclonal antibody (mAb) therapy, and known or suspected allergy or intolerance to any medication administered during the course of the study. h. Subjects who discontinued treatment due to disease progression within 6 weeks of initiating a CPI-containing treatment. i. History of treatment with 4-1BB (CD137) targeted drugs. j. Prior treatment with T-cell agonists or anti-cytotoxic T-lymphocyte-associated protein 4 targeted agents within 12 weeks prior to initiation of treatment.

[0491] 4. Toxicities from previous anticancer therapy have not resolved to baseline levels or below Grade 1, with the exception of alopecia, anorexia, vitiligo, fatigue, hyperthyroidism, hypothyroidism, and peripheral neuropathy. Anorexia, hyperthyroidism, hypothyroidism, and peripheral neuropathy must have resolved to Grade 2 or below.

[0492] 5. Known past or present malignancy other than the enrolling diagnosis, except: Cervical cancer stage 1B or below. b. Noninvasive basal cell carcinoma or squamous cell carcinoma. c. Noninvasive superficial bladder cancer. d. Prostate cancer currently undetectable by PSA. e. Breast cancer in subjects with BRCA1 or BRCA2 positive ovarian cancer (not applicable to breast cancer expansion cohort). f. Curable cancer with CR >2 years.

[0493] 6. Subject has known allergy, hypersensitivity, or intolerance to GEN1046 or any of its excipients.

[0494] 7. The subject has a condition where, in the opinion of the investigator, participation would not be in the subject's best interest (e.g., would compromise health and well-being) or which may interfere with, limit, or disrupt the evaluations specified in the protocol.

[0495] 8. The subject has had major surgery (e.g., requiring general anesthesia) within 4 weeks prior to screening, has not fully recovered from surgery, or has surgery scheduled at the time the subject is expected to participate in the study. NOTE: Subjects whose scheduled surgery will be performed under local anesthesia may participate.

[0496] 9. History of seropositivity for human immunodeficiency virus (HIV).

[0497] 10. History of hepatitis B / positive serology (except for immunity through vaccination or recovery from natural infection, or passive immunity through immune globulin therapy): a. Positive antibody test for hepatitis B core antigen and b. Tested negative for antibodies to hepatitis B surface antigen.

[0498] 11. History of uncured, active Hepatitis C infection.

[0499] 12. Substance abuse or medical, psychological, or social conditions that may interfere with the subject's participation in the study or the assessment of the study results.

[0500] 13. Subject has previously been exposed to this study.

[0501] 14. The subject is a pregnant or lactating female.

[0502] 15. Subject has a contraindication to the use of pembrolizumab according to local prescribing information.

[0503] Administration of GEN1046 GEN1046 will be administered by intravenous infusion over a minimum of 60 minutes on Day 1 of each 21- or 42-day treatment cycle after all procedures and evaluations are completed.

[0504] In ECA and ECB, subjects will receive GEN1046 100 mg 1Q3W in combination with pembrolizumab 200 mg 1Q3W (ECA) or GEN1046 100 mg 1Q6W in combination with pembrolizumab 400 mg 1Q6W (ECB).

[0505] Pembrolizumab Administration ECA and ECB: After completion of all pre-dose procedures and evaluations, pembrolizumab 200 mg or 400 mg, respectively, will be administered on day 1 of each 3-week or 6-week dosing cycle.

[0506] Pembrolizumab is administered first, followed by GEN1046. Pembrolizumab is administered as an intravenous infusion over 30 minutes. After administration of pembrolizumab, immediate saline flushing is required to clear the infusion line before initiating the GEN1046 infusion. The interval between infusions should be approximately 30 minutes or longer, depending on the situation. Dose reduction of pembrolizumab is not recommended.

[0507] Study Drug Information GEN1046 is supplied as a clear to milky, colorless to pale yellow solution formulated at 20 mg / mL in 20 mM histidine, 250 mM sucrose, 0.02% polysorbate 80, pH 5.5, which can be diluted (extemporaneously) with 0.9% NaCl (saline) for infusion.

[0508] Pembrolizumab (KEYTRUDA®) intravenous infusion is a sterile, preservative-free, clear to slightly opalescent, colorless to pale yellow solution that requires dilution for intravenous infusion.

[0509] GEN1046 Infusion-Related Reactions (IRR) For subjects who experience an IRR following administration of GEN1046: - Grade 1: If a grade 1 IRR occurs, the infusion does not need to be interrupted and may be continued at half the rate at the investigator's discretion under close medical supervision. - Grade 2-3: If an IRR grade 2 or 3 occurs, the infusion must be interrupted and appropriate medical management initiated. If symptoms resolve to grade 1 or less within 1 hour, the infusion may be resumed at half the rate under close medical supervision at the investigator's discretion. o Subjects in clinical trials who have previously experienced a grade 2 or 3 infusion reaction should be premedicated. Premedication to prevent IRR in subsequent infusions can be at the investigator's discretion according to local guidelines, but should preferably include an antihistamine (e.g., diphenhydramine 50 mg or equivalent antihistamine), acetaminophen / paracetamol (e.g., acetaminophen 500-1000 mg or equivalent), and, if deemed necessary, subjects should receive the maximum recommended dose of 100 mg prednisone or equivalent corticosteroid. If a subject experiences a second grade 3 IRR despite premedication, the infusion should be discontinued and the subject should be withdrawn from treatment. - Grade 4: If anaphylaxis or a Grade 4 IRR occurs, administration of GEN1046 should be immediately discontinued and appropriate medical treatment should be administered.

[0510] Pembrolizumab infusion reactions Pembrolizumab can cause serious or life-threatening IRRs, including severe hypersensitivity or anaphylaxis. Signs and symptoms usually occur during or shortly after infusion and generally resolve completely within 24 hours after the end of the infusion.

[0511] [Table 11]

[0512] Discontinuation of treatment Subjects will receive GEN1046 on day 1 of each 3-week or 6-week dosing cycle until they meet one of the predefined discontinuation criteria (below).

[0513] Subjects in the ECA and ECB cohorts will receive pembrolizumab in combination with GEN1046 on day 1 of each 3-week dosing cycle or day 1 of each 6-week dosing cycle, respectively, until disease progression or one of the predefined discontinuation criteria is met. Both GEN1046 and pembrolizumab should be discontinued. Subjects may continue on GEN1046 or pembrolizumab monotherapy only if approved by the sponsor's medical monitor. Upon discontinuation of both drugs, subjects will enter the safety follow-up period. - Radiographic progression or confirmation of radiographic progression by iRECIST - clinical progression - Untraceable - Subject requests discontinuation - death - Unacceptable adverse effects requiring discontinuation of study treatment - The investigator determines that it is in the subject's best interest to discontinue study treatment. - Withdrawal of consent - Pregnancy

[0514] Efficacy evaluation All subjects undergo imaging of the brain, chest, abdomen, and pelvis at screening. All subjects with SCCHN require imaging of the head and neck.

[0515] Tumor imaging will preferably be obtained by computed tomography (CT). Up to five target lesions (maximum two per organ) will be identified at screening and followed throughout the study. Non-target lesions will also be evaluated throughout the study. The first tumor imaging at screening will be performed within 21 days prior to the first dose date. The site will review the screening images to confirm that subjects have measurable lesions per RECIST 1.1.

[0516] Imaging during the study will be performed every 6 weeks (± 7 days) for 50 weeks from the date of first treatment, and then every 12 weeks (± 7 days) until investigator assessment of disease progression (unless the investigator chooses to continue treatment and follow iRECIST), initiation of new anti-cancer therapy, withdrawal of consent, or death, whichever occurs first.

[0517] RECIST 1.1 criteria will be used to assess response to secondary endpoints (Eisenhauer et al., 2009, Eur J Cancer 45, 228-247.); iRECIST will be used to assess response to exploratory endpoints (Seymour et al., 2017, Lancet Oncol 18, e143-e152). If the investigator chooses to apply iRECIST, treatment should continue until progressive disease (PD) is confirmed.

[0518] Additional CT or MRI scans may be performed at the investigator's discretion to confirm response or new symptoms. Tumor imaging to confirm partial response (PR) or complete response (CR) will be performed at least 4 weeks after the first confirmed response.

[0519] Disease activity assessment by iRECIST iRECIST is based on RECIST 1.1 but has been modified to account for the unique response patterns observed with immunotherapy. iRECIST will be evaluated as an exploratory endpoint in this study (Seymour et al., 2017, Lancet Oncol 18, e143-e152).

[0520] iRECIST disease progression must be confirmed at least 4-7 weeks after the first radiographic evidence of PD in clinically stable participants. Subjects without confirmed disease progression may continue receiving GEN1046 until confirmed disease progression as long as they remain clinically stable. Clinically stable subjects must meet the following criteria: - Subjects must be experiencing clinical benefit from continued GEN1046 treatment (as assessed by the investigator) and not be showing rapid disease progression - Subjects tolerate GEN1046 - Subjects must demonstrate stable ECOG status - Administration after progression does not delay urgent intervention to prevent serious complications of disease progression (e.g., central nervous system metastases that require prompt treatment).

[0521] Clinically unstable subjects will discontinue GEN1046 at the first evidence of radiographic progression. If repeat imaging reveals iRECIST-confirmed disease progression (iCPD), subjects will discontinue GEN1046.

[0522] ECOG performance status ECOG performance status will be assessed by the investigator at Screening, Day 1 of each cycle, and at the discontinuation visit. Performance status will be scored using the ECOG Performance Status Scale Index (Table 12).

[0523] [Table 12]

[0524] Preliminary Results and Conclusions Doses of 25 to 1200 mg Q3W evaluated in the titration phase of the FIH study were safe and generally well tolerated. The maximum tolerated dose (MTD) was not reached. - Preliminary evaluation of safety data showed no dose-dependency and no dose-response in frequency of adverse events (AEs). - Responses based on RECIST v1.1 were observed with GEN1046 doses of 80 to 200 mg Q3W during the dose escalation phase of the FIH study. Additionally, responses were observed in a dose expansion of 100 mg Q3W. - Sustained modulation of pharmacodynamic markers (proliferation of [Ki67+] effector memory CD8+ T cells and total CD8+ T cells, and increased IFNγ and IP-10 levels) was observed in peripheral blood at dose levels up to and including 200 mg. Decreased modulation of these endpoints was observed at higher dose levels (≥ 400 mg). - A semi-mechanistic PK / pharmacodynamic model (see Example 13 of WO 2021 / 156326) predicted a bell-shaped response for trimer formation, which peaked around 100mg Q3W. To balance trimer levels and target binding for PD-L1 RO, a dose of 100mg Q3W was selected that could provide an optimal initial response to GEN1046. - GEN1046 monotherapy resulted in longer progression-free survival (PFS) in subjects previously treated with a checkpoint inhibitor (Figure 9). - In checkpoint inhibitor-pretreated NSCLC subjects, clinical response to GEN1046 monotherapy was associated with time since last prior anti-PD-1 therapy (Figure 12). NSCLC subjects who responded to GEN1046 monotherapy tended to have received more recent prior anti-PD-1 therapy. The shorter time since treatment with anti-PD-1 drugs may suggest that residual anti-PD-1 activity drives the response to GEN1046. In support of this, patients treated with anti-PD-1 drugs in the clinic showed long-term PD-1 receptor occupancy by therapeutic antibodies, which could persist for more than 200 days (Brahmer et al., JCO 2010; 28(19): 3167-3175). The persistence of anti-PD-1 therapeutics bound to the PD-1 receptor could lead to an increase in the number of free PD-L1 molecules available for binding to GEN1046. Residual anti-PD-1 activity may allow for more complete blockade of the PD-1 pathway (blocking the interaction of PD-1 with both PD-L1 and PD-L2), which may be important for the biological activity of GEN1046 in the post-CPI setting. More recent anti-PD-1 therapies may have a direct effect on the tumor microenvironment, for example, by initiating an anti-tumor immune response, which may be enhanced by GEN1046 if administered immediately or shortly after progression on anti-PD-1 treatment. Responders demonstrated a "low" proportion of PD-1+ CD8 T cells, which may reflect receptor occupancy (RO) from prior anti-PD-1 therapy. Conversely, non-responders generally demonstrated a higher proportion of PD-1+ CD8 T cells, which may indicate a more exhausted phenotype.

[0525] [Example 8] A Phase 2, Multicenter, Randomized, Open-Label Study of GEN1046 as Monotherapy and in Combination with Pembrolizumab in Patients with Relapsed / Refractory Metastatic Non-Small Cell Lung Cancer After Standard of Care with Immune Checkpoint Inhibitors Study design This is a Phase 2, multicenter, randomized, open-label study evaluating the safety and efficacy of GEN1046 as monotherapy and in combination with pembrolizumab in adult patients with locally advanced or metastatic NSCLC following treatment with a CPI-containing regimen.

[0526] The study will enroll approximately 126 subjects, with 120 eligible subjects (40 per group) randomized to one of the treatment arms described below. Subjects must provide fresh and / or archived tumor tissue for future central laboratory confirmation of tumor PD-L1 expression. The proportion of subjects with non-squamous histology will be capped at approximately 70%. Randomization will be stratified by PD-L1 expression (TPS ≥ 50% vs 1%-49%) and histology (squamous vs non-squamous).

[0527] A. GEN1046 100mg Q3W for the first 2 cycles, followed by GEN1046 500mg Q6W for subsequent cycles. B. GEN1046 100mg Q3W in combination with pembrolizumab 200mg Q3W C. GEN1046 100mg Q6W in combination with pembrolizumab 400mg Q6W

[0528] During a preliminary safety run-in, six subjects will be enrolled into arms B and C (three in each group). These subjects will be closely monitored and followed for a minimum of three weeks. After the completion of the safety run-in for these cohorts, the collected data (including but not limited to all relevant safety and clinical data) will be evaluated. After this review, if the combination regimen is deemed well tolerated, randomization of arms A, B, and C will begin.

[0529] Treatment of a subject should continue until the subject meets one of the dosing criteria defined below.

[0530] Computed tomography (CT) or magnetic resonance imaging (MRI) with contrast will be performed at baseline before the first dose, and at 6, 12, 18, and 24 weeks (± 7 days) after the first dose of study medication, and then every 9 weeks (± 7 days) thereafter. CT or MRI will be obtained until disease progression (investigator assessment), initiation of next anticancer treatment, withdrawal of consent, or death, whichever occurs first. Disease progression as defined by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 must be confirmed by an additional confirmatory scan after the first documented disease progression (PD). Clinically unstable subjects will discontinue study treatment at the first evidence of radiographic disease progression and will not need to undergo repeated imaging to confirm PD. If a delayed response to treatment is suspected, the investigator may continue treatment beyond the time of RECIST v1.1-defined progression if the subject is experiencing clinical benefit. After a subject experiences PD, vital status will be collected every 12 weeks until death, withdrawal of consent, loss to follow-up, or end of study, whichever occurs first. Subsequent anticancer treatment and the subject's response to it will also be collected. iRECIST will be used to assess subsequent progression while administering past progression as defined by RECIST v1.1.

[0531] Study design rationale This is a randomized, open-label study evaluating the safety and efficacy of GEN1046 as monotherapy or in combination with pembrolizumab in adult patients with relapsed / refractory metastatic NSCLC after treatment with a CPI-containing regimen. Randomization eliminates potential allocation bias, while the open-label design allows for efficient management of AEs / SAEs. PD-L1 expression levels are associated with pembrolizumab efficacy, and histology is an important baseline disease characteristic. Randomization will be stratified to ensure balance between arms for these two factors.

[0532] The primary objective is to evaluate the objective response rate (ORR) of antitumor activity of GEN1046 as monotherapy and in combination with pembrolizumab, an established efficacy parameter for evaluating antitumor activity in proof-of-concept trials in NSCLC.

[0533] Rationale for Dosing and Schedule The choice of 100 mg Q3W dose for GEN1046 was based on clinical data from the FIH study, GCT1046-01, which evaluated doses from 25 to 1200 mg Q3W in 61 subjects in the dose escalation phase.Furthermore, to understand the PK / pharmacodynamics / efficacy relationship, a PK / pharmacodynamics model was developed to predict 4-1BB, GEN1046, PD-L1 trimolecular complex (trimer) formation, and RO (receptor occupancy) of PD-L1 in tumors (see Example 9).

[0534] In summary, a dose of GEN1046, 100 mg Q3W, was selected based on the dose escalation study in Example 7.

[0535] In Arm A, a regimen of an activation dose of GEN1046 (100 mg Q3W for two cycles) followed by a higher maintenance dose of GEN1046 (500 mg Q6W for subsequent cycles) will be tested based on the following: - Semi-mechanistic PK / pharmacodynamic model PK / pharmacodynamic model showed that trimer formation in tumors peaked with the 100mg Q3W regimen of GEN1046, which was chosen as the activation dose for the first 2 cycles because it is predicted to result in sustained 4-1BB activation. In the GCT1046-01 study, clinical data from the expansion cohort showed that the 100mg Q3W dose produced responses within the first 2 cycles. - After the first two cycles, a maintenance regimen of GEN1046 500mg Q6W will be used, which is predicted to result in a higher PD-L1 RO throughout the dosing cycle and to a lesser extent intermittent 4-1BB activation via bound trimers compared to 100mg Q3W. This dose is expected to result in improved duration of response (DOR).

[0536] Arms B and C will evaluate the combination of GEN1046 and pembrolizumab in two dosing schedules: - In arm B, GEN1046 100mg Q3W regimen will be tested alongside pembrolizumab 200mg Q3W regimen. In this regimen, GEN1046 is expected to provide the highest trimer formation and sustained 4-1BB activation, allowing optimal target / pathway binding and improved antitumor efficacy in combination with pembrolizumab. - In arm C, GEN1046 100mg Q6W regimen will be evaluated along with pembrolizumab 400mg Q6W regimen. Based on PK / pharmacodynamic modeling, GEN1046 is expected to provide sustained 4-1BB activation in 3-week dosing cycles, versus intermittent / transient 4-1BB activation in 6-week dosing cycles. Transient activation of 4-1BB is expected to allow resetting of T cell responses to reduce long-term interferon signaling (Weber, EW, et al. (2021), Science 372 (6537)), which may prevent exhaustion of tumor-infiltrating CD8+ T cells due to sustained 4-1BB activation, and in combination with pembrolizumab, may result in improved depth of invasion and DOR.

[0537] Pembrolizumab 200mg Q3W and 400mg Q6W regimens are approved as first-line and second-line SOC treatment for NSCLC, respectively. Pembrolizumab 200mg Q3W and 400mg Q6W are expected to provide comparable efficacy and safety profiles (Lala et al., 2020, Eur J Cancer 131, 68-75).

[0538] Selection Criteria 1. Subjects must be 18 years of age or older.

[0539] 2. Subjects must have histologically or cytologically confirmed stage 4 NSCLC and have received at least one prior systemic therapy for metastatic disease, including an anti-PD-1 / PD-L1 mAb. NOTE: Subjects must have received at least two prior doses of an anti-PD-1 / PD-L1 mAb approved for NSCLC. a. Subjects progressed on or after treatment with a single dose of anti-PD-1 / PD-L1 mAb as monotherapy or in combination with SOC. b. Subject progressed during or after platinum combination therapy following anti-PD-1 / PD-L1 mAb. c. Subject experienced disease progression during or after platinum combination therapy followed by anti-PD-1 / PD-L1 mAb.

[0540] 3. Subjects must have a central laboratory-assessed PD-L1 tumor expression score of ≥ 1% on the TPS during screening.

[0541] 4. Subjects must have measurable disease by RECIST v1.1.

[0542] 5. ECOG (Eastern Cooperative Oncology Group) performance status (PS) must be 1 or less.

[0543] 6. Subjects must have a life expectancy of 3 months or greater.

[0544] 7. Subjects must have the following organ and bone marrow function: Absolute neutrophil count (ANC) ≥ 1500 / L. b. Platelet count ≥100,000 / L. c. Hemoglobin ≥ 9.0 g / dL (if no transfusion within 4 weeks prior to randomization). d. Total bilirubin ≤ 1.5 x institutional upper limit of normal (ULN) (except in cases of Gilbert syndrome, in which case direct bilirubin ≤ 2 x institutional ULN and total bilirubin < 3 mg / dL). e. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 3 x ULN. If alkaline phosphatase is concomitantly elevated above 2.5 x ULN, ALT and AST values ​​should be ≤ 1.5 x ULN. f. Glomerular filtration rate is 45mL / min / 1.73m according to the simplified MDRD (Modification of Diet in Renal Disease) formula. 2 That's all. g. Prothrombin time (PT) / International normalized ratio (INR) ≤ 1.5 x ULN (unless the subject is receiving anticoagulant therapy, in which case PT should be within the therapeutic range for the intended use of the anticoagulant). h. Activated partial thromboplastin time (aPTT) ≤ 1.5 x ULN (unless the subject is receiving anticoagulant therapy, in which case the aPTT should be within the therapeutic range for the intended use of the anticoagulant).

[0545] Exclusion crit...

Claims

Claim 1: A pharmaceutical composition comprising a binding agent for use in a method for reducing or preventing tumor progression or treating cancer in a human subject, the method comprising administering the binding agent to the subject prior to, concurrently with, or following administration of an antibody that binds to programmed death-1 (PD-1), or an antigen-binding fragment thereof; where the binding agent comprises a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1; (a) the first binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second antigen-binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; and the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; or the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively; The pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO:

52.

3. 2. The pharmaceutical composition of claim 1, wherein the antibody that binds to PD-1 is pembrolizumab or a biosimilar thereof.

4. The following are true: (a) and (b), i.e. (a) the first binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 5; and (b) the second binding region of the binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 15; The pharmaceutical composition of claim 1.

5. 2. The pharmaceutical composition of claim 1, wherein the binding agent is akasunlimab or a biosimilar thereof.

6. the binding agent is in the form of a composition or formulation comprising histidine, sucrose and polysorbate-80, and has a pH of 5 to 6; or the binding agent is in the form of a composition or formulation comprising about 20 mM histidine, about 250 mM sucrose, about 0.02% polysorbate-80, and having a pH of about 5.5; The pharmaceutical composition of claim 1.

7. The pharmaceutical composition of claim 1, wherein the tumor or cancer is a solid tumor or solid cancer, or the tumor or cancer is a PD-L1-positive tumor.

8. the tumor or cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, renal cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma; or The tumor or cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urothelial carcinoma (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)), and squamous cell carcinoma of the head and neck (SCCHN) (e.g., cancer of the oral cavity, pharynx, or larynx), or the tumor or cancer is lung cancer, particularly non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC; The pharmaceutical composition of claim 1.

9. The pharmaceutical composition of claim 8, wherein the tumor or cancer is metastatic.

10. 9. The pharmaceutical composition of claim 8, wherein the lung cancer, particularly NSCLC, comprises cancer cells and PD-L1 is expressed in 1% or more of the cancer or tumor cells, e.g., as assessed by immunohistochemistry (IHC).

11. 2. The pharmaceutical composition of claim 1, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., a PD-1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

12. 10. The pharmaceutical composition of claim 1, wherein the tumor or cancer has recurred and / or is refractory after treatment, such as systemic treatment with a checkpoint inhibitor.

13. 2. The pharmaceutical composition of claim 1, wherein the cancer or tumor has recurred and / or is refractory, or the subject has progressed, following treatment with a PD-1 or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

14. 10. The pharmaceutical composition of claim 1, wherein one dose of the binding agent and one dose of the antibody that binds PD-1, or antigen-binding fragment thereof, are administered every three weeks (1Q3W) or every six weeks (1Q6W).

15. a 100 mg dose of the binding agent and a 200 mg dose of the antibody that binds PD-1, or antigen-binding fragment thereof, administered every three weeks (1Q3W); or a 100 mg dose of the binding agent and a 400 mg dose of the antibody that binds PD-1, or antigen-binding fragment thereof, are administered every six weeks (1Q6W); The pharmaceutical composition of claim 1.

16. The pharmaceutical composition of claim 1, wherein the interval between the end of administration of the binding agent and the start of administration of the antibody that binds to PD-1, or the interval between the end of administration of the antibody that binds PD-1 and the start of administration of the binding agent, is at most about 14 days.

17. The following (i) and (ii), namely (i) a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1; (a) the first binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; (b) the second antigen-binding region comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; a binding agent comprising (ii) an antibody, or antigen-binding fragment thereof, that binds to PD-1, wherein the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; or the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively; Kit including:

18. A combination drug comprising (i) and (ii), i.e., the combination drug comprises: (i) a binding agent comprising a first binding domain that binds to CD137 and a second antigen-binding domain that binds to PD-L1, (a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. and (ii) an antibody or antigen-binding fragment thereof that binds to PD-1, the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 43, 44 and 45, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 46, 47 and 48, respectively; or the antibody comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 62, 63 and 64, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 65, 66 and 67, respectively; The combination drug comprising:

19. 19. The kit of claim 17 or the pharmaceutical combination of claim 18, wherein the binding agent and / or the antibody or antigen-binding fragment thereof that binds to PD-1 is as defined in any one of claims 1 to 16.

20. 19. The kit of claim 17 or the pharmaceutical combination of claim 18, wherein the binding agent and the antibody that binds PD-1, or antigen-binding fragment thereof, are for systemic administration, in particular for injection or infusion, such as intravenous injection or infusion.

21. 19. The kit of claim 17 or the pharmaceutical combination of claim 18 for use in a method for reducing or preventing tumor progression or treating cancer in a subject.

22. The kit or pharmaceutical combination according to claim 21, wherein the tumor or cancer and / or the subject and / or the method is as defined in any one of claims 1 to 16.

23. The kit of claim 17, or the pharmaceutical combination of claim 18, wherein the binding agent is formulated to be administered before, simultaneously with, or after the antibody or antigen-binding fragment thereof.

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