Multispecific binding agents to CD40 and CD137 in combination therapy for cancer

Abstract

The present invention relates to combination therapies using binding agents that bind human CD40 and human CD137, in combination with checkpoint inhibitors to reduce or prevent tumor progression or to treat cancer.

Description

[Technical Field]

[0001] The present invention relates to combination therapies using binding agents that bind to human CD40 and human CD137 in combination with checkpoint inhibitors to reduce or prevent tumor progression or to treat cancer. [Background technology]

[0002] CD40 is a member of the tumor necrosis factor (TNF) receptor (TNFR) family and is known as a costimulatory protein found on various cell types. CD40 is constitutively expressed by antigen-presenting cells (APCs) such as dendritic cells (DCs), B cells, and macrophages. It can also be expressed by endothelial cells, platelets, smooth muscle cells, fibroblasts, and epithelial cells. Consistent with its widespread expression on normal cells, CD40 is also expressed on a variety of tumor cells.

[0003] antigen-specific CD4 + Presentation of peptide antigens in association with MHC class II molecules to T cells is mediated by CD4, together with costimulatory signals (from CD80 and / or CD86). + This results in T cell activation and upregulation of the DC licensing factors CD40 ligand (CD40L) and lymphotoxin-α1β2 (LTα1β2). Expression of CD40L and LTα1β2 on activated antigen-specific CD4+ T cells induces signaling through CD40 and the LTβ receptor (LTβR), which in turn induces DCs to express CD8 +It provides a license to induce T cell responses. CD40 signaling leads to the production of interleukin-12 (IL-12) and upregulation of CD70, CD86, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), and GITR ligand (GITRL), whereas LTβR signaling leads to the production of type I interferon (IFN). The signaling system that controls the activity of nuclear factor kappa B (NF-κB) responds to virtually all TNFR superfamily members. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) also contribute to these events. MHC class I-restricted peptide-mediated CD8 + T cell priming results in upregulation of CD27, 4-1BB, OX40, and glucocorticoid-inducible TNFR-related protein (GITR). + Stimulation of these receptors in T cells with their cognate TNF superfamily ligands, in combination with IL-12 and type I IFN, results in robust CD8 + T cell activation, proliferation and effector function, as well as CD8 +CD40 antibodies can exert various effects, including the death of CD40-expressing tumor cells by inducing antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cell-mediated phagocytosis (ADCP), as well as direct apoptosis or cell signaling to induce growth arrest, independent of CD40 expression on tumor cells via APC licensing to stimulate anti-cancer immune responses. Antibodies that bind to CD40 can initiate the priming of effector cytotoxic T lymphocytes (CTLs) via CD40 on APCs, induce the release of IL-2 by these cells, and indirectly activate NK cells. Antibodies that stimulate CD40 have been disclosed in the prior art, including the human IgG2 antibody CP-870,893 (WO03 / 040170), the humanized IgG1 antibody dacetuzumab (WO00 / 075348), and the chimeric IgG1 antibody Chi Lob7 / 4 (US2009 / 0074711). Additionally, a CD40 antagonist antibody, the human IgG1 antibody lucatumumab (WO02 / 028481), has been disclosed.

[0004] CD137 (4-1BB) is also a member of the TNFR family. +CD137 is a costimulatory molecule on CD4+ T cells, regulatory T cells (Tregs), natural killer T cells (NK(T) cells), B cells, and neutrophils. In T cells, CD137 is not constitutively expressed but is induced upon T cell receptor (TCR) activation, 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 agonist antibodies triggers signal transduction using TRAF-2 and TRAF-1 as adaptors. Early signaling by CD137 involves K-63 polyubiquitination, which ultimately leads to activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways. Signaling leads to increased T cell costimulation, proliferation, cytokine production, maturation, and long-term 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 can induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art, including the human IgG4 antibody urelumab (AU2004279877) and the human IgG2 antibody utomilumab (Fisher et al., 2012, Cancer Immunol. Immunother. 61:1721-1733).

[0005] Westwood JA, et al., Leukemia Research 38 (2014), 948-954, discloses "Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers." WO2018 / 011421 provides binding agents, such as bispecific antibodies, that bind to human CD40 and human CD137. Such bispecific antibodies crosslink CD40 on antigen-presenting cells (APCs) with 4-1BB on activated T cells, thereby inducing conditional stimulatory and costimulatory activity in both cell types, useful for the treatment of solid tumors.

[0006] PD-1, CTLA4, PD-L1, TIM-3, KIR, and LAG-3 are inhibitory checkpoint molecules that regulate the immune system and enable self-tolerance, making them ideal targets for cancer immunotherapy.

[0007] In tumor-draining lymph nodes and within the tumor microenvironment, 4-1BB is expressed by subsets of CD4+ and CD8+ T cells characterized by coexpression of multiple TCR-inducible molecules, including high levels of programmed cell death 1 (PD-1) (Gros et al., J. Clin Invest 2014;124(5):2246-59; Seifert et al., Cancers (Basel) 12; Simoni et al., Nature 557: 575-579). Upregulation of PD-1 on T cells may contribute to T cell exhaustion and reduce T cell activation upon binding to its ligand, programmed cell death 1 ligand 1 (PD-L1) (Yu et al., Eur J Pharmacol 881: 173240). PD-L1 expression is often upregulated by tumor cells, especially in inflamed tumors (Teng, et al., Cancer Res 75: 2139-2145). This allows tumor cells to provide inhibitory signals to activated T cells, allowing them to escape T cell-mediated cytotoxicity. Antibodies that block the PD-1 / PD-L1 inhibitory axis can restore T cell function (Boussiotis et al., N Engl J Med 375: 1767-1778; Chen et al., Nature 541: 321-330). Summary of the Invention [Problem to be solved by the invention]

[0008] However, despite these advances in the art, there remains a significant need for improved therapies to prevent tumor progression or treat cancer. [Means for solving the problem]

[0009] The inventors surprisingly found that the combination of (i) stimulation with a binding agent that binds to human CD40 and human CD137, and (ii) checkpoint inhibition (particularly inhibition of the PD-1 / PD-L1 axis) amplifies the immune response.

[0010] Accordingly, in a first aspect, the present disclosure provides a binding agent for use in a method for reducing or preventing tumor progression or treating cancer in a subject, said method comprising administering the binding agent to said subject prior to, simultaneously with, or after administration of a checkpoint inhibitor, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

[0011] In a second aspect, the present disclosure provides a kit comprising (i) a binding agent comprising a first binding region that binds to CD40 and a second binding region that binds to CD137, (ii) a checkpoint inhibitor, and optionally (iii) one or more additional therapeutic agents.

[0012] In a third aspect, the present disclosure provides a kit of the second aspect for use in a method for reducing or preventing the progression of a tumor or for treating cancer in a subject.

[0013] In a fourth aspect, the present disclosure provides a method for reducing or preventing tumor progression or treating cancer in a subject, said method comprising administering to said subject a binding agent prior to, concurrently with, or after administration of a checkpoint inhibitor, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137. [Brief explanation of the drawings]

[0014] [Figure 1]Figure 1 shows a schematic diagram of the expected mechanism of action of the CD40x4-1BB bispecific antibody. CD40 is expressed on antigen-presenting cells (APCs) as well as tumor cells. 4-1BB (CD137) is expressed on activated T cells. DuoBody-CD40x4-1BB (GEN1042 / BNT312) is a bispecific antibody that crosslinks CD40 on antigen-presenting cells (APCs) with 4-1BB on activated T cells, thereby conditionally stimulating both cell types. The CD40x4-1BB bispecific antibody can thereby enhance DC licensing, T cell clonal expansion, cytokine production, T cell survival, and T cell- and NK cell-mediated cytotoxicity. [Figure 2] Figure 2 shows IFNγ production induced by bsIgG1-CD40x4-1BB in combination with pembrolizumab in a mixed lymphocyte reaction (MLR) of mature dendritic cells (mDCs) and purified CD8+ T cells. Purified CD8+ T cells were cocultured with allogeneic LPS-matured DCs for 5 days in the presence of bsIgG1-CD40x4-1BB (0.001–30 μg / mL), pembrolizumab (0.1–30 μg / mL), or both, alone or in combination. IFNγ secretion was analyzed by ELISA. Data shown are the mean IFNγ ± standard deviation (SD) of duplicate wells from one representative donor pair of five donor pairs included in three experiments. The horizontal lines in the graph, from top to bottom, represent IFNγ production for 10 μg / mL bsIgG1-CD40×4-1BB without pembrolizumab (dashed line), 10 μg / mL pembrolizumab without bsIgG1-CD40×4-1BB (dashed and dotted line), and no treatment (thin plain line). [Figure 3]Figure 3 shows IFNγ production induced by bsIgG1-CD40x4-1BB in combination with pembrolizumab in a mixed lymphocyte reaction (MLR) of mature dendritic cells (mDCs) and purified CD8+ T cells. Purified CD8+ T cells were cocultured with allogeneic LPS-matured DCs for 5 days in the presence of bsIgG1-CD40x4-1BB (0.001–30 μg / mL), pembrolizumab (0.1–30 μg / mL), or both, alone or in combination. IFNγ secretion was analyzed by ELISA. Data shown are the mean IFNγ ± standard deviation (SD) of duplicate wells in three experiments. Each individual graph represents one of five donor pairs. [Figure 4] Figure 4 shows IFNγ production induced by DuoBody-CD40×4-1BB in combination with pembrolizumab in a mixed lymphocyte reaction (MLR) of mature dendritic cells (mDCs) and purified CD8+ T cells. Purified CD8+ T cells were cocultured with allogeneic LPS-matured DCs for 5 days in the presence of DuoBody-CD40×4-1BB (0.001–30 μg / mL), pembrolizumab (0.1–100 μg / mL), or control antibodies bsIgG1-CD40×ctrl, bsIgG1-ctrl×4-1BB, IgG1-ctrl-FEAL (all at 30 μg / mL), or IgG4 isotype control (100 μg / mL), either alone or in combination. IFNγ secretion was analyzed by ELISA. Data shown are the mean IFNγ ± standard deviation (SD) of duplicate wells from one donor pair (n=1). [Figure 5]Figure 5 shows IFNγ production induced by DuoBody-CD40x4-1BB or bsIgG1-CD40x4-1BB, either alone or in combination with pembrolizumab, in a mixed lymphocyte reaction (MLR) of mature dendritic cells (mDCs) and purified CD8+ T cells. Purified CD8+ T cells were cocultured with allogeneic LPS-matured DCs for 5 days in the presence of DuoBody-CD40x4-1BB (0.001–30 μg / mL), bsIgG1-CD40x4-1BB (0.001–30 μg / mL), either alone or in combination with pembrolizumab (1 μg / mL). An additional monotherapy control treatment of 30 μg / mL IgG1-ctrl-FEAL was evaluated. IFNγ secretion was analyzed by ELISA. Data shown are the mean IFNγ±standard deviation (SD) of duplicate wells from one donor pair (n=1). [Figure 6] Figure 6 shows IFNγ production induced by in-house derived bsIgG1-CD40x4-1BB in combination with nivolumab in a mixed lymphocyte reaction (MLR) of mature dendritic cells (mDCs) and purified CD8+ T cells. Purified CD8+ T cells were cocultured with allogeneic LPS-matured DCs for 5 days in the presence of bsIgG1-CD40x4-1BB (0.001–10 μg / mL), nivolumab (α-PD-1; 0.0005–5 μg / mL), or both, alone or in combination. IFNγ secretion was analyzed by ELISA. Data shown are the mean IFNγ ± standard deviation (SD) of duplicate wells from one donor pair. [Figure 7]Figure 7 shows IFNγ secretion induced by IgG1-PD1 in combination with DuoBody-CD40x4-1BB in an allogeneic mixed lymphocyte reaction (MLR) assay. Two unique donor pairs of allogeneic human mature dendritic cells (mDCs) and CD8+ T cells were co-cultured for 5 days in the presence of IgG1-PD1 (0.001–100 μg / mL), DuoBody-CD40x4-1BB (0.001–30 μg / mL), or a combination of IgG1-PD1 and DuoBody-CD40x4-1BB. Controls included IgG1-ctrl-FERR (100 μg / mL), bsIgG1-CD40xctrl (30 μg / mL), bsIgG1-ctrlx4-1BB (30 μg / mL), and IgG1-ctrl-FEAL (30 μg / mL). IFNγ secretion in supernatants was analyzed using an IFNγ-specific AlphaLISA immunoassay. Data shown are the mean IFNγ levels ± standard error of the mean (SEM) for two unique allogeneic donor pairs treated with one representative concentration of 1 μg / mL IgG1-PD1. [Figure 8] Figure 8 shows synergy analysis of IFNγ secretion induced by the combination of IgG1-PD1 and DuoBody-CD40x4-1BB in an allogeneic mixed lymphocyte reaction (MLR) assay. Synergy of the treatment combination of IgG1-PD1 and DuoBody-CD40x4-1BB in a 7x7 dose-response matrix in an allogeneic MLR assay was determined using the Bliss and Highest Single Agent (HSA) synergy scoring models for donor pairs 1 (A) and 2 (B). A score of ≥ 10 indicates synergy. [Figure 9]Figure 9 shows the enhancement of CD8+ T cell proliferation by IgG1-PD1 in combination with DuoBody-CD40x4-1BB in an antigen-specific T cell stimulation assay. Human CD8+ T cells were electroporated with RNA encoding the claudin 6 (CLDN6)-specific T cell receptor (TCR) and RNA encoding programmed cell death protein 1 (PD-1) and labeled with carboxyfluorescein succinimidyl ester (CFSE). T cells were then cocultured with CLDN6-electroporated immature dendritic cells (iDCs) in the presence of 0.8 μg / mL of IgG1-PD1, pembrolizumab, or IgG1-ctrl-FERR, either alone or in combination with the indicated concentrations of DuoBody-CD40x4-1BB. After 4 days, CFSE dilution in T cells was analyzed by flow cytometry and used to calculate the expansion index. Data from one representative donor out of four donors evaluated in two independent experiments is shown. Error bars represent the standard deviation (SD) of duplicate wells. The dotted line indicates the expansion index of CD8+ T cells co-cultured with mock-electroporated (i.e., not expressing CLDN6) iDCs. [Figure 10] Figure 10 shows enhanced cytokine secretion by IgG1-PD1 in combination with DuoBody-CD40x4-1BB after antigen-specific CD8+ T cell stimulation. Human CD8+ T cells expressing claudin 6 (CLDN6)-specific T cell receptors (TCRs) and programmed cell death protein 1 (PD-1) were cocultured with CLDN6-expressing iDCs in the presence of 0.8 μg / mL of IgG1-PD1, pembrolizumab, or IgG1-ctrl-FERR, either alone or in combination with the indicated concentrations of DuoBody-CD40x4-1BB, as described in Figure 9. Cytokine concentrations in the culture supernatants were determined after 4 days. Data from one representative donor out of four donors evaluated in two independent experiments are shown. Error bars represent the standard deviation (SD) of duplicate wells. [Figure 11]Figure 11 shows the binding of IgG1-PD1 to PD-1 from different species. CHO-S cells transiently transfected with different species of PD-1 were incubated with IgG1-PD1, pembrolizumab, or the non-binding control antibodies IgG1-ctrl-FERR and IgG4-ctrl, and binding was analyzed using flow cytometry. Non-transfected CHO-S cells incubated with IgG1-PD1 were included as negative controls. (A-B) Data shown are the geometric mean fluorescence intensity (gMFI) ± SD of duplicate wells from one representative experiment out of four. (C-D) Data shown are the gMFI ± SD of duplicate wells from one representative experiment out of two. (E) Data shown are the geometric mean fluorescence intensity (gMFI) ± SD of duplicate wells from one representative experiment out of four. Abbreviations: gMFI = geometric mean fluorescence intensity; PD-1 = programmed cell death protein 1; PE = R-phycoerythrin. [Figure 12]Figure 12 shows the competitive binding of IgG1-PD1 to PD-L1 and PD-L2 for human PD-1. CHO-S cells transiently transfected with human PD-1 were incubated with 1 μg / mL of biotinylated recombinant human PD-L1 (A) or PD-L2 (B) in the presence of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR was included as a negative control. Cells were stained with streptavidin-allophycocyanin, and the percentage of cells binding to biotinylated PD-L1 or PD-L2 was determined by measuring the percentage of streptavidin-allophycocyanin+ cells using flow cytometry. The percentage of streptavidin-allophycocyanin+ cells in the no antibody control and untransfected samples is indicated by a dashed line. Data shown are from a single replicate from one representative experiment of three separate experiments. Abbreviations: Ab = antibody; CHO-S = Chinese hamster ovary, suspension; ctrl = control; FERR = L234F / L235E / G236R-K409R; PD-1 = programmed cell death protein 1; PD-L1 = programmed cell death 1 ligand 1; PD-L2 = programmed cell death 1 ligand 2. [Figure 13] Figure 13 shows functional inhibition of the PD-1 / PD-L1 checkpoint by IgG1-PD1. Blockade of the PD-1 / PD-L1 axis was tested using a cell-based bioluminescent PD-1 / PD-L1 blockade reporter assay. Data shown are the mean luminescence ± SD of duplicate wells in one representative experiment of five (pembrolizumab and IgG1-PD1), three (IgG1-ctrl-FERR), or two (nivolumab) experiments. Abbreviations: FERR = L234F / L235E / G236R-K409R; PD1 = programmed cell death protein 1; PD-L1 = programmed cell death 1 ligand 1; RLU = relative light units; SD = standard deviation. [Figure 14]Figure 14 shows the enhancement of CD8+ T cell proliferation by IgG1-PD1 in an antigen-specific T cell proliferation assay. Human CD8+ T cells were electroporated with RNA encoding a CLDN6-specific TCR and RNA encoding PD-1 and labeled with CFSE. T cells were then cocultured with iDCs electroporated with RNA encoding CLDN6 in the presence of IgG1-PD1, pembrolizumab, nivolumab, or IgG1-ctrl-FERR. After 4 days, CFSE dilution in T cells was analyzed by flow cytometry and used to calculate the expansion index. Data from one representative donor (26268_B) out of four donors evaluated in three independent experiments are shown. Error bars represent the SD of duplicate wells. Curves were fitted using a four-parameter logarithmic fit using GraphPad Prism. Abbreviations: CFSE = carboxyfluorescein succinimidyl ester; FERR = L234F / L235E / G236R-K409R; PD1 = programmed cell death protein 1; SD = standard deviation. [Figure 15] Figure 15 shows IFNγ secretion induced by IgG1-PD1 in an allogeneic MLR assay. Three unique donor pairs of allogeneic human mDCs and CD8+ T cells were cocultured for 5 days in the presence of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR and an IgG4 isotype control were included as negative controls. IFNγ secretion in the supernatants was analyzed using an IFNγ-specific immunoassay. Data shown are the mean ± standard error (SEM) concentrations of three unique allogeneic donor pairs. Abbreviations: FERR = L234F / L235E / G236R-K409R; IFN = interferon; IgG = immunoglobulin G; mDC = mature dendritic cells; MLR = mixed lymphocyte reaction; SEM = standard error. [Figure 16]Figure 16 shows cytokine secretion induced by IgG1-PD1 in an allogeneic MLR assay. Three unique donor pairs of allogeneic human mDCs and CD8+ T cells were cocultured for 5 days in the presence of 1 μg / mL of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR was included as a negative control. Cytokine secretion in supernatants was analyzed using Luminex. (A) Cytokine levels are expressed as the mean fold change relative to cytokine levels measured in untreated cocultures. (B) Levels of cytokine production for three unique allogeneic donor pairs are shown; horizontal lines indicate the mean, upper, and lower limits. Abbreviations: FC = fold change; FERR = L234F / L235E / G236R-K409R; GM-CSF = granulocyte-macrophage colony-stimulating factor; IgG = immunoglobulin G; IL = interleukin; MCP-1 = monocyte chemotactic protein 1; mDC = mature dendritic cell; MLR = mixed lymphocyte reaction; TNF = tumor necrosis factor. [Figure 17] Figure 17 shows C1q binding to membrane-bound IgG1-PD1. C1q binding to IgG1-PD1 was analyzed using stimulated human CD8+ T cells. After incubation with IgG1-PD1, IgG1-ctrl-FERR, IgG1-ctrl, or the positive control antibody IgG1-CD52-E430G (without inactivating mutations and with hexamerization-enhancing mutations), cells were incubated with human serum as a source of C1q. C1q binding was detected using a FITC-conjugated rabbit anti-C1q antibody. Data shown are the geometric mean fluorescence intensity (gMFI) ± standard deviation (SD) from duplicate wells from one representative donor out of seven across three equivalent experiments. Abbreviations: FITC = fluorescein isothiocyanate; gMFI = geometric mean fluorescence intensity; PE = R-phycoerythrocyanin. [Figure 18]Figure 18 shows FcγR binding of IgG1-PD1. Binding of IgG1-PD1 to immobilized human recombinant FcγR constructs was analyzed by SPR in a qualified assay (n=1). Binding of IgG1-PD1 to FcγRIa (A), FcγRIIa-H131 (B), FcγRIIa-R131 (C), FcγRIIb (D), FcγRIIIa-F158 (E), and FcγRIIIa-V158 (F). Antibody IgG1-ctrl (without FER inactivating mutations) was included as a positive control for binding. Abbreviations: ctrl = control; FcγR = Fc gamma receptor; IgG = immunoglobulin G; PD-1 = programmed cell death protein 1; RU = resonance units. [Figure 19] Figure 19 shows FcγR binding of IgG1-PD1 and several other anti-PD-1 antibodies. Binding of IgG1-PD1, nivolumab, pembrolizumab, dostallimab, and cemiplimab to immobilized human recombinant FcγR constructs was analyzed by SPR (n=3). Binding of the test antibodies to FcγRIa (A), FcγRIIa-H131 (B), FcγRIIa-R131 (C), FcγRIIb (D), FcγRIIIa-F158 (E), and FcγRIIIa-V158 (F) is shown. IgG1-ctrl and IgG4-ctrl antibodies were included as positive controls for FcγR binding of IgG1 and IgG4 molecules with wild-type Fc regions. Binding responses ± SD from three separate experiments are shown. Abbreviations: ctrl = control; FcγR = Fc gamma receptor; IgG = immunoglobulin G; PD-1 = programmed cell death protein 1; RU = resonance units. [Figure 20]Figure 20 shows FcγRIa binding of IgG1-PD1 and several other anti-PD-1 antibodies. Binding of IgG1-PD1, nivolumab, pembrolizumab, dostallimab, and cemiplimab to CHO-S cells transiently expressing human FcγRIa was analyzed by flow cytometry. IgG1-ctrl and IgG1-ctrl-FERR were included as positive and negative controls, respectively. Abbreviations: ctrl = control; FcγR = Fc gamma receptor; FERR = L234F / L235E / G236R-K409R; huIgG = human immunoglobulin G; PD-1 = programmed cell death protein 1; PE = R-phycoerythrin. [Figure 21] Figure 21 shows total human IgG in mouse plasma samples. At t=0, mice were intravenously injected with 1 or 10 mg / kg IgG1-PD1, and serial plasma samples were taken at 10 minutes, 4 hours, 1 day, 2 days, 8 days, 14 days, and 21 days post-injection. Total huIgG in plasma samples was determined for each mouse by ECLIA. Data are presented as the mean huIgG concentration ± SD of three individual mice. The dashed line indicates the plasma concentration of wild-type (wt) huIgG predicted by a two-compartment model based on IgG clearance in humans (Bleeker et al., 2001, Blood. 98(10):3136-42). The dotted lines indicate the LLOQ and ULOQ. Abbreviations: huIgG = human IgG; IgG = immunoglobulin G; LLOQ = lower limit of quantification; PD-1 = programmed cell death protein 1; SD = standard deviation; ULOQ = upper limit of quantification. [Figure 22]Figure 22 shows the anti-tumor activity of IgG1-PD1 in human PD-1 knock-in mice. An MC38 colon cancer syngeneic tumor model was established by SC implantation in hPD-1 KI mice. Mice were administered 0.5, 2, or 10 mg / kg of IgG1-PD1 or pembrolizumab, or 10 mg / kg of IgG1-ctrl-FERR, 2QW x 3 (9 mice per group). (A) Mean tumor volume ± SEM in each group until the last time point at which the group was terminated. (B) Tumor volume of different groups on the last day (day 11) at which all groups were terminated. Data shown are tumor volumes of individual mice in each treatment group, as well as the mean tumor volume ± SEM per treatment group. Tumor volumes were compared between the IgG1-ctrl-FERR-treated group and the untreated groups using Mann-Whitney analysis. *p<0.05, **p<0.01, and ***p<0.001. C. Progression-free survival, defined as the percentage of mice with tumor volumes smaller than 500 mm3, is shown as a Kaplan-Meier curve. The analysis excluded one mouse from the 2 mg / kg IgG1-PD1 group that was found to have died of unclear causes on day 16, before the tumor volume exceeded 500 mm3. Abbreviations: 2QWx3 = twice weekly for 3 weeks; ctrl = control; FERR = L234F / L235E / G236R / K409R mutations; IgG = immunoglobulin G; KI = knock-in; PD-1 = programmed cell death protein 1; SC = subcutaneous; SEM = standard error of mean. [Figure 23]Figure 23 shows the secretion of IFNγ (A), GM-CSF (B), TNFα (C), IL-2 (D), and IL-6 (E) induced by DuoBody-CD40x4-1BB in combination with atezolizumab, nivolumab, or pembrolizumab in a mixed lymphocyte reaction (MLR) of mature dendritic cells (mDCs) and purified CD8+ T cells. Purified CD8+ T cells were cocultured with allogeneic LPS-matured DCs for 5 days in the presence of DuoBody-CD40x4-1BB (0.001–30 μg / mL), atezolizumab (1 μg / mL), nivolumab (1 μg / mL), or pembrolizumab (1 μg / mL) alone, or DuoBody-CD40x4-1BB in combination with atezolizumab, nivolumab, or pembrolizumab. Cocultures without treatment (no Tx) or treated with bsIgG1-CD40xctrl (30 μg / mL), bsIgG1-ctrlx4-1BB (30 μg / mL), or IgG1-ctrl-FEAL (30 μg / mL) were included as controls. IFNγ secretion was analyzed by ELISA, and GM-CSF, TNFα, IL-2, and IL-6 secretion were analyzed by Luminex. Data shown are the mean + standard deviation (SD) of duplicate wells of one representative donor pair out of four pairs tested in one experiment. [Figure 24]Figure 24 shows the effect of DuoBody-CD40x4-1BB in combination with an anti-PD-(L)1 antibody on T cell proliferation in vitro. Human CD8+ T cells were electroporated with RNA encoding a CLDN6-specific TCR together with RNA encoding PD-1 and labeled with CFSE. T cells were then co-cultured for 4 days with iDCs electroporated with RNA encoding CLDN6 in the presence or absence of DuoBody-CD40x4-1BB (0.2, 0.0067, or 0.0022 μg / mL) and the anti-PD-1 antibodies IgG1-PD1 (0.8 μg / mL), pembrolizumab (0.8 μg / mL), or nivolumab (1.6 μg / mL), the anti-PD-L1 antibody atezolizumab (0.4 μg / mL), or the negative control antibody IgG1-ctrl-FERR (0.8 μg / mL). CFSE dilution in T cells was analyzed by flow cytometry and used to calculate the expansion index. Data from one representative donor out of four tested are shown. Error bars indicate the SD of duplicate wells. The dotted line represents the expansion index of CD8+ T cells co-cultured with iDCs without antibody treatment. CFSE = carboxyfluorescein succinimidyl ester; CLDN6 = claudin-6; iDC = immature dendritic cell; PD-(L)1 = programmed cell death protein (ligand) 1; SD = standard deviation; TCR = T cell receptor. [Figure 25]Figure 25 shows the effect of DuoBody-CD40x4-1BB in combination with an anti-PD-(L)1 antibody on cytokine secretion in vitro. Human CD8+ T cells expressing CLDN6-specific TCR and PD-1 were cocultured with CLDN6-expressing iDCs for 4 days in the presence of DuoBody-CD40x4-1BB (0.2, 0.0067, or 0.0022 μg / mL) and the anti-PD-1 antibodies IgG1-PD1 (0.8 μg / mL), pembrolizumab (0.8 μg / mL), or nivolumab (1.6 μg / mL), the anti-PD-L1 antibody atezolizumab (0.4 μg / mL), or the negative control antibody IgG1-ctrl-FERR (0.8 μg / mL), as in Figure 24. Cytokine concentrations in the supernatants were determined by multiplex ECLIA. Data from one representative donor out of four tested are shown. Error bars indicate SD of duplicate wells. CLDN6 = claudin-6; ECLIA = electrochemiluminescence immunoassay; GM-CSF = granulocyte / macrophage colony-stimulating factor; iDC = immature dendritic cells; IFN = interferon; IL = interleukin; PD-(L)1 = programmed cell death protein (ligand) 1; SD = standard deviation; TCR = T cell receptor; TNF = tumor necrosis factor. [Figure 26]Figure 26 shows the effect of DuoBody-CD40x4-1BB in combination with anti-PD-(L)1 antibody on T cell proliferation in vitro. CellTrace Violet-labeled human PBMCs were stimulated for 4 days with anti-CD3 antibody (0.09 μg / mL) in the presence of DuoBody-CD40x4-1BB (0.2 μg / mL), either alone or in combination, and the anti-PD-1 antibodies pembrolizumab or nivolumab, or the anti-PD-L1 antibody atezolizumab (all at 0.05, 0.5, or 5 μg / mL), or the negative control antibody IgG1-ctrl-FEAL (0.2 μg / mL). CellTrace Violet dilution in CD8+ (Panel A) and CD4+ T cells (Panel B) was analyzed by flow cytometry and used to calculate the expansion index. Data from one representative donor of three donors tested is shown. Error bars indicate SD of triplicate wells. The dotted line represents the expansion index of cells treated with IgG1-ctrl-FEAL. The dashed line represents the expansion index of cells treated with single-agent DuoBody-CD40x4-1BB. PD-(L)1 = programmed cell death protein (ligand) 1; PBMC = peripheral blood mononuclear cells; SD = standard deviation. [Figure 27] Figure 27 shows the characterization of the exhaustion-like phenotype of CD3+ T cells after two rounds of CD3 / CD28 stimulation. (A) LAG3 expression in in vitro exhausted CD3+ T cells was determined by flow cytometry. Data shown are median fluorescence intensity corrected for background fluorescence (ΔMFI). (B) In vitro exhausted CD3+ T cells were cocultured with allogeneic LPS-matured DCs without treatment or in the presence of 1 μg / mL pembrolizumab. IFNγ secretion was analyzed by AlphaLISA, and IL-2 secretion was analyzed by MSD multiplex. Data shown are the mean + standard deviation (SD) of duplicate wells of one representative donor pair of two pairs tested in two experiments. [Figure 28]Figure 28 shows IFNγ (A) and IL-2 (B) secretion induced by DuoBody-CD40x4-1BB in combination with pembrolizumab in a mixed lymphocyte reaction (MLR) between mature dendritic cells (mDC) and in vitro exhausted CD3+ T cells (Tex). Tex were cocultured with allogeneic LPS-matured DCs for 5 days in the presence of DuoBody-CD40x4-1BB (0.001–30 μg / mL) or pembrolizumab (1 μg / mL), alone or in combination. Cocultures without treatment (no Tx) or treated with bsIgG1-CD40xctrl (30 μg / mL), bsIgG1-ctrlx4-1BB (30 μg / mL), or IgG1-ctrl-FEAL (30 μg / mL) were included as controls. IFNγ secretion was analyzed by AlphaLISA and IL-2 secretion was analyzed by MSD multiplex. Data shown are the mean + standard deviation (SD) of duplicate wells of one representative donor pair out of two tested in two experiments. DETAILED DESCRIPTION OF THE INVENTION

[0015] Table 1 - Sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0016] SEQ ID NO: 63 HCDR1 (MAB-19-0618) Kabat and IMGT consensus SEQ ID NO: 64HCDR1 (MAB-19-0618) Kabat SEQ ID NO: 45HCDR1 (MAB-19-0618) IMGT SEQ ID NO: 65 HCDR2 (MAB-19-0618) Kabat and IMGT consensus (=IMGT) SEQ ID NO: 66HCDR2 (MAB-19-0618) Kabat SEQ ID NO: 47 HCDR3 (MAB-19-0618) Kabat and IMGT common region (=Kabat) SEQ ID NO: 67HCDR3 (MAB-19-0618) IMGT SEQ ID NO: 48 LCDR1 (MAB-19-0618) Kabat and IMGT consensus (=IMGT) SEQ ID NO: 68LCDR1 (MAB-19-0618) Kabat SEQ ID NO: 49 LCDR2 (MAB-19-0618) Kabat and IMGT consensus (=IMGT) SEQ ID NO: 69LCDR2 (MAB-19-0618) Kabat SEQ ID NO: 50LCDR3 (MAB-19-0618) Intersection = Kabat = IMGT

[0017] The present disclosure is further described in more detail below, but it is understood that this disclosure is not limited to the particular methodologies, protocols, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any order to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with various disclosed and / or preferred elements. Furthermore, all permutations and combinations of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise. For example, if in a preferred embodiment of the binding agent used herein the first heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR] and in another preferred embodiment of the binding agent used herein the second heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL], then in a further preferred embodiment of the binding agent used herein the first heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR] and the second heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL].

[0019] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G. W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0020] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques 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; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. See Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0021] All methods described herein may be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "for example," etc.), are intended merely to better illustrate the present disclosure and do not otherwise impose limitations on the scope of the claimed present disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0022] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise stated herein, each separate value is incorporated into the specification as if it were individually listed herein.

[0023] Numerous documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0024] definition The following definitions are provided and apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise specified. All terms not defined have their art-recognized meanings.

[0025] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to mean the inclusion of a stated element, integer, or step or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step or group of elements, integers, or steps. The term "consisting essentially of" means the exclusion of other elements, integers, or steps of some essential importance. The term "comprising" encompasses the term "essentially consisting of," which in turn encompasses the term "consisting of." Thus, at each occurrence in this application, the term "comprising" can be replaced with the term "essentially consisting of" or "consisting of." Similarly, at each occurrence in this application, the term "essentially consisting of" can be replaced with the term "consisting of."

[0026] As used in the context of describing the present disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references shall be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0027] "And / or," as used herein, shall be construed as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" shall be construed as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0028] In the context of the present disclosure, the term "about" refers to a range of accuracy that a person skilled in the art would understand to still ensure the technical function of the characteristic in question. This term typically refers to a deviation from the specified 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 understood by a person skilled in the art, the specific deviation from the numerical value for such a given technical function will depend on the nature of the technical function. For example, a natural or biological technical function may generally have a greater deviation than an artificial or engineered technical function.

[0029] The term "binding agent," in the context of the present disclosure, refers to any substance 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, particularly non-peptide moieties. Such moieties can, for example, link a desired antigen-binding functional group or region, such as an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0030] "Immune checkpoint," as used herein, refers to regulators of the immune system, particularly costimulatory and inhibitory signals that modulate the extent and quality of T cell receptor recognition of antigens. 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 to displace CD28 binding. 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 KIR 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. 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 part of the adenosinergic pathway, for example, the interaction of A2AR and / or A2BR produced by CD39 and CD73 with adenosine. In certain embodiments, the inhibitory signal is the interaction of B7-H3 with its receptor and / or B7-H4 with its receptor.In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX, or TDO.

[0031] The terms "checkpoint inhibitor" (CPI) and "immune checkpoint (ICP) inhibitor" are used synonymously herein. The terms refer to molecules, such as binding agents, that completely or partially reduce, inhibit, interfere with, or negatively modulate one or more checkpoint proteins, or to molecules, such as binding agents, that completely or partially reduce, inhibit, interfere with, or negatively modulate the expression of one or more checkpoint proteins, such as molecules that inhibit immune checkpoints, particularly binding agents that inhibit the inhibitory signals of immune checkpoints. In one embodiment, an immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, an immune checkpoint inhibitor binds to one or more molecules that regulate checkpoint proteins. In one embodiment, an immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins, e.g., at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor can be used in accordance with the present disclosure. The term "partial" 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% at a level, e.g., at a level of inhibition of a checkpoint protein.

[0032] In one embodiment, the checkpoint inhibitor may 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 to displace CD28 binding; 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 several KIR with their ligands; the interaction of TIGIT with one or more of its ligands, PVR, PVRL2, and PVRL3; CD94 / NK The checkpoint inhibitor is selected from the group consisting of the interaction between G2A and HLA-E, the interaction between VISTA and its binding partner, the interaction between one or more Siglecs and their ligands, the interaction between GARP and one or more of its ligands, the interaction between CD47 and SIRPα, the interaction between PVRIG and PVRL2, the interaction between CSF1R and CSF1, the interaction between BTLA and HVEM, the interaction between a part of the adenosinergic pathway, for example, the interaction between A2AR and / or A2BR and adenosine produced by CD39 and CD73, the interaction between B7-H3 and its receptor and / or B7-H4 and its receptor, and inhibitory signals mediated by IDO, CD20, NOX, or TDO. In one embodiment, the checkpoint inhibitor is at least one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a KIR inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a VISTA inhibitor, and a GARP inhibitor. In one embodiment, the checkpoint inhibitor may 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.

[0033] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 43, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 44.

[0034] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47 Includes; The light chain variable region is (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50 Includes.

[0035] In one embodiment of the anti-PD-1 antibodies described herein, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:43 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:44.

[0036] In one embodiment, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antagonists of inhibitory signals, such as antibodies targeting PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, B7-H3, or B7-H4. These ligands and receptors are reviewed in Pardoll, D., Nature. 12: 252-264, 2012. Additional immune checkpoint proteins that can be targeted in accordance with the present disclosure are described herein.

[0037] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antigen receptors, such as antibodies or B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains with a characteristic immunoglobulin (Ig) fold. The term encompasses soluble immunoglobulins as well as membrane-bound immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally called antibodies.

[0038] The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2 nd ed. Raven Press, NY (1989). Briefly, immunoglobulins generally comprise a number of chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are primarily composed of 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 C H or CH (constant heavy chain) domain / region C H 1(CH1), C H 2(CH2), C H3(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 a VL and a CL. The light chain constant region is typically composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions that may be highly variable in sequence and / or in the form of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more highly conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the 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 specified or contradicted by context, CDR sequences herein are identified according to the rules of IMGT 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 otherwise specified or contradicted by context, references to amino acid positions in constant regions in the present disclosure are according to EU numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0039] There are five types of mammalian immunoglobulin heavy chains: α, δ, ε, γ, and μ, which constitute the different classes of antibodies: 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: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is substantially conserved among different immunoglobulin isotypes, while the variable region is highly diverse and is responsible for antigen recognition.

[0040] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and are not to be understood as limiting. Amino acids are organic compounds containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids can be classified based on their structure and chemical characteristics. Thus, the classes of amino acids may be reflected in one or both of the following tables:

[0041] Table 2: Major classifications based on the structure and general chemical characteristics of the R group [Table 2]

[0042] Table 3: Physical and functional classification of amino acid residue alternatives [Table 3]

[0043] For purposes of the present disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring in nature. The term "variant" particularly includes fragments of an amino acid sequence.

[0044] Amino acid insertion variants include the insertion of a single, two or more amino acids into a specific 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, but random insertions with appropriate screening of the resulting product are also possible.

[0045] 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.

[0046] 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 deletions can be at any position in the protein. Amino acid deletion mutants containing deletions at the N-terminus and / or C-terminus of the protein are also referred to as N-terminal and / or C-terminal truncation mutants.

[0047] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Substitution of one amino acid for another may be classified as conservative or non-conservative substitution. Preferably, the amino acid sequence is modified at a position that is not conserved between homologous proteins or peptides, and / or the amino acid is replaced with 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 an amino acid with an amino acid with a similar charge or no charge. Conservative amino acid changes involve the substitution of a member of a family of amino acids that are related in their side chains. In the context of the present disclosure, a "conservative substitution" refers to the substitution of one amino acid with another amino acid with similar structural and / or chemical characteristics, e.g., the substitution of one amino acid residue with another amino acid residue of the same class as defined in either of the two tables above, e.g., leucine may be substituted with isoleucine, since both are aliphatic, branched, hydrophobic substances. Similarly, aspartic acid may be substituted with glutamic acid because both are residues with small negative charges. Naturally occurring amino acids can also generally be divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: - glycine, alanine; - valine, isoleucine, leucine; - Aspartic acid, glutamic acid; - Asparagine, glutamine; - serine, threonine; - lysine, arginine; and - Phenylalanine, tyrosine.

[0048] The term "amino acid corresponding to position" and similar expressions, as used herein, refers to the number of an amino acid position in a 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 "corresponds to" an amino acid or segment in another sequence means that the amino acid or segment in one sequence, when aligned with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW, or similar, typically with default settings, has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. It is considered well known in the art how to align sequences or segments in sequences to determine positions in sequences that correspond to amino acid positions according to the present disclosure.

[0049] 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 either thereof, 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 of a significant duration, 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 period of time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a period of 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 more highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the 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), and 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 a binding domain that interacts with an antigen.The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from the amino terminus to the 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, such as C1q. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies can exist in various forms, such as polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0050] The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The terms "binding region" and "antigen-binding region" are used interchangeably herein and refer to the region that interacts with antigens and includes both VH and VL regions. Antibodies as used herein include not only monospecific antibodies, but also multispecific antibodies that contain multiple, for example, two or more, for example, three or more different antigen-binding regions.

[0051] As indicated above, the term antibody as used herein, unless otherwise specified or clearly contradicted by the context, includes fragments of antibodies that are antigen-binding fragments, i.e., fragments of antibodies 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, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO2007 / 059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al., Nature 341, 544-546 (1989)), also called domain antibodies (Holt et al.; Trends Biotechnol. 2003 Nov;21(11):484-90); (vi) camelid or nanobody molecules (Revets et al.; Expert Opin Biol Ther. 2005 Jan;5(1):111-24); and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined using recombinant methods with a synthetic linker that allows them to be produced as a single protein chain in which 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 by the term antibody unless otherwise specified or clearly indicated by the context.Although such fragments are generally included within the meaning of "antibody," they are collectively and independently a unique feature of the present disclosure, exhibiting various biological properties and usefulness. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific versions of such fragments, are discussed further herein. Unless otherwise specified, the term "antibody" should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant technology, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen.

[0052] Antibodies as produced may have a certain isotype. The term "isotype," as used herein, refers to the class of immunoglobulin (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. When a particular isotype, e.g., IgG1, is mentioned herein, the term 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 that 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 changes in the constant region.

[0053] IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some of the embodiments described herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments described herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0054] 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." Multispecific antibodies, e.g., bispecific antibodies, may have any format, including any of the bispecific or multispecific antibody formats described herein below.

[0055] 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 in that isotype in nature, e.g., in the case of an IgG1 antibody, the VH, CH1, CH2, CH3, hinge, VL, and CL domains.

[0056] 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 domains. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions 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, such as a mouse, have been grafted onto human framework sequences.

[0057] The term "chimeric antibody," as used herein, refers to an antibody whose variable region is derived from a non-human species (e.g., rodent) and whose constant region is derived from a different species, e.g., human. Chimeric antibodies may be produced by antibody engineering. "Antibody engineering" is a term commonly used to describe various types of antibody modifications, and processes for antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques, such as those described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies can be genetically or enzymatically engineered recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibody production can be performed by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic use in humans have been developed to reduce the expected antibody immunogenicity of non-human antibodies, e.g., rodent antibodies. These typically contain a non-human (e.g., mouse or rabbit) variable region specific for an antigen of interest, and may contain human constant antibody heavy and light chain domains. The terms "variable region" or "variable domain," when used in the context of a chimeric antibody, refer to the region comprising the CDRs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.

[0058] 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 have 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) that together form the antigen-binding site onto homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). Substitution (backmutation) of framework residues from the parent antibody (i.e., non-human antibody) with human framework regions may be necessary to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions that may contain one or more amino acid backmutations relative to the non-human amino acid sequences, and a fully human constant region. To obtain humanized antibodies with favorable characteristics, such as affinity and biochemical properties, additional amino acid modifications may be applied, and such modifications may not necessarily be back mutations.

[0059] 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 to or similar to one or more amino acid sequences in the other or parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, etc. derived from another or parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical to or similar to the amino acid sequence of the other or 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 one or more or all of the VH and VL CDRs and / or framework regions, VH, VL, CL, hinge, 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 to or similar to the VH and VL CDR sequences of said non-human parent antibody. Chimeric antibodies may be described herein as "derived from" a non-human parent antibody, which typically means that the VH and VL sequences may be identical or similar to those of the 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, which typically means that the binding arm or antigen-binding region comprises VH and / or VL CDRs or VH and / or VL sequences that are identical to or similar to those of the binding arm or antigen-binding region of the parent antibody. However, as described elsewhere herein, amino acid modifications, e.g., mutations, may be made in the CDRs, constant regions, or elsewhere in the antibody, binding arm, antigen-binding region, 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.

[0060] Non-human antibodies can be produced in a variety of different species, for example, mice, rabbits, chickens, guinea pigs, llamas, and goats.

[0061] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody techniques, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of antibody genes, may also be employed; such methods are well known to those skilled in the art.

[0062] Hybridoma production in such non-human species is a very well-established procedure. Immunization protocols and techniques for isolation of splenocytes from immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0063] As used herein, unless contradictory in context, the term "Fab arm" or "arm" refers to one heavy chain-light chain pair and is used synonymously herein with "half molecule."

[0064] The term "binding arm comprising an antigen-binding region" refers to an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm may comprise, for example, six VH and VL CDR sequences, a VH and VL sequence, a Fab or Fab' fragment, or a Fab arm.

[0065] As used herein, unless contradicted by context, the term "Fc region" refers to the region of an antibody consisting of two Fc sequences of an immunoglobulin heavy chain, wherein the Fc sequences include at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the term "Fc region" as used herein refers to the region of an antibody that includes, from the N-terminus to the C-terminus, at least the hinge region, the CH2 domain, and the CH3 domain. The Fc 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.

[0066] In the context of the present disclosure, the term "induce Fc-mediated effector functions to a lesser extent" as used in relation to antibodies, including multispecific antibodies, means that the antibody induces Fc-mediated effector functions, such as functions selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC or CDC, to a lesser extent compared to a human IgG1 antibody that (i) has the same CDR sequences as said antibody, in particular CDR sequences comprising the same first and second antigen-binding regions, and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions.

[0067] Fc-mediated effector function can be measured by binding to FcγR, binding to C1q, or induction of Fc-mediated cross-linking through FcγR.

[0068] 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 to 230 according to the EU numbering system set forth in Kabat (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689 (1991)). However, the hinge region may also be of any of the other subtypes described herein.

[0069] 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 system set forth in Kabat (ibid.). However, the CH1 region may also be of any of the other subtypes described herein.

[0070] 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 system set forth in Kabat (ibid.). However, the CH2 region may also be of any of the other subtypes described herein.

[0071] 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 system set forth in Kabat (ibid.). However, the CH3 region may also be of any of the other subtypes described herein.

[0072] The term "monovalent antibody", in the context of the present disclosure, means that the antibody molecule is capable of binding to a single molecule of antigen and is therefore not capable of cross-linking antigens.

[0073] A "CD40 antibody" or "anti-CD40 antibody" is an antibody that specifically binds to the antigen CD40, as described above.

[0074] A "CD137 antibody" or "anti-CD137 antibody" is an antibody that specifically binds to the antigen CD137 as described above.

[0075] A "CD40xCD137 antibody" or "anti-CD40xCD137 antibody" is a bispecific antibody that contains two different antigen-binding regions, one of which specifically binds to the antigen CD40 and the other of which specifically binds to the antigen CD137.

[0076] As used herein, the terms "binding" or "capable of binding" refer to the binding of an antibody to a predetermined antigen or epitope, typically as determined using biolayer interferometry (BLI) or, for example, as determined using surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using an antigen as the ligand and an antibody as the analyte, typically in the range of about 10 to about 100. -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 K of M D , or even lower K D An antibody will bind with an affinity equivalent to its K for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). D at least 10 times lower, such as at least 100 times lower, for example at least 1,000 times lower, such as at least 10,000 times lower, for example at least 100,000 times lower DThe antibody binds to a predetermined antigen with an affinity equivalent to the K D It is therefore dependent on the K D is very low (i.e., the antibody is highly specific), the affinity for the antigen can be at least 10,000 times lower than the affinity for a non-specific antigen.

[0077] The term "k" d ”(seconds -1 ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. The value is also referred to as k off Also called the value.

[0078] The term “K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0079] Two antibodies have the "same specificity" if they bind to the same antigen or the same epitope. Whether an antibody being tested recognizes the same epitope as a particular antigen-binding antibody, i.e., whether the antibodies bind to the same epitope, can be tested by various methods well known to those skilled in the art.

[0080] Competition between antibodies can be detected by cross-blocking assay. For example, competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the well of a microtiter plate, and an antibody that binds to the antigen and a candidate competing test antibody can be added. The amount of antigen-bound antibody in the well is indirectly correlated 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 less antibody will bind to the antigen bound to the well coated with the antigen. The amount of antibody bound to the antigen bound to the well can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0081] An antibody that competes for binding to an antigen with another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, or with an antibody having specificity for the antigen of another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, may be an antibody comprising a variant of the heavy and / or light chain variable regions described herein, e.g., an antibody comprising modifications in the CDRs and / or a particular degree of identity as described herein.

[0082] An "isolated multispecific antibody," as used herein, 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 to CD40 and CD137 is substantially free of monospecific antibodies that specifically bind to CD40 or CD137).

[0083] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition, displaying a single binding specificity and affinity for a particular epitope.

[0084] The term "heterodimeric interaction between the first and second CH3 regions" as used herein refers to the interaction between the first CH3 region and the second CH3 region in a first CH3 / second CH3 heterodimeric antibody.

[0085] The term "homodimeric interaction of the first and second CH3 regions," as used herein, refers to the interaction of a first CH3 region with another first CH3 region in a first CH3 / first CH3 homodimeric antibody, and the interaction of a second CH3 region with another second CH3 region in a second CH3 / second CH3 homodimeric antibody.

[0086] The term "homodimeric antibody," as used herein, refers to an antibody comprising two first Fab arms or half molecules, wherein the amino acid sequences of said Fab arms or half molecules are the same.

[0087] The term "heterodimeric antibody," as used herein, refers to an antibody comprising a first and a second Fab arm or half molecule, wherein the amino acid sequences of said first and second Fab arms or half molecules are different. In particular, the CH3 region or the antigen-binding region, or the CH3 region and the antigen-binding region, of said first and second Fab arms / half molecules are different.

[0088] The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, such as a cysteine ​​residue in the hinge region of an antibody, is more likely to become reduced than oxidized.

[0089] The present disclosure also describes multispecific antibodies, e.g., bispecific antibodies, that comprise functional variants of one or more of the VL regions, VH regions, or CDRs of the bispecific antibodies of the examples. Functional variants of VL, VH, or CDRs used in the context of bispecific antibodies still enable 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.

[0090] Such functional variants typically retain significant sequence identity with the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account 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 × 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0091] In the context of the present disclosure, unless otherwise specified, the following notation is used to describe mutations: i) substitution of an amino acid at a given position is designated, for example, as K409R, which means substitution of lysine with arginine at position 409 of the protein; and ii) for specific variants, specific three-letter or one-letter codes are used, including the codes Xaa and X, to indicate any amino acid residue. Thus, substitution of lysine with arginine at position 409 is designated as K409R, and substitution of lysine at position 409 with any amino acid residue is designated as K409X. In the case of deletion of lysine at position 409, this is designated by K409*.

[0092] Exemplary variants include those that differ from the VH and / or VL and / or CDRs of a parent sequence primarily by conservative substitutions; for example, 12, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.

[0093] In the context of the present disclosure, conservative substitutions can be defined by substitutions within the classes of amino acids defined in Tables 2 and 3.

[0094] The term "CD40," as used herein, refers to CD40, also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), which is a receptor for the ligand TNFSF5 / CD40L. CD40 is known to activate ERK in macrophages and B cells and transduce TRAF6- and MAP3K8-mediated signals, leading to the induction of immunoglobulin secretion by B cells. Other synonyms used for CD40 include, but are not limited to, B-cell surface antigen CD40, Bp50, CD40L receptor, and CDw40. In one embodiment, CD40 is human CD40, having UniProt accession number P25942. The sequence of human CD40 is also set forth in SEQ ID NO: 35. Amino acids 1-20 of SEQ ID NO:35 correspond to the signal peptide of human CD40; while amino acids 21-193 of SEQ ID NO:35 correspond to the extracellular domain of human CD40; the remainder of the protein, i.e., amino acids 194-215 and 216-277 of SEQ ID NO:35, are the transmembrane and cytoplasmic domains, respectively.

[0095] The term "CD137," as used herein, refers to CD137(4-1BB), also known 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 set forth in SEQ ID NO: 37. Amino acids 1 to 23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; while 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.

[0096] The "programmed death-1 (PD-1)" receptor refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 (also known as CD279) is predominantly expressed on preactivated T cells 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). The term "PD-1," as used herein, includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1. The sequence of human PD-1 is also set forth in SEQ ID NO: 39. "Programmed death-ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1," as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, such as macaque (cynomolgus), African elephant, wild boar, and mouse PD-L1 (see, for example, Genbank accession numbers NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), as well as analogs that share at least one epitope in common with hPD-L1. The sequence of human PD-L1 is also set forth in SEQ ID NO: 40, in which amino acids 1-18 are predicted to be the signal peptide. The sequence of macaque (cynomolgus) PD-L1 is also set forth in SEQ ID NO: 41, in which amino acids 1-18 are predicted to be the 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 epitope with hPD-L2. The 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 expressing PD-L1 and / or PD-L2 can switch off PD-1-expressing T cells, resulting in suppression of anti-cancer immune responses. The interaction of PD-1 with its ligands results in a reduction in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked.

[0097] Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) (also known as CD152) is a T-cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 (B7-1) and CD86 (B7-2). The term "CTLA-4," as used herein, includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one epitope with hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28, which has higher binding affinity to CD80 and CD86. CTLA4 is expressed on the surface of activated T cells, and its ligand is expressed on the surface of professional antigen-presenting cells. Binding of CTLA4 to its ligand prevents the costimulatory signal of CD28 and produces an inhibitory signal. Thus, CTLA-4 downregulates T-cell activation. The sequence of human CTLA-4 is also shown in SEQ ID NO:42.

[0098] "T cell immunoreceptor with Ig and ITIM domains" (TIGIT, also known as WUCAM or Vstm3) is an immunoreceptor on T cells and natural killer (NK) cells that binds to PVR (CD155) on DCs and macrophages, as well as PVRL2 (CD112; nectin-2) and PVRL3 (CD113; nectin-3), and regulates T cell-mediated immunity. The term "TIGIT," as used herein, includes human TIGIT (hTIGIT), variants, isoforms, and species homologs of hTIGIT, and analogs that share at least one common epitope with hTIGIT. The term "PVR," as used herein, includes human PVR (hPVR), variants, isoforms, and species homologs of hPVR, and analogs that share at least one common epitope with hPVR. The term "PVRL2," as used herein, includes human PVRL2 (hPVRL2), variants, isoforms, and species homologs of hPVRL2, and analogs that share at least one epitope in common with hPVRL2. The term "PVRL3," as used herein, includes human PVRL3 (hPVRL3), variants, isoforms, and species homologs of hPVRL3, and analogs that share at least one epitope in common with hPVRL3.

[0099] "B and T lymphocyte attenuator" (BTLA, also known as CD272) is a TNFR family member expressed in Th1 but not Th2 cells. BTLA expression is induced during T cell activation, particularly on the surface of CD8+ T cells. As used herein, the term "BTLA" includes human BTLA (hBTLA), variants, isoforms, and species homologs of hBTLA, as well as analogs that share at least one epitope with hBTLA. BTLA expression is gradually downregulated during differentiation of human CD8+ T cells into an effector cell phenotype. Tumor-specific human CD8+ T cells express high levels of BTLA. BTLA binds to "herpesvirus entry mediator" (HVEM, also known as TNFRSF14 or CD270) and is involved in T cell inhibition. The term "HVEM" as used herein includes human HVEM (hHVEM), variants, isoforms, and species homologs of hHVEM, as well as analogs that share at least one epitope with hHVEM. The BTLA-HVEM complex negatively regulates T cell immune responses.

[0100] "Killer cell immunoglobulin-like receptors" (KIRs) are receptors for MHC class I molecules on NK T cells and NK cells that are involved in the differentiation between healthy and diseased cells. KIRs bind to human leukocyte antigens (HLA) A, B, and C, which suppress normal immune cell activation. The term "KIR," as used herein, includes human KIR (hKIR), variants, isoforms, and species homologs of hKIR, as well as analogs that share at least one epitope with hKIR. The term "HLA," as used herein, includes variants, isoforms, and species homologs of HLA, as well as analogs that share at least one epitope with HLA. KIR, as used herein, specifically refers to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0101] "Lymphocyte activation gene-3 (LAG-3)" (also known as CD223) is an inhibitory receptor involved in the inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits the function of CD8+ effector T cells, which leads to suppression of the immune response. LAG-3 is expressed on activated T cells, NK cells, B cells, and DCs. The term "LAG-3," as used herein, includes human LAG-3 (hLAG-3), variants, isoforms, and species homologs of hLAG-3, as well as analogs that share at least one common epitope.

[0102] "T-cell membrane protein-3 (TIM-3)" (also known as HAVcr-2) is an inhibitory receptor involved in inhibiting lymphocyte activity by inhibiting Th1 cell responses. Its ligand is galectin 9 (GAL9), which is upregulated in various types of cancer. Other TIM-3 ligands include phosphatidylserine (PtdSer), high-mobility group protein 1 (HMGB1), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). The term "TIM-3," as used herein, includes human TIM3 (hTIM-3), variants, isoforms, and species homologs of hTIM-3, as well as analogs that share at least one common epitope. The term "GAL9," as used herein, includes human GAL9 (hGAL9), variants, isoforms, and species homologs of hGAL9, as well as analogs that share at least one common epitope. The term "PdtSer" as used herein includes mutants and analogs that share at least one common epitope. The term "HMGB1" as used herein includes human HMGB1 (hHMGB1), mutants, isoforms, and species homologs of hHMGB1, and analogs that share at least one common epitope. The term "CEACAM1" as used herein includes human CEACAM1 (hCEACAM1), mutants, isoforms, and species homologs of hCEACAM1, and analogs that share at least one common epitope.

[0103] "CD94 / NKG2A" is an inhibitory receptor predominantly expressed on the surface of natural killer cells and CD8+ T cells. As used herein, the term "CD94 / NKG2A" includes human CD94 / NKG2A (hCD94 / NKG2A), hCD94 / NKG2A variants, isoforms, and species homologs, as well as analogs sharing at least one common epitope. The CD94 / NKG2A receptor is a heterodimer containing CD94 and NKG2A. It inhibits NK cell activation and CD8+ T cell function, possibly through binding to ligands such as HLA-E. CD94 / NKG2A limits cytokine release and cytotoxic responses of natural killer cells (NK cells), natural killer T cells (NK-T cells), and T cells (α / β and γ / δ). NKG2A is frequently expressed on tumor-infiltrating cells, while HLA-E is overexpressed in numerous cancers.

[0104] "Indoleamine 2,3-dioxygenase" (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. As used herein, the term "IDO" includes human IDO (hIDO), hIDO variants, isoforms, and species homologs, as well as analogs sharing at least one common epitope. IDO is the rate-limiting enzyme in tryptophan degradation, catalyzing its conversion to kynurenine. Therefore, IDO is involved in the depletion of essential amino acids. It is known to be involved in the suppression of T and NK cells, the generation and activation of Tregs and myeloid-derived suppressor cells, and the promotion of tumor angiogenesis. IDO is overexpressed in many cancers and has been shown to promote immune system evasion by tumor cells and, when induced by local inflammation, facilitate chronic tumor progression.

[0105] As used herein, the "adenosinergic pathway" or "adenosine signaling pathway" involves the conversion of ATP to adenosine by the ectonucleotidases CD39 and CD73, resulting in inhibitory signaling via adenosine binding by one or more of the inhibitory adenosine receptors, the "adenosine A2A receptor" (A2AR, also known as ADORA2A) and the "adenosine A2B receptor" (A2BR, also known as ADORA2B). Adenosine is a nucleoside with immunosuppressive properties, present at high concentrations in the tumor microenvironment, limiting immune cell infiltration, cytotoxicity, and cytokine production. Thus, adenosine signaling is a strategy used by cancer cells to evade clearance from the host immune system. Adenosine signaling via A2AR and A2BR is a key checkpoint in cancer therapy, typically activated by the high adenosine concentrations present in the tumor microenvironment. CD39, CD73, A2AR, and A2BR are expressed by most immune cells, including T cells, invariant natural killer cells, B cells, platelets, mast cells, and eosinophils. Adenosine signaling via A2AR and A2BR counteracts immune cell activation mediated by the T cell receptor, resulting in an increase in the number of Tregs and a decrease in the activation of DCs and effector T cells. The term "CD39," as used herein, includes human CD39 (hCD39), hCD39 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. The term "CD73," as used herein, includes human CD73 (hCD73), hCD73 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. The term "A2AR," as used herein, includes human A2AR (hA2AR), hA2AR variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. The term "A2BR," as used herein, includes human A2BR (hA2BR), variants, isoforms, and species homologs of hA2BR, and analogs that share at least one common epitope.

[0106] "V-domain Ig suppressor of T-cell activation" (VISTA, also known as C10orf54) shares homology with PD-L1 but displays a unique expression pattern restricted to the hematopoietic compartment. The term "VISTA," as used herein, includes human VISTA (hVISTA), variants, isoforms, and species homologs of hVISTA, and analogs that share at least one common epitope. VISTA induces T-cell suppression and is expressed by leukocytes within tumors.

[0107] Members of the "sialic acid-binding immunoglobulin-type lectin" (Siglec) family recognize sialic acid and are involved in the discrimination between "self" and "non-self." The term "Siglec," as used herein, includes human Siglecs (hSiglecs), variants, isoforms, and species homologs of hSiglecs, as well as analogs that share at least one epitope with one or more hSiglecs. The human genome contains 14 Siglecs, some of which are involved in immunosuppression, including, but not limited to, Siglec-2, Siglec-3, Siglec-7, and Siglec-9. Siglec receptors bind to sialic acid-containing glycans but differ in their recognition of the linkage regiochemistry and spatial distribution of sialic acid residues. Family members also have distinct expression patterns. A wide range of malignant tumors overexpress one or more Siglecs.

[0108] "CD20" is an antigen expressed on the surface of B and T cells. High expression of CD20 can be found in cancers such as B cell lymphoma, hairy cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells. The term "CD20," as used herein, includes human CD20 (hCD20), hCD20 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope.

[0109] Glycoprotein A repeats predominant (GARP) play a role in immune tolerance and the tumor's ability to evade the patient's immune system. The term "GARP," as used herein, includes human GARP (hGARP), hGARP variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. GARP is expressed on lymphocytes, such as Tregs in peripheral blood and tumor-infiltrating T cells at tumor sites. It can bind to latent transforming growth factor-β (TGF-β). Disruption of GARP signaling in Treg cells leads to a decrease in tolerance and inhibits Treg migration to the gastrointestinal tract and increased proliferation of cytotoxic T cells.

[0110] "CD47" is a transmembrane protein that binds to the ligand "signal regulatory protein alpha" (SIRPα). As used herein, the term "CD47" includes human CD47 (hCD47), variants, isoforms, and species homologs of hCD47, as well as analogs that share at least one epitope with hCD47. As used herein, the term "SIRPα" includes human SIRPα (hSIRPα), variants, isoforms, and species homologs of hSIRPα, as well as analogs that share at least one epitope with hSIRPα. CD47 signaling is involved in various cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 is overexpressed in many cancers and functions as a "don't eat me" signal to macrophages. Blocking CD47 signaling via inhibitory anti-CD47 or anti-SIRPα antibodies allows macrophage phagocytosis of cancer cells and promotes the activation of cancer-specific T lymphocytes.

[0111] "Poliovirus receptor-related immunoglobulin domain containing" (PVRIG, also known as CD112R) binds to "poliovirus receptor-related 2" (PVRL2). PVRIG and PVRL2 are overexpressed in many cancers. PVRIG expression also induces TIGIT and PD-1 expression, and PVRL2 and PVR (TIGIT ligand) are co-overexpressed in a number of cancers. Blockade of the PVRIG signaling pathway results in increased T cell function and CD8+ T cell responses, thus reducing immunosuppression and increasing interferon responses. The term "PVRIG," as used herein, includes human PVRIG (hPVRIG), variants, isoforms, and species homologs of hPVRIG, as well as analogs that share at least one epitope with hPVRIG. "PVRL2," as used herein, includes hPVRL2, as defined above.

[0112] The "colony-stimulating factor 1" (CSF1) pathway is another checkpoint that can be targeted according to the present disclosure. CSF1R is a myeloid growth factor receptor that binds to CSF1. Blocking CSF1R signaling can functionally reprogram macrophage responses, thereby enhancing antigen presentation and anti-tumor T cell responses. The term "CSF1R," as used herein, includes human CSF1R (hCSF1R), variants, isoforms, and species homologs of hCSF1R, as well as analogs that share at least one common epitope with hCSF1R. The term "CSF1," as used herein, includes human CSF1 (hCSF1), variants, isoforms, and species homologs of hCSF1, as well as analogs that share at least one common epitope with hCSF1.

[0113] "Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase" refers to an enzyme in the NOX family of enzymes in myeloid cells that generates immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (NOX1-NOX5) have been found to be involved in cancer development and immunosuppression. Elevated ROS levels have been detected in almost all cancers and promote many aspects of tumor development and progression. ROS produced by NOX impairs NK and T cell function, and inhibition of NOX in myeloid cells improves the antitumor function of neighboring NK and T cells. As used herein, the term "NOX" includes human NOX (hNOX), hNOX mutants, isoforms, and species homologs, as well as analogs that share at least one epitope with hNOX.

[0114] Another immune checkpoint that can be targeted according to the present disclosure is signaling mediated by "tryptophan-2,3-dioxygenase" (TDO). TDO represents an alternative pathway to IDO in tryptophan degradation and is involved in immunosuppression. Because tumor cells can catabolize tryptophan via TDO instead of IDO, TDO may represent an additional target for checkpoint blockade. Indeed, numerous cancer cell lines have been found to upregulate TDO, and TDO can complement IDO inhibition. As used herein, the term "TDO" includes human TDO (hTDO), mutants, isoforms, and species homologs of hTDO, as well as analogs that share at least one epitope with hTDO.

[0115] Many immune checkpoints are regulated by the interaction of specific receptors with ligand pairs, such as those described above. Thus, immune checkpoint proteins mediate immune checkpoint signaling. For example, checkpoint proteins directly or indirectly regulate T cell activation, T cell proliferation, and / or T cell function. Cancer cells often utilize these checkpoint pathways to protect themselves from immune system attack. Therefore, the function of checkpoint proteins is typically to regulate T cell activation, T cell proliferation, and / or T cell function. Immune checkpoint proteins thus regulate and maintain self-tolerance and the duration and scope of physiological immune responses. Many immune checkpoint proteins belong to the B7:CD28 family or the tumor necrosis factor receptor (TNFR) superfamily and activate signaling molecules recruited to their cytoplasmic domains upon binding to specific ligands (Suzuki et al., 2016, Jap J Clin Onc, 46:191-203).

[0116] The term "dysfunctional," as used herein, refers to immune cells in a state of reduced immune responsiveness to antigenic stimulation, including unresponsiveness to antigen recognition and impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0117] The term "anergy," as used herein, refers to a state of unresponsiveness 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 becoming resistant to subsequent activation by the antigen, even in the presence of costimulation. The state of unresponsiveness can often be negated by the presence of IL-2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0118] The term "exhaustion," as used herein, refers to immune cell exhaustion, such as the T cell failure caused by persistent TCR signaling that occurs during many chronic infections and cancers. It is distinct from anergy in that it results from persistent signaling, rather than from defective or deficient signaling. Exhaustion is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional landscape that differs from that of functional effector or memory T cells. Exhaustion prevents optimal control of disease (e.g., infection or tumor). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) as well as cell-intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways, such as those described herein).

[0119] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have sustained or amplified biological function, or reviving or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include increased gamma interferon secretion from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor clearance) compared to their levels before the intervention. In one embodiment, 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 skilled in the art.

[0120] The term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule," as used herein, refers to a nucleic acid molecule, e.g., DNA or RNA, that reduces, inhibits, interferes with, or negatively modulates, fully or partially, one or more checkpoint 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).

[0121] The term "oligonucleotide," as used herein, refers to a nucleic acid molecule capable of reducing protein expression, particularly the expression of a checkpoint protein, such as a checkpoint protein described herein. Oligonucleotides are short DNA or RNA molecules, typically containing 2 to 50 nucleotides. Oligonucleotides may be single-stranded or double-stranded. Checkpoint inhibitor oligonucleotides may be antisense oligonucleotides.

[0122] Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, particularly the nucleic acid sequence (or fragments thereof) of a checkpoint protein. Antisense RNA is typically used to prevent the protein translation of mRNA, for example, mRNA encoding a checkpoint protein, by binding to the mRNA. Antisense DNA is typically used to target specific, complementary (coding or non-coding) RNA. Upon binding, such DNA / RNA hybrids can be degraded by the enzyme RNase H. Furthermore, morpholino antisense oligonucleotides can 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 B7-H4 expression in macrophages, resulting in increased T cell proliferation and reduced tumor volume in mice bearing tumor-associated antigen (TAA)-specific T cells.

[0123] The terms "siRNA," "short interfering RNA," or "small inhibitory RNA" are used interchangeably herein to refer to double-stranded RNA molecules, typically 20-25 base pairs in length, that interfere with the expression of specific genes, such as genes encoding checkpoint proteins with complementary nucleotide sequences. In one embodiment, siRNA interferes with mRNA, thereby blocking translation, for example, of immune checkpoint proteins. Transfection of exogenous siRNA can be used for gene knockdown, but this effect may be only transient, especially in rapidly dividing cells. Stable transfection can be achieved, for example, by RNA modification or by using an expression vector. Useful modifications and vectors for stable transfection of cells with siRNA are known in the art. siRNA sequences can also be modified to introduce a short loop between the two strands, resulting in "small hairpin RNAs" or "shRNAs." shRNAs can be processed into functional siRNAs by Dicer. shRNAs have relatively slow rates of degradation and turnover. Thus, the immune checkpoint inhibitor can be an shRNA.

[0124] The term "aptamer," as used herein, refers to a single-stranded nucleic acid molecule, typically 25-70 nucleotides in length, e.g., DNA or RNA, capable of binding to a target molecule, such as a polypeptide. In one embodiment, an aptamer binds to an immune checkpoint protein, e.g., an immune checkpoint protein described herein. For example, an aptamer according to the present disclosure can specifically bind to an immune checkpoint protein or polypeptide, or to a molecule in a signaling pathway that modulates the expression of an immune checkpoint protein or polypeptide. The generation and therapeutic use of aptamers is well known in the art (see, e.g., U.S. Pat. No. 5,475,096).

[0125] The term " small molecule inhibitor " or " small molecule " is used interchangeably herein and refers to a low molecular weight organic compound, usually up to 1000 daltons, that can completely or partially reduce, inhibit, interfere with or negatively modulate one or more checkpoint proteins as mentioned above.Such small molecule inhibitors are usually synthesized by organic chemistry, but can also be isolated from natural sources such as plants, fungi and microorganisms.Small molecular weight allows small molecule inhibitors to penetrate cell membranes and diffuse quickly.For example, various A2AR antagonists known in the art are organic compounds with molecular weights of less than 500 daltons.

[0126] The term "cell-based therapy" refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells, or stem cells) that express an immune checkpoint inhibitor into a subject for the purpose of treating a disease or disorder (e.g., a cancer disease).

[0127] The term "oncolytic virus" as used herein refers to a virus that selectively replicates in cancerous or hyperproliferative cells, either in vitro or in vivo, with no or minimal effect on normal cells, and can slow the growth of or induce the death of such cells. Oncolytic viruses for the delivery of immune checkpoint inhibitors include expression cassettes that can encode immune checkpoint inhibitors, such as inhibitory nucleic acid molecules such as siRNA, shRNA, oligonucleotides, antisense DNA or RNA, aptamers, antibodies or fragments thereof, or soluble immune checkpoint proteins or fusions. 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, such as Seneca Valley virus; SVV-001), coxsackieviruses, parvoviruses, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza virus), measles virus, reovirus, Sindbis virus, vaccinia virus, such as those representatively described in WO2017 / 209053 (including the Copenhagen, Western Reserve, and Wyeth strains), and adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF). Methods for producing and using recombinant oncolytic viruses comprising soluble forms of immune checkpoint inhibitors are disclosed in WO2018 / 022831, which is incorporated herein by reference in its entirety. The oncolytic virus can be used as an attenuated virus.

[0128] A "treatment cycle" is defined herein as the period within which separate dosages of a binding agent are added due to their pharmacodynamics, or in other words, the period after the administered binding agent has been substantially eliminated from the subject's body. Multiple smaller doses over a short time frame, e.g., within 2-24 hours, e.g., within 2-12 hours, or on the same day, may be equivalent to a larger single dose.

[0129] In the context of the present invention, the terms "treatment," "treating," or "therapeutic intervention" refer to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. This term is intended to include all areas of treatment for a given condition from which a subject is afflicted, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, delay the progression of the disease, disorder, or condition, reduce or alleviate symptoms and complications, and / or cure or eliminate the disease, disorder, or condition, as well as to prevent the condition, where prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition, or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications. In one embodiment, "treatment" refers to the administration of an effective amount of a therapeutically active binding agent, such as the administration of a therapeutically active antibody of the present disclosure, for the purpose of alleviating, alleviating, arresting, or eradicating (curing) the symptoms or pathology.

[0130] Resistance to, failure to respond to, and / or recurrence from treatment with the binding agents of the present disclosure can be determined according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST criteria v1.1). The RECIST criteria are listed in the table below (LD: longest dimension).

[0131] Table 4: Response definitions (RECIST criteria v1.1) [Table 4]

[0132] "Best overall response" is the best response recorded from the start of treatment until disease progression / recurrence (the minimum measurement recorded from the start of treatment will be used as the reference for PD). Subjects with CR or PR are considered to have an objective response. Subjects with CR, PR, or SD are considered to have disease controlled. Subjects with NE are counted as non-responders. "Best overall response" is the best response recorded from the start of treatment until disease progression / recurrence (the minimum measurement recorded from the start of treatment will be used as the reference for PD). Subjects with CR, PR, or SD are considered to have disease controlled. Subjects with NE are counted as non-responders.

[0133] "Duration of response (DOR)" applies only to subjects whose confirmed best overall response is CR or PR and is defined as the time from first evidence of objective tumor response (CR or PR) to the date of first PD or death from the underlying cancer.

[0134] "Progression-free survival (PFS)" is defined as the number of days from Day 1 of Cycle 1 to first documented progression or death from any cause.

[0135] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 to death from any cause. If it is not known whether the subject died, OS will be adjusted to the latest date the subject is known to be alive (on or before the date of censoring).

[0136] In the context of the present disclosure, the term "treatment regimen" refers to a structured treatment plan designed to improve and maintain health.

[0137] The term "effective amount" or "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount of a binding agent, e.g., an antibody such as a multispecific antibody or a monoclonal antibody, may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the binding agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the binding agent or fragment thereof. If the patient does not respond adequately to the initial dose, a higher dose (or a higher dose, effectively achieved by a different, more localized route of administration) may be used. If unwanted side effects occur in a patient using a given dose, a lower dose (or a lower dose, effectively achieved by a different, more localized route of administration) may be used.

[0138] As used herein, the term "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" refer to abnormal cells that develop by rapid and uncontrolled cell proliferation and continue to grow after the stimulus that initiated the new growth has ceased.

[0139] The term "cancer" according to the present disclosure includes leukemia, seminoma, melanoma, sarcoma, myeloma, teratoma, lymphoma, mesothelioma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, renal cancer, urothelial cancer, adrenal cancer, adrenocortical cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastric cancer, digestive cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, penile cancer, uterine cancer, ovarian cancer and lung cancer, and metastases thereof. Examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer or metastases of the above mentioned cancer types or tumors.

[0140] 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 relies on the detachment of malignant cells from the primary tumor, infiltration of the extracellular matrix, penetration of the endothelial basement membrane into body cavities and blood vessels, and subsequent infiltration of the target organ after transport by blood. Ultimately, 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 because tumor cells or elements remain and may develop metastatic potential. In one embodiment, the term "metastasis," according to the present disclosure, refers to "distant metastasis," which refers to metastasis far from the primary tumor and regional lymph node system.

[0141] Terms such as "reduce," "inhibit," "interfere," and "negatively modulate," as used herein, refer to the ability to cause an overall decrease in levels, for example, 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 or substantially complete inhibition, i.e., a reduction to zero or substantially to zero.

[0142] Terms such as "increase" or "enhance" in one embodiment relate 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%.

[0143] "Physiological pH," as used herein, refers to a pH of about 7.5.

[0144] "Wt. %," as used in disclosing the present invention, refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g), expressed as a percentage of the total weight of the total composition in grams (g).

[0145] The term "freeze" refers to the solidification of a liquid, usually by the removal of heat.

[0146] The term "lyophilize" or "freeze-drying" refers to freeze-drying a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to cause the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0147] The term "recombinant," in the context of the present disclosure, means "made by genetic engineering." In one embodiment, "recombinant," in the context of the present disclosure, is non-naturally occurring.

[0148] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a naturally occurring peptide or nucleic acid is a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory. The term "naturally occurring" means "naturally occurring", and includes not only known objects, but also 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.

[0149] The term "peptide," according to the present disclosure, includes oligopeptides and polypeptides, and refers to a substance comprising 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, and 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 generally used interchangeably herein.

[0150] A "therapeutic protein," when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's condition or pathology. In one embodiment, a therapeutic protein has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset, or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein may also have prophylactic properties and can be used to delay the onset of a disease or lessen the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides and may also refer to therapeutically active fragments thereof. It 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.

[0151] The term "portion" refers to a given portion. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous portion of said structure.

[0152] The terms "portion" and "fragment" are used interchangeably herein 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 the 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 between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0153] "Fragment", when referring to an amino acid sequence (peptide or protein), refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. C-terminally truncated fragments (N-terminal fragments) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. N-terminally truncated fragments (C-terminal fragments) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves 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%, or at least 90% of the amino acid residues 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 from the amino acid sequence.

[0154] According to the present disclosure, a portion or fragment of a peptide or protein preferably possesses 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 of nucleic acids. For example, a pharmacologically active fragment of a peptide or protein possesses at least one pharmacological activity 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, particularly 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, particularly up to 10, at most 12, at most 15, at most 20, at most 30, or at most 55 consecutive amino acids of the peptide or protein.

[0155] "Mutant" as used herein 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 or wild-type (WT) amino acid sequence, or may be a modified version of the wild-type amino acid sequence. Preferably, the mutant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid modifications, preferably 1 to about 10 or 1 to about 5 amino acid modifications, compared to the parent.

[0156] "Wild-type" or "WT" or "native," as used herein, refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0157] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is expected to 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 expressed over 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 expressed 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, and in some embodiments for consecutive such amino acids. In some embodiments, the degree of similarity or identity is expressed for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0158] "Sequence similarity" refers to the percentage of either identical amino acids or amino acids that 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.

[0159] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of identical nucleotides or amino acids between the sequences to be compared in an optimally aligned state. The percentage is purely statistical, although the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually carried out by comparing the sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison may be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs using 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 at the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi).In some embodiments, the algorithm parameters used for the BLASTN algorithm at the NCBI website include: (i) an expected threshold set to 10; (ii) a character size set to 28; (iii) maximum match in the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter if low complexity regions are used. In some embodiments, the algorithm parameters used for the BLASTP algorithm at the NCBI website include: (i) an expected threshold set to 10; (ii) a character size set to 3; (iii) maximum match in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence:11, extension:1; and (vi) an adjustment of the conditional compositional score matrix.

[0160] Percentage identity is obtained by determining the number of identical positions where the sequences being compared match, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0161] In some embodiments, the degree of similarity or identity is expressed 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 expressed 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, for consecutive such amino acid residues. In some embodiments, the degree of similarity or identity is expressed over the entire length of the reference sequence.

[0162] Homologous amino acid sequences exhibit, according to the present disclosure, at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0163] The amino acid sequence variants described herein can be readily prepared by those 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 peptides 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.

[0164] In one embodiment, a 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 refers to any fragment or variant that exhibits one or more functional properties identical to or similar to those of the amino acid sequence from which it is derived, i.e., 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, particularly refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been altered compared to the amino acid sequence of a parent molecule or sequence, yet is still capable of performing one or more of the functions of the parent molecule or sequence, e.g., capable of inducing an immune response. In one embodiment, the alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function 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 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.

[0165] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a designated 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, substantially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence can be a variant of the particular sequence or a fragment thereof. For example, one of skill in the art will understand that antigens suitable for use herein may be altered to have a sequence that differs from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0166] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell. In a preferred embodiment, the binding agent used in the present disclosure is in a substantially purified form.

[0167] The term "genetic modification" or simply "modification" includes the transfection of a cell with a nucleic acid. The term "transfection" refers to the introduction of a nucleic acid, particularly RNA, into a cell. For purposes of the present 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, according to the present disclosure, cells for transfection of nucleic acids described herein may be present in vitro or in vivo; for example, the cells may form part of an organ, tissue, and / or organism of a patient. According to the present disclosure, transfection may be transient or stable. For some applications of transfection, transient expression of the transfected genetic material is sufficient. RNA may be transiently transfected into cells to express its encoded protein. Typically, nucleic acids introduced during the transfection process are not integrated into the nuclear genome, resulting in the exogenous nucleic acid being diluted or degraded through mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must be performed.Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding the antigen is transiently transfected into the cell.RNA can also be transfected into the cell to transiently express its encoded protein.

[0168] According to the present disclosure, a peptide or protein analog is a modified form of the peptide or protein from which it is derived, retaining at least one functional property of the peptide or protein. For example, a pharmacologically active analog of a peptide or protein retains at least one pharmacological activity of the peptide or protein from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule associated with the protein or peptide, such as carbohydrates, lipids, and / or proteins or peptides. In one embodiment, a protein or peptide "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the proteins and peptides.

[0169] "Activation" or "stimulation" as used herein refers to the state of immune effector cells, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cells" refers, inter alia, to immune effector cells that have undergone cell division.

[0170] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0171] The term "clonal expansion" or "expansion" refers to the process by which a specific entity multiplies. In the context of the present disclosure, the term is preferably used in the context of an immune response, in which immune effector cells are stimulated by an antigen and proliferate, resulting in the amplification of specific immune effector cells that recognize said antigen. Preferably, clonal expansion leads to the differentiation of immune effector cells.

[0172] "Antigen" according to the present disclosure encompasses any substance expected to elicit an immune response and / or any substance against which an immune response or immune mechanism, e.g., a cellular response, is directed. This also includes situations in which an antigen is processed into antigenic peptides, particularly when presented in the context of MHC molecules, and an immune response or immune mechanism is directed against one or more antigenic peptides. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). The term "antigen," according to the present disclosure, includes any molecule that contains at least one epitope, e.g., a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that, after appropriate processing, preferably induces an immune response specific to the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0173] According to the present disclosure, any suitable antigen that is a candidate for an immune response can be used, and in this case, the immune response may be both a humoral and a cellular immune response. In the context of some embodiments of the present disclosure, the antigen is preferably presented by cells, preferably by antigen-presenting cells in the context of MHC molecules, thereby generating an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents, and the pathogen or antigen may also be a tumor antigen. According to the present disclosure, the antigen may be a naturally occurring product, such as a viral protein, or a portion thereof.

[0174] The term "disease-associated antigen" is used in its broadest sense and refers to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that is expected to stimulate the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

[0175] In a preferred embodiment, the antigen is a tumor antigen, i.e., a part of a tumor cell, particularly one that is present primarily intracellularly or as a surface antigen of the tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, for example, an antigen derived from a virus, bacterium, unicellular organism, or parasite, for example, a viral antigen such as a viral ribonucleoprotein or capsid protein. In particular, the antigen is capable of inducing the activation of cells of the immune system, preferably CD4, in particular through modulation of the activity of T cell receptors. + and CD8 + It must be presented by MHC molecules leading to lymphocyte modulation, particularly activation.

[0176] The term "tumor antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface, or cell nucleus. This particularly refers to an antigen produced intracellularly or as a surface antigen on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, α2-H-iron protein, and γ-fetoprotein, as well as various viral tumor antigens. According to the present disclosure, tumor antigens preferably include any antigen characteristic of tumors or cancers in terms of type and / or expression level, as well as tumor or cancer cells.

[0177] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0178] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A bacterial antigen may be derived from the bacterial cell wall or cytoplasmic membrane.

[0179] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen; that is, a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, 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 groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents, but not to the latter. Epitopes may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, e.g., amino acid residues that are effectively blocked or covered by the specifically antigen-binding peptide (in other words, amino acid residues within the footprint of the specifically antigen-binding peptide).

[0180] An epitope of a protein preferably comprises a contiguous or discontinuous portion of said protein and is preferably about 5 to about 100, preferably about 5 to about 50, more preferably about 8 to about 0, and most preferably about 10 to about 25 amino acids in length; for example, the epitope may be preferably 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 the epitope in the context of the present disclosure is a T cell epitope.

[0181] Terms such as "epitope," "antigen fragment," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and preferably refer to an incomplete representation of an antigen that is preferably capable of eliciting an immune response against an antigen or a cell that expresses or contains the antigen, preferably a cell that presents the antigen. Preferably, these terms refer to the immunogenic site of an antigen. Preferably, the immunogenic site is a part of the antigen that is recognized by (i.e., specifically binds to) a T cell receptor, especially when presented in the context of an MHC molecule. Certain preferred immunogenic sites bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen may include a contiguous or discontinuous portion of the antigen and may be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length. For example, an epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, the epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0182] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses; in this case, MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the binding peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides can also be effective. For class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may also be effective.

[0183] Peptide and protein antigens may be 2 to 100 amino acids in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides may be greater than 50 amino acids. In some embodiments, peptides may be greater than 100 amino acids.

[0184] The peptide or protein antigen can be any peptide or protein that is capable of inducing or increasing the ability of the immune system to generate antibody and T cell responses against the peptide or protein.

[0185] In one embodiment, the vaccine antigen, i.e., the antigen against which an immune response is induced upon inoculation into a subject, is recognized by immune effector cells. Preferably, when recognized by immune effector cells, the vaccine antigen can, in the presence of an appropriate costimulatory signal, induce the stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of the disclosed embodiments of the present invention, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen-presenting cell. In one embodiment, the antigen is presented by a diseased cell (e.g., a tumor cell or an infected cell). In one embodiment, the antigen receptor is a TCR that binds to an epitope of an antigen presented in the context of an MHC. In one embodiment, when the TCR is expressed by and / or present on a T cell, binding to an antigen presented by a cell, such as an antigen-presenting cell, results in the stimulation, priming, and / or expansion of the T cell. In one embodiment, when a TCR is expressed by and / or present on a T cell, binding to an antigen presented on the diseased cell results in cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors such as perforin and granzymes.

[0186] In one embodiment, the antigen receptor is an antibody or B cell receptor that binds to an epitope in the antigen. In one embodiment, the antibody or B cell receptor binds to a native epitope of the antigen.

[0187] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is associated with and positioned on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from outside the cell, e.g., by an antibody located outside the cell. In this context, the portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be via one or more transmembrane domains, one or more lipid anchors, or via interaction with any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion, or may be a protein that associates with the surface of a cell through interaction with another protein that is a transmembrane protein.

[0188] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is available for binding by proteins and other molecules. An antigen is expressed on the surface of a cell when it is on the surface of said cell and is available for binding, for example, by an antigen-specific antibody added to the cell.

[0189] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and that is preferably available from the outside of said cell for binding to a molecule, such as an antibody, that is on the outside of said cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0190] The terms "T cell" and "T lymphocyte" are used interchangeably herein, examples of which include T helper cells (CD4 + T cells), and cytotoxic T cells (CTL, CD8 +T cells). The term "antigen-specific T cells" or similar terms particularly relates to T cells that recognize the antigen that targets the T cell when presented on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, in the context of an MHC molecule, and preferably exert T cell effector function. A T cell is considered antigen-specific if it kills a target cell expressing the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) may be measured. In certain embodiments of the present disclosure, RNA (particularly mRNA) encodes at least one epitope.

[0191] The term "target" is intended to mean a substance, such as a cell or tissue, that is the target for an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigenic epitope, i.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses the antigen, preferably a cell that presents said antigen in association with class I MHC.

[0192] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of said antigen (e.g., degradation of a protein into peptides), and the association (e.g., via binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by a cell, preferably an antigen-presenting cell.

[0193] "Antigen-responsive CTL" refers to a CD8 CTL that is responsive to an antigen or a peptide derived from the antigen presented together with class I MHC on the surface of an antigen-presenting cell. + It means T cells.

[0194] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFNγ and TNFα, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0195] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to an integrated body response to an antigen, preferably a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against" a substance, such as an antigen, cell, or tissue, relates to an immune response, such as a cellular response, directed against such substance. An immune response involves the development of antibodies against one or more antigens, as well as the activation of antigen-specific T lymphocytes, preferably CD4 + and CD8 + T-lymphocytes, more preferably CD8 + The expansion of T-lymphocytes may include one or more responses selected from the group consisting of: expansion of T-lymphocytes, which can be detected by various in vitro proliferation or cytokine production tests.

[0196] The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, preferably a cellular immune response, a humoral immune response, or both. The immune response may be protective / preventative / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigenic peptide, preferably a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B, or C), CMV, or RSV)). "Inducing" in this context can mean that prior to induction, there was no immune response against a particular antigen or pathogen, but it can also mean that prior to induction, there was a certain level of immune response against a particular antigen or pathogen, and that after induction, said immune response is enhanced. Thus, "inducing an immune response" in this context also includes "enhancing an immune response." Preferably, after inducing an immune response in an individual, said individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease state is alleviated by inducing an immune response.

[0197] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are meant to include cellular responses directed against cells characterized by antigen expression and / or antigen presentation with class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are responsible for the production of killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells such as diseased cells.

[0198] The term "humoral immune response" refers to the process in an organism in which antibodies are produced in response to substances or organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T and / or B cells through membrane-bound receptors that bind to a single antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide, thereby producing antibody-secreting plasma cell clones in addition to memory B cells, each of which produces antibodies that recognize the same antigen epitope as that recognized by its antigen receptor. Memory B lymphocytes remain quiescent until later activated by their specific antigen. These lymphocytes provide the cellular basis of memory, resulting in the recruitment of antibody responses upon re-exposure to the specific antigen.

[0199] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering to an individual one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA (particularly mRNA) encoding them as described herein, to stimulate an immune response against said one or more immunogens or antigens or cells characterized by the presentation of said one or more immunogens or antigens.

[0200] "Cells characterized by antigen presentation" or "cells presenting antigens" or "MHC molecules presenting antigens on the surface of antigen-presenting cells" or similar expressions refer to cells such as diseased cells, in particular tumor or infected cells, or antigen-presenting cells that present antigens or antigenic peptides, either directly or after processing, in the environment of MHC molecules, preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules.

[0201] The term "transcription", in the context of the present disclosure, relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA), which can then be translated into peptides or proteins.

[0202] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence. With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes where a strand of mRNA directs the assembly of a sequence of amino acids to produce a peptide or protein.

[0203] The terms "optionally" or "optionally," as used herein, mean 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 and cases where it does not occur.

[0204] "Endogenous," as used herein, refers to any substance that is from or produced within an organism, cell, tissue, or system.

[0205] 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.

[0206] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition affecting an individual's body. Disease is often understood as a medical condition associated with specific symptoms and signs. Diseases may be caused by factors originally from external sources, such as infectious diseases, or by internal malfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death in the affected individual, or similar problems in those who come into contact with that individual. In this broader sense, "disease" sometimes includes injury, disability, disorder, syndrome, infection, isolated symptoms, deviant behavior, and abnormal changes in structure or function, while in other contexts and for other purposes, these may be considered distinct categories. Diseases typically affect individuals not only physically but also emotionally, as suffering from and living with many illnesses can alter a person's outlook on life and personality.

[0207] The term "therapeutic treatment" refers to any treatment that improves the health and / or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in an individual, stop or slow the onset of the disease in an individual, inhibit or slow the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or previously had the disease.

[0208] The term "prophylactic treatment" or "preventive treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein. Similarly, the term "method for preventing" in the context of disease progression, such as tumor or cancer progression, relates to any method intended to prevent a disease from progressing in an individual.

[0209] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate), or any other non-mammal, such as a bird (chicken), fish, or any other animal species, that may or may not have a disease or disorder, or that may be in need of preventative intervention such as vaccination, or in need of intervention such as protein replacement, that may be susceptible to or susceptible to a disease or disorder (e.g., cancer, infectious disease). In many embodiments, an individual is a human being. Unless otherwise specified, the terms "individual" and "subject" do not designate a particular age, and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0210] The term "patient" refers to an individual or subject for treatment, in particular an affected individual or subject.

[0211] Aspects and embodiments of the present disclosure In a first aspect, the present disclosure provides a binding agent for use in a method for reducing or preventing tumor progression or treating cancer in a subject, said method comprising administering the binding agent to said subject prior to, simultaneously with, or after administration of a checkpoint inhibitor, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

[0212] As demonstrated in the present disclosure, the combination of (i) stimulation with a binding agent that binds to human CD40 and human CD137, and (ii) checkpoint inhibition (particularly inhibition of the PD-1 / PD-L1 axis) amplifies the immune response. Without being bound by any theory, the rationale behind this surprising discovery may be as follows: CD137 binds to PD-1 +PD-L1 / PD-1 signaling is co-expressed on T cells. Therefore, blockade of PD-L1 / PD-1 signaling and costimulation via CD137 can synergize to enhance T cell effector function and improve response duration. Through conditional activation of CD40 and CD137, binding agents targeting CD40 and CD137 induce potent antitumor activity through enhanced T cell priming, cytokine and chemokine production, and expansion and survival of antigen-experienced T cells. The PD-(L)1 pathway is expected to be activated during priming as well as during continuous antigen exposure, which may reduce the magnitude of the immune response induced by binding agents targeting CD40 and CD137.

[0213] Binding agents that bind to CD40 and CD137 In one embodiment, the CD40 is human CD40, particularly human CD40 comprising the sequence set forth in SEQ ID NO: 36. 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 CD40 is human CD40 and CD137 is human CD137. In one embodiment, the CD40 is human CD40 comprising the sequence set forth in SEQ ID NO: 36 and the CD137 is human CD137 comprising the sequence set forth in SEQ ID NO: 38.

[0214] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 7 or 9, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 8 or 10; b) The second antigen-binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17 or 19, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18 or 20.

[0215] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; b) The second antigen-binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 11, 12, and 13, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively.

[0216] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD40 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: 7 or 9, and a light chain variable region (VL) region 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: 8 or 10; b) The second 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: 17 or 19, 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: 18 or 20.

[0217] In one embodiment of the binder according to the first aspect, a) the first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7 or 9, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; b) The second 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: 17 or 19, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 18 or 20.

[0218] In one embodiment of the binder according to the first aspect, a) the first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 10; b) The second 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: 19, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0219] The binding agent may in particular be an antibody, such as a multispecific antibody, e.g., a bispecific antibody. The binding agent may also be in the form of a full-length antibody or an antibody fragment.

[0220] It is further preferred that the binding agent is a human or humanized antibody.

[0221] Each variable region may comprise three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0222] 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.

[0223] In one embodiment of the first aspect, the binder 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 second heavy chain variable region (VH) and the second heavy chain constant region (CH). Includes.

[0224] In one embodiment of the first aspect, the binder is 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). Includes.

[0225] In one embodiment of the first aspect, the binding agent is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises: 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). Includes; The second binding arm is 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). Includes.

[0226] In one embodiment of the first aspect, the binding agent comprises i) a first heavy chain and a first light chain comprising the antigen-binding region capable of binding to CD40, 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 second light chain comprising the antigen-binding region capable of binding to CD137, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region.

[0227] Each of the first and second heavy chain constant regions (CH) may comprise one or more of the constant heavy chain 1 (CH1) 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.

[0228] Each of the first and second heavy chain constant regions (CHs) may comprise a CH3 region, and the two CH3 regions may 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 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 position 409 in the human IgG1 heavy chain according to EU numbering.

[0229] In the first heavy chain constant region (CH), at least one amino acid 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 may be substituted, and in the second heavy chain constant region (CH), at least one amino acid 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 may be substituted. In certain embodiments, the first and second heavy chains are not substituted at the same positions (i.e., the first and second heavy chains contain asymmetric mutations).

[0230] 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.

[0231] In one embodiment of the first aspect, the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2 and CH3 regions.

[0232] In one particular embodiment of the binding agent according to the first aspect, the first and second heavy chain constant regions (CH) are modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an otherwise identical antibody comprising the unmodified first and second heavy chain constant regions (CH). In particular, each or both of the 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: 21 or 29.

[0233] Fc-mediated effector function can be determined by measuring the binding of a binding agent to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. In particular, Fc-mediated effector function can be determined by measuring the binding of a binding agent to C1q.

[0234] The first and second heavy chain constant regions of the binding agent may be modified such that binding of C1q to said antibody is reduced compared to the 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.

[0235] 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.

[0236] 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 in said first and second heavy chains, respectively.

[0237] 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 in the first and second heavy chain constant regions (HC), respectively.

[0238] 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 in both the first and second heavy chain constant regions are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0239] In one embodiment of the binding agent according to the first aspect, the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in 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 in the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0240] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain comprises: a) the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 29 [IgG1-FC]; 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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having 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 comprises an amino acid sequence selected from the group consisting of:

[0241] In one embodiment of the binding agent according to the first aspect, the constant region of the first or second heavy chain, e.g. of the second heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 30 [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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having 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:

[0242] In one embodiment of the binding agent according to the first aspect, the constant region of the first or second heavy chain, e.g. the first heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 23 or 31 [IgG1-F409R]; 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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having 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:

[0243] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain comprises: a) the sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 32 [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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having 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:

[0244] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain, e.g. of the second heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 33 [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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions, e.g. at most 5 substitutions, 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:

[0245] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain, e.g. of the first heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 34 [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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions, e.g. at most 5 substitutions, 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:

[0246] In one embodiment of the first aspect, the binding agent comprises a kappa (κ) light chain constant region.

[0247] In one embodiment of the first aspect, the binding agent comprises a lambda (λ) light chain constant region.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] In one embodiment of the binding agent according to the first aspect, the kappa (κ) light chain is a) the sequence set forth in SEQ ID NO: 27; 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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having 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 comprises an amino acid sequence selected from the group consisting of:

[0252] In one embodiment of the binding agent according to the first aspect, the lambda (λ) light chain is a) the sequence set forth in SEQ ID NO: 28; 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 starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having 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 comprises an amino acid sequence selected from the group consisting of:

[0253] The binding agent (particularly the 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 the antibody) is of the IgG1m(f) allotype.

[0254] Preferably, the binding agent is administered in a suitable amount, i.e., the amount of binding agent administered, e.g., in each dose and / or treatment cycle, is an amount capable of inducing intracellular signaling upon binding to CD137 expressed on another cell. Thus, a suitable amount of a binding agent according to the present disclosure can transactivate two different cell types. In humans, CD40 is expressed on numerous cells, such as antigen-presenting cells (APCs), e.g., dendritic cells, whereas CD137 is expressed on T cells and other cells. Therefore, a suitable amount of a binding agent that binds to CD40 and CD137 according to the present disclosure can simultaneously bind to APCs and T cells expressing these receptors. Therefore, without being bound by theory, the binding agent can (i) mediate cell-to-cell interactions between APCs and T cells through receptor binding and (ii) activate both CD40 and CD137 simultaneously, which is primarily induced by cross-linking and receptor clustering upon cell-to-cell interaction and does not necessarily depend on the agonistic activity of the parent monospecific bivalent antibody. Thus, these transactivating binding agents exert costimulatory activity in the context of APC:T cell interactions, enabling T cell responses against tumor cells. Therefore, this mechanism of action may mirror natural T cell activation through antigen presentation by activated APCs, allowing APCs to present various tumor-specific antigens to T cells. Without being limited by theory, costimulatory activity may result in one or more of the following: (i) activation of specific T cells only (i.e., those in contact with APCs), as opposed to any T cells; (ii) reactivation of exhausted T cells through strong costimulation via activated APCs and triggering of CD137; and (iii) priming of T cells by inducing antigen presentation by activated APCs and simultaneously triggering CD137.

[0255] The amount of binding agent administered in each dose and / or treatment cycle may be within a range in which, inter alia, 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 binding agent binds to both CD40 and CD137.

[0256] In a preferred embodiment, the amount of binding agent administered, e.g., the amount of binding agent administered in each dose and / or each treatment cycle, is: a) about 0.01 to 2.5 (e.g., about 0.04 to 2.5) mg / kg body weight, or about 1 to 200 (e.g., about 3 to 200) mg in total; and / or b) Approximately 0.07×10 -9 ~16.9×10 -9 (For example, about 0.25 × 10 -9 ~16.9×10 -9 ) mol / kg body weight, or a total of approximately 8 × 10 -9 ~1350×10 -9 (For example, about 20 × 10 -9 ~1350×10 -9 )mol is.

[0257] In some embodiments, the amount of binding agent administered, e.g., the amount of binding agent administered in each dose and / or each treatment cycle, is a) about 0.62 to 1.88 (e.g., about 1.0 to 1.5) mg / kg body weight, or about 50 to 150 (e.g., about 80 to 120) mg in total; and / or b) Approximately 4.1×10 -9 ~12.7×10 -9 (For example, about 6.7 × 10 -9 ~10.1×10 -9 ) mol / kg body weight, or a total of approximately 335 × 10 -9 ~1020×10 -9 (For example, about 535 × 10 -9 ~810×10-9 )mol is.

[0258] According to these embodiments, the dose defined in mg / kg may be converted to a flat dose based on the median body weight of subjects to whom the binding agent is administered being 80 kg, or vice versa.

[0259] The binding agent can be administered by any method and by any route known in the art, hi a preferred embodiment, the binding agent is administered systemically, e.g., parenterally, particularly intravenously.

[0260] The binding agent can be administered in the form of any suitable pharmaceutical composition described herein. In a preferred embodiment, the binding agent is administered in the form of an infusion.

[0261] The binding agent can be administered prior to, simultaneously with, or after administration of the checkpoint inhibitor.

[0262] In one embodiment, the binding agent is administered before the administration of the checkpoint inhibitor. For example, the gap between the end of administration of the binding agent and the beginning of administration of the checkpoint inhibitor can be at least about 10 minutes, for example, 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, or can be up to about 14 days (up to about 2 weeks), for example, 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.

[0263] In one embodiment, the binding agent is administered after the administration of the checkpoint inhibitor. For example, the gap between the end of the administration of the checkpoint inhibitor and the beginning of the administration of the binding agent can be at least about 10 minutes, for example, 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, or up to about 14 days (up to about 2 weeks), for example, 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.

[0264] In one embodiment, the binding agent is administered simultaneously with the checkpoint inhibitor.For example, the binding agent and the checkpoint inhibitor can be administered using the composition that comprises both drugs.Alternatively, the binding agent can be administered to one limb of the subject, and the checkpoint inhibitor can be administered to another limb of the subject.

[0265] Checkpoint inhibitors In one embodiment, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antagonists of inhibitory signals, such as antibodies targeting, for example, PD-1, PD-L1, CTLA-4, LAG-3, or TIM-3. These ligands and receptors are reviewed in Pardoll, D., Nature. 12: 252-264, 2012. Additional immune checkpoint proteins that can be targeted in accordance with the present disclosure are described herein.

[0266] In one embodiment, the immune checkpoint inhibitor prevents inhibitory signals associated with an immune checkpoint. In one embodiment, the immune checkpoint inhibitor is an antibody or fragment thereof that disrupts or inhibits inhibitory signaling associated with an immune checkpoint. In one embodiment, the immune checkpoint inhibitor is a small molecule inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts or inhibits inhibitory signaling.

[0267] Inhibiting or blocking inhibitory immune checkpoint signaling results in the prevention or reversal of immune suppression and the establishment or enhancement of T cell immunity against cancer cells, as described herein. In one embodiment, inhibition of immune checkpoint signaling reduces or inhibits immune system dysfunction, as described herein. In one embodiment, inhibition of immune checkpoint signaling results in less dysfunction of impaired immune cells, as described herein. In one embodiment, inhibition of immune checkpoint signaling results in less dysfunction of impaired T cells, as described herein.

[0268] In one embodiment, the immune checkpoint inhibitor inhibits interactions between checkpoint blocker proteins, such as the interaction of PD-1 with PD-L1 or PD-L2; the interaction of CTLA-4 with CD80 or CD86; the interaction of LAG-3 with one or more of its ligands; the interaction of one or more KIRs with their respective ligands; the interaction of TIM-3 with one or more of its ligands (e.g., galectin-9, PtdSer, HMGB1, and CEACAM1); the interaction of TIGIT with one or more of its ligands (e.g., PVR, PVRL2, and PVRL3); the interaction of VISTA with one or more of its binding partners; GARP and its interaction with one or more of its ligands; inhibitory signaling via CD39 and / or CD73 and / or the interaction of A2AR and / or A2BR with adenosine; the interaction of B7-H3 with its receptor and / or the interaction of B7-H4 with its receptor; the interaction of BTLA with its ligand HVEM; the interaction of CD94 / NKG2A with HLA-E; the interaction of one or more Siglecs with their respective ligands; CD20 signaling; the interaction of CD47 with SIRPα, the interaction of PVRIG with PVRL2; the interaction of CSF1R with CSF1; NOX signaling; and / or IDO and / or TDO signaling.

[0269] The immune checkpoint inhibitor may be an antibody, an antigen-binding fragment thereof, or a construct thereof comprising a portion of an antibody together with an antigen-binding fragment having the required specificity. The antibody or antigen-binding fragment thereof is as described herein. Antibodies or antigen-binding fragments thereof that are immune checkpoint inhibitors include, in particular, antibodies or antigen-binding fragments thereof that bind to immune checkpoint proteins, such as immune checkpoint receptors or immune checkpoint receptor ligands. The antibody or antigen-binding fragment may also be conjugated to a further moiety as described herein. In particular, the antibody or antigen-binding fragment thereof is a chimerized, humanized, or human antibody. Preferably, the immune checkpoint inhibitor antibody or antigen-binding fragment thereof is an immune checkpoint receptor antagonist or an immune checkpoint receptor ligand antagonist.

[0270] In a preferred embodiment, the antibody that is an immune checkpoint inhibitor is an isolated antibody.

[0271] In one embodiment, the immune checkpoint inhibitor is an antibody, fragment, or construct that prevents an interaction between checkpoint blocker proteins, for example, an antibody or fragment thereof that prevents the interaction of PD-1 with PD-L1 or PD-L2; an antibody, fragment, or construct that prevents the interaction of CTLA-4 with CD80 or CD86; an antibody, fragment, or construct that prevents the interaction of LAG-3 with its ligand; an antibody, fragment, or construct that prevents the interaction of TIM-3 with one or more of its ligands, Galectin-9, PtdSer, HMGB1, and CEACAM1; an antibody, fragment, or construct that prevents the interaction of one or more KIR with their respective ligands; an antibody, fragment, or construct that prevents the interaction of TIGIT with one or more of its ligands, PVR, PVRL2, and PVRL3; an antibody, fragment, or construct that prevents the interaction of VISTA with one or more of its binding partners; antibodies, fragments or constructs thereof that prevent inhibitory signaling via CD39 and / or CD73 and / or that prevent the interaction of A2AR and / or A2BR with adenosine; antibodies, fragments or constructs thereof that prevent the interaction of B7-H3 with its receptor and / or B7-H4 with its receptor; antibodies, fragments or constructs thereof that prevent the interaction of BTLA with its ligand HVEM; antibodies, fragments or constructs thereof that prevent the interaction of LAG-3 with one or more of its ligands or more; an antibody, fragment or construct thereof that prevents the interaction of CD94 / NKG2A with HLA-E; an antibody, fragment or construct thereof that prevents the interaction of one or more Siglecs with their respective ligands; an antibody, fragment or construct thereof that prevents CD20 signaling; an antibody, fragment or construct thereof that prevents the interaction of CD47 with SIRPα; an antibody, fragment or construct thereof that prevents the interaction of PVRIG with PVRL2;an antibody, fragment, or construct thereof that prevents the interaction between CSF1R and CSF1; an antibody, fragment, or construct thereof that prevents NOX signaling; and / or an antibody, fragment, or construct thereof that prevents IDO and / or TDO signaling;

[0272] Immune checkpoint inhibitors may be inhibitory nucleic acid molecules, such as oligonucleotides, siRNAs, shRNAs, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers), particularly antisense oligonucleotides. In one embodiment, immune checkpoint inhibitors that are siRNAs interfere with mRNA and thus block translation, e.g., translation of immune checkpoint proteins.

[0273] The checkpoint inhibitor may also be a soluble form of the molecule (or a variant thereof) itself, such as a soluble PD-L1 or a PD-L1 fusion.

[0274] In the context of the present disclosure, more than one checkpoint inhibitor can be used, and in this case, more than one checkpoint inhibitor targets different checkpoint pathways or targets the same checkpoint pathway.Preferably, more than one checkpoint inhibitor is a different checkpoint inhibitor.Preferably, when more than one different checkpoint inhibitor is used, particularly at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different checkpoint inhibitors are used, preferably 2, 3, 4 or 5 different checkpoint inhibitors are used, more preferably 2, 3 or 4 different checkpoint inhibitors are used, even more preferably 2 or 3 different checkpoint inhibitors are used, and most preferably 2 different checkpoint inhibitors are used.

[0275] In one embodiment, the inhibitory immunomodulator (immune checkpoint blocker) is a component of the PD-1 / PD-L1 or PD-1 / PD-L2 signaling pathway. Accordingly, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the PD-1 signaling pathway. In a specific embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In a specific embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 ligand inhibitor, for example, a PD-L1 inhibitor or a PD-L2 inhibitor. In a preferred embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is an antibody, antigen-binding portion thereof, or construct thereof that disrupts or inhibits the interaction of the PD-1 receptor with one or more of its ligands, PD-L1 and / or PD-L2. Antibodies that bind to PD-1 and disrupt or inhibit the interaction of PD-1 with one or more of its ligands are known in the art. In a specific embodiment, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-1. In certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-L1 and disrupts or inhibits its interaction with PD-1, thereby increasing immune activity. In certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-L2 and disrupts or inhibits its interaction with PD-1, thereby increasing immune activity.

[0276] In one embodiment, the inhibitory immunomodulator is a component of the CTLA-4 signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the CTLA-4 signaling pathway. In a specific embodiment, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 inhibitor. In a specific embodiment, the checkpoint inhibitor of the CTLA-4 signaling pathway is a CTLA-4 ligand inhibitor.

[0277] In one embodiment, the inhibitory immunomodulator is a component of the TIGIT signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the TIGIT signaling pathway. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT inhibitor. In certain embodiments, the checkpoint inhibitor of the TIGIT signaling pathway is a TIGIT ligand inhibitor.

[0278] In one embodiment, the inhibitory immunomodulator is a component of the B7 family signaling pathway. In a specific embodiment, the B7 family members are B7-H3 and B7-H4. In one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of B7-H3 and / or B7-4. Although the B7 family does not have any distinct receptors, these ligands are upregulated in tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blocking these ligands can enhance anti-tumor immunity.

[0279] In one embodiment, the inhibitory immunomodulator is a component of the BTLA signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the BTLA signaling pathway. In certain embodiments, the checkpoint inhibitor of the BTLA signaling pathway is a BTLA inhibitor. In certain embodiments, the checkpoint inhibitor of the BTLA signaling pathway is an HVEM inhibitor.

[0280] In one embodiment, the inhibitory immunomodulator is a component of one or more KIR signaling pathways. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of one or more KIR signaling pathways. In certain embodiments, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR inhibitor. In certain embodiments, the checkpoint inhibitor of one or more KIR signaling pathways is a KIR ligand inhibitor. For example, a KIR inhibitor according to the present disclosure may be an anti-KIR antibody that binds to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0281] In one embodiment, the inhibitory immunomodulator is a component of the LAG-3 signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of LAG-3 signaling. In certain embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 inhibitor. In certain embodiments, the checkpoint inhibitor of the LAG-3 signaling pathway is a LAG-3 ligand inhibitor.

[0282] In one embodiment, the inhibitory immunomodulator is a component of the TIM-3 signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the TIM-3 signaling pathway. In a specific embodiment, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 inhibitor. In a specific embodiment, the checkpoint inhibitor of the TIM-3 signaling pathway is a TIM-3 ligand inhibitor.

[0283] In one embodiment, the inhibitory immunomodulator is a component of the CD94 / NKG2A signaling pathway. Accordingly, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the CD94 / NKG2A signaling pathway. In a specific embodiment, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94 / NKG2A inhibitor. In a specific embodiment, the checkpoint inhibitor of the CD94 / NKG2A signaling pathway is a CD94 / NKG2A ligand inhibitor.

[0284] In one embodiment, the inhibitory immunomodulator is a component of the IDO signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the IDO signaling pathway, e.g., an IDO inhibitor.

[0285] In one embodiment, the inhibitory immunomodulator is a component of the adenosine signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the adenosine signaling pathway. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD39 inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is a CD73 inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2AR inhibitor. In certain embodiments, the checkpoint inhibitor of the adenosine signaling pathway is an A2BR inhibitor.

[0286] In one embodiment, the inhibitory immunomodulator is a component of the VISTA signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the VISTA signaling pathway. In a specific embodiment, the checkpoint inhibitor of the VISTA signaling pathway is a VISTA inhibitor.

[0287] In one embodiment, the inhibitory immunomodulator is a component of one or more Siglec signaling pathways. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of one or more Siglec signaling pathways. In certain embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec inhibitor. In certain embodiments, the checkpoint inhibitor of one or more Siglec signaling pathways is a Siglec ligand inhibitor.

[0288] In one embodiment, the inhibitory immunomodulator is a component of the CD20 signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the CD20 signaling pathway. In a specific embodiment, the checkpoint inhibitor of the CD20 signaling pathway is a CD20 inhibitor.

[0289] In certain embodiments, the inhibitory immunomodulator is a component of the GARP signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the GARP signaling pathway. In certain embodiments, the checkpoint inhibitor of the GARP signaling pathway is a GARP inhibitor.

[0290] In one embodiment, the inhibitory immunomodulator is a component of the CD47 signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the CD47 signaling pathway. In a specific embodiment, the checkpoint inhibitor of the CD47 signaling pathway is a CD47 inhibitor. In a specific embodiment, the checkpoint inhibitor of the CD47 signaling pathway is a SIRPα inhibitor.

[0291] In certain embodiments, the inhibitory immunomodulator is a component of the PVRIG signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the PVRIG signaling pathway. In certain embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG inhibitor. In certain embodiments, the checkpoint inhibitor of the PVRIG signaling pathway is a PVRIG ligand inhibitor.

[0292] In certain embodiments, the inhibitory immunomodulator is a component of the CSF1R signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the CSF1R signaling pathway. In certain embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1R inhibitor. In certain embodiments, the checkpoint inhibitor of the CSF1R signaling pathway is a CSF1 inhibitor.

[0293] In certain embodiments, the inhibitory immunomodulator is a component of the NOX signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the NOX signaling pathway, e.g., a NOX inhibitor.

[0294] In certain embodiments, the inhibitory immunomodulator is a component of the TDO signaling pathway. Thus, in one embodiment of the present disclosure, the checkpoint inhibitor is an inhibitor of the TDO signaling pathway, e.g., a TDO inhibitor.

[0295] Exemplary PD-1 inhibitors include, but are not limited to, BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606 and US 2015 / 0079109), lambrolizumab (e.g., as disclosed in WO 2008 / 156712 as hPD109A and its humanized derivatives h409A1, h409A16 and h409A17), AB137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivolumab (Opdivo, BMS-936558; Bristol Myers Squibb), and ribozyme inhibitors (Rx100, Rx101, Rx102, Rx103, Rx104, Rx105, Rx106, Rx107, Rx108, Rx110, Rx119, Rx120, Rx121, Rx122, Rx123, Rx124, Rx125, Rx126, Rx127, Rx128, Rx129, Rx130, Rx131, Rx132, Rx133, Rx134, Rx135, Rx136, Rx137, Rx138, Rx139, Rx140, Rx141, Rx142, Rx143, Rx144, Rx145, Rx146, Rx147, Rx148, Rx149, Rx149, Rx149, Rx149, Rx14 Squibb; see U.S. Patent No. 8,008,449; WO2013 / 173223; WO2006 / 121168), pembrolizumab (Keytruda; MK-3475; Merck; see WO2008 / 156712), pidilizumab (CT-011; CureTech; Hardy et al., 1994, Cancer Res., 54(22):5793-6 and WO2009 / 101611), PDR001 (Novartis; see WO2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see WO2012 / 145493), TSR-042 (see WO2014 / 179664), cemiplimab (REGN-2810; Regeneron; H4H7798N; see US2015 / 0203579 and WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70: 136), AMP-224 (GSK-2661380; Li et al., 2016, Int J Mol Sci 17(7):1151 and WO2010 / 027827 and WO2011 / 066342), PF-06801591 (Pfizer), tislelizumab (BGB-A317; BeiGene; see WO2015 / 35606, U.S. Patent No. 9,834,606, and US2015 / 0079109), BI754091, SHR-1210 (see WO2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, INCSHR1210 (Jiangsu Hengrui), as described in WO2006 / 121168. Medicine; also known as SHR-1210; see WO2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang et al., 2017, J. Hematol. Oncol. 70: 136), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics; see WO2017 / 19846), IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540), cetrelimab (JNJ-63723283; JNJ-3283; see Calvo et al., J. Clin. Oncol. 36, no. 5_suppl (2018) 58), genolizumab (CBT-501; Patel et al., J. ImmunoTher. Cancer, 2017, 5(Suppl 2):P242), sasanlimab (PF-06801591; Youssef et al., Proc. Am. Assoc. Cancer Res. Ann.Meeting 2017; Cancer Res 2017;77(13 Suppl):see Abstract), toripalimab (JS-001; see US2016 / 0272708), camrelizumab (SHR-1210; INCSHR-1210; see US2016 / 376367; Huang et al., Clin. Cancer Res. 2018;24(6):1296-1304), spartalizumab (PDR001; see WO2017 / 106656; see Naing et al., J. Clin. Oncol. 34, no. 15_suppl (2016) 3060-3060), BCD-100 (JSC BIOCAD, Russia; see WO2018 / 103017), balstilimab (AGEN2034; see WO2017 / 040790), sintilimab (IBI-308; see WO2017 / 024465 and WO2017 / 133540), and ezabenlimab (BI-754091; US2017 / 334995; Johnson et al., J. Clin. Oncol. 36, no.5_suppl (2018) 212-212), zimberelimab (GLS-010; see WO2017 / 025051), LZM-009 (see US2017 / 210806), AK-103 (see WO2017 / 071625, WO2017 / 166804, and WO2018 / 036472), retifanlimab (MGA-012; see WO2017 / 019846), Sym-021 (see WO2017 / 055547 anti-PD-1 antibodies such as CS1003 (see CN107840887), anti-PD-1 antibodies such as the IgG1-PD1 disclosed herein (i.e., comprising a VH sequence as defined in SEQ ID NO: 43, a VL sequence as defined in SEQ ID NO: 44, an Fc sequence as defined in SEQ ID NO: 61, and a kappa sequence as defined in SEQ ID NO: 27), e.g., those disclosed in U.S. Pat. No. 7,488,802, U.S. Pat. No. 8,008,449, U.S. Pat. No. 8,168,757, , WO03 / 042402, WO2010 / 089411 (which further disclose anti-PD-L1 antibodies), WO2010 / 036959, WO2011 / 159877 (which further disclose antibodies against TIM-3), WO2011 / 082400, WO2011 / 161699, WO2009 / 014708, WO03 / 099196, WO2009 / 114335, WO2012 / 145493 (which further disclose antibodies against PD-L1 and anti-PD-1 antibodies described in WO2015 / 035606, WO2014 / 055648 (which further disclose anti-KIR antibodies), US2018 / 0185482 (which further discloses anti-PD-L1 and anti-TIGIT antibodies), US8,008,449, US8,779,105, US6,808,710, US8,168,757, US2016 / 0272708, and US8,354,509, e.g., Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, small molecule antagonists against the PD-1 signaling pathway, e.g., siRNAs directed against PD-1 disclosed in WO2019 / 000146 and WO2018 / 103501, soluble PD-1 proteins disclosed in WO2018 / 222711, and oncolytic viruses containing soluble forms of PD-1, e.g., described in WO2018 / 022831.

[0296] In certain embodiments, the PD-1 inhibitor is nivolumab (Opdivo; BMS-936558), pembrolizumab (Keytruda; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI754091, or SHR-1210. In one embodiment, the PD-1 inhibitor is an IgG1-PD1 as disclosed herein.

[0297] In certain embodiments, the inhibitory immunomodulatory agent is an anti-PD-1 antibody or antigen-binding fragment thereof comprising the complementarity-determining regions (CDRs) of one of the anti-PD-1 antibodies or antigen-binding fragments described above, e.g., an anti-PD-1 antibody or antigen-binding fragment thereof comprising the CDRs of one anti-PD-1 antibody or antigen-binding fragment selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, and IgG1-PD1.

[0298] In some embodiments, the CDRs of an anti-PD-1 antibody are delineated using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242).

[0299] In certain embodiments, the inhibitory immunomodulatory agent is an anti-PD-1 antibody or antigen-binding fragment thereof comprising the heavy chain variable region and light chain variable region of one of the anti-PD-1 antibodies or antigen-binding fragments described above, e.g., an anti-PD-1 antibody or antigen-binding fragment thereof comprising the heavy chain variable region and light chain variable region of one anti-PD-1 antibody or antigen-binding fragment selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, and IgG1-PD1.

[0300] In certain embodiments, the inhibitory immunomodulatory agent is an anti-PD-1 antibody or antigen-binding fragment thereof selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, IgG1-PD1.

[0301] The anti-PD-1 antibodies of the present disclosure are preferably monoclonal, and may be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an 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 may belong to 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.

[0302] In certain embodiments of the present disclosure, the anti-PD-1 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) described herein, including, but not limited to, Fab, Fab' and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Antigen-binding fragments include fragments comprising any of the variable domains. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region alone or in combination with all or part of the following: hinge region, CH1, CH2, CH3, and CL domains. Also included within the present disclosure are antigen-binding fragments comprising any combination of the variable region with the hinge region, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0303] The anti-PD-1 antibodies disclosed herein may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of PD-1, or may be specific for both PD-1 and a heterologous protein. See, e.g., PCT Publications WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547-1553.

[0304] The anti-PD-1 antibodies disclosed herein may be described or identified in terms of the particular CDRs they contain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including, but not limited to, Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, the CDRs or individual identified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., its variable region) shall be understood to encompass (or be specific for) the CDRs defined in any of the foregoing schemes. For example, a particular CDR (e.g., CDR-H3) may be a CDR of a given V. H or V L When a variable region is described as containing the amino acid sequence of a corresponding CDR in the amino acid sequence of the variable region, it is understood that such CDR has the sequence of a corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the aforementioned schemes. A particular CDR or scheme for identifying a CDR may be specified, for example, a CDR defined by the Kabat, Chothia, AbM, or IMGT method.

[0305] In some embodiments, the numbering of amino acid residues in the CDR sequences of the anti-PD-1 antibodies or antigen-binding fragments thereof provided herein is according to the IMGT numbering scheme described in Lefranc, MP et al., Dev. Comp. Immunol., 2003, 27, 55-77.

[0306] In some embodiments, the anti-PD-1 antibodies disclosed herein comprise the CDRs of the antibody nivolumab. See WO2006 / 121168. In some embodiments, the CDRs of the antibody nivolumab are detailed using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242). The present disclosure encompasses anti-PD-1 antibodies or derivatives thereof comprising a heavy or light chain variable domain, the variable domain comprising (a) a set of three CDRs, the set of CDRs being from the monoclonal antibody nivolumab, and (b) a set of four framework regions, the set of framework regions being different from the set of framework regions in the monoclonal antibody nivolumab, and the anti-PD-1 antibody or derivative thereof binds to PD-1. In a specific embodiment, the anti-PD-1 antibody is nivolumab.

[0307] The anti-PD-1 antibodies disclosed herein may also be described or specified in terms of their binding affinity to PD-1 (e.g., human PD-1). Preferred binding affinities include those with a binding affinity of 5×10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 These include those with a dissociation constant or Kd less than M.

[0308] Anti-PD-1 antibodies also include modified derivatives and constructs, i.e., derivatives and constructs modified by the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not interfere with the antibody's binding to PD-1. For example, without limitation, anti-PD-1 antibody derivatives include modified antibodies, such as those modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. 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, etc. In addition, the derivatives or constructs may contain one or more non-classical amino acids.

[0309] Exemplary PD-1 ligand inhibitors are PD-L1 inhibitors and PD-L2 inhibitors, including, but not limited to, MEDI4736 (durvalumab; AstraZeneca; see WO2011 / 066389), MSB-0010718C (see US2014 / 0341917), YW243.55.S70 (see SEQ ID NO: 20 in WO2010 / 077634 and US8,217,149), MIH1 (Affymetrix eBioscience; see EP3230319), MDX-1105 (Roche / Genentech; see WO2013019906 and US8,217,149), STI-1014 (Sorrento; see WO2013 / 181634), CK-301 (checkpoint therapy), KN035 (3D Med / Alphamab; see Zhang et al., 2017, Cell Discov. 3:17004), atezolizumab (Tecentriq; RG7446; MPDL3280A; R05541267; see US9,724,413), BMS-936559 (Bristol Myers Squibb; US 7,943,743, see WO2013 / 173223), avelumab (Bavencio; see US 2014 / 0341917), LY3300054 (Eli Lilly Co.), CX-072 (Proclaim-CX-072; also known as CytomX; see WO2016 / 149201), FAZ053, KN035 (see WO2017020801 and WO2017020802), MDX-1105 (see US2015 / 0320859), anti-PD-L1 antibodies such as those disclosed in US 7,943,743, e.g., 3G10, 12A4 (BMS -936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, etc., WO2010 / 077634, US8,217,149, WO2010 / 036959, WO2010 / 077634, WO2011 / 066342, US8,217,149, US7,943,743, WO2010 / 089411, US7,635,757, US8,2 17,149, US2009 / 0317368, WO2011 / 066389, WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801 , WO2016 / 111645, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 160792, WO2016 Examples of such antibodies include the anti-PD-L1 antibodies described in WO2015 / 022630, WO2016 / 007235, WO2015 / 179654, WO2015 / 173267, WO2015 / 181342, WO2015 / 109124, WO2018 / 222711, WO2015 / 112805, WO2015 / 061668, WO2014 / 159562, WO2014 / 165082, and WO2014 / 100079.

[0310] In certain embodiments, the PD-L1 inhibitor is atezolizumab (Tecentriq; RG7446; MPDL3280A; R05541267; see US Pat. No. 9,724,413).

[0311] In a specific embodiment, the inhibitory immunomodulator is an anti-PD-L1 antibody or antigen-binding fragment thereof that comprises the complementarity-determining region (CDR) of one of the anti-PD-L1 antibodies or antigen-binding fragments described above, such as the CDRs of atezolizumab or its antigen-binding fragment.

[0312] In some embodiments, the CDRs of an anti-PD-L1 antibody are delineated using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242).

[0313] In a specific embodiment, the inhibitory immunomodulator is an anti-PD-L1 antibody or antigen-binding fragment thereof that comprises the heavy chain variable region and light chain variable region of one of the anti-PD-L1 antibodies or antigen-binding fragments described above, for example, the heavy chain variable region and light chain variable region of atezolizumab or its antigen-binding fragment.

[0314] The anti-PD-L1 antibodies of the present disclosure are preferably monoclonal, and may be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, and PD-L1-binding fragments of any of the above. In some embodiments, the anti-PD-L1 antibodies described herein specifically bind to PD-L1 (e.g., human PD-L1). The immunoglobulin molecules of the present disclosure may belong to 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.

[0315] In certain embodiments of the present disclosure, the anti-PD-L1 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) described herein, including, but not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V HAntigen-binding fragments include fragments comprising any of the variable domains. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with all or part of the following: hinge region, CH1, CH2, CH3, and CL domains. Also included within the disclosure are antigen-binding fragments comprising any combination of the variable region(s) with the hinge region, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0316] The anti-PD-L1 antibodies disclosed herein may be monospecific, bispecific, trispecific, or may have greater multispecificity. Multispecific antibodies may be specific for different epitopes of PD-L1, or may be specific for both PD-L1 and a heterologous protein. See, e.g., PCT Publications WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60 69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547 1553.

[0317] The anti-PD-L1 antibodies disclosed herein may be described or identified in terms of the particular CDRs that they contain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including, but not limited to, Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, the CDRs or individual identified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., its variable region) shall be understood to encompass (or be specific for) the CDRs defined in any of the foregoing schemes. For example, a particular CDR (e.g., CDR-H3) may be a CDR of a given V. H or V L When a variable region is described as containing the amino acid sequence of a corresponding CDR in the amino acid sequence of the variable region, it is understood that such CDR has the sequence of a corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the aforementioned schemes. A particular CDR or scheme for identifying a CDR may be specified, for example, a CDR defined by the Kabat, Chothia, AbM, or IMGT method.

[0318] In some embodiments, the numbering of the amino acid residues in the CDR sequences of the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein is according to the IMGT numbering scheme described in Lefranc, MP et al., Dev. Comp. Immunol., 2003, 27, 55-77.

[0319] In some embodiments, the anti-PD-L1 antibodies disclosed herein comprise the CDRs of the antibody atezolizumab. See US 9,724,413. In some embodiments, the CDRs of the antibody atezolizumab are detailed using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242). The present disclosure encompasses anti-PD-L1 antibodies or derivatives thereof comprising a heavy or light chain variable domain comprising (a) a set of three CDRs, wherein the set of CDRs is from the monoclonal antibody atezolizumab, and (b) a set of four framework regions, wherein the set of framework regions differs from the set of framework regions in the monoclonal antibody atezolizumab, and wherein the anti-PD-L1 antibody or derivative thereof binds to PD-L1. In a specific embodiment, the anti-PD-L1 antibody is atezolizumab.

[0320] The anti-PD-L1 antibodies disclosed herein may also be described or specified in terms of their binding affinity to PD-L1 (e.g., human PD-L1). Preferred binding affinities include those with a binding affinity of 5×10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10-9 Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 These include those with a dissociation constant or Kd less than M.

[0321] Anti-PD-L1 antibodies also include modified derivatives and constructs, i.e., derivatives and constructs that have been modified by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not interfere with the binding of the antibody to PD-L1. For example, without limitation, anti-PD-L1 antibody derivatives include modified antibodies, such as those modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. 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, etc. In addition, the derivatives or constructs may contain one or more non-classical amino acids.

[0322] Exemplary CTLA-4 inhibitors include, but are not limited to, the monoclonal antibodies ipilimumab (Yervoy; Bristol Myers Squibb) and tremelimumab (Pfizer / MedImmune), trevilizumab, AGEN-1884 (Agenus), and ATOR-1015, WO2001 / 014424, US2005 / 0201994, EP1212422, US5,811,097, US5,855,887, US6,051,2 27, US6,682,736, US6,984,720, WO01 / 14424, WO00 / 37504, US2002 / 0039581, US2002 / 086014, WO98 / 42752, US6,207,156, US5,977,318, US7,109,003, and US7,132,281, and the anti-CTLA4 antibodies, CTLA-4 These include the dominant negative proteins abatacept (Orencia; see EP 2855533), which contain the Fe region of IgG1 fused to the ECD, and belatacept (Nulojix; see WO 2014 / 207748), a second generation higher affinity CTLA-4-Ig variant with two amino acid substitutions in the CTLA-4 ECD compared to abatacept, soluble CTLA-4 polypeptides such as RG2077 and CTLA4-IgG4m (see US 6,750,334), anti-CTLA-4 aptamers, and CTLA-4-directed siRNAs such as those disclosed in US 2015 / 203848. Exemplary CTLA-4 ligand inhibitors are described in Pile et al., 2015 (Encyclopedia of Inflammatory Diseases, M. Parnham (ed.), doi: 10.1007 / 978-3-0348-0620-6_20).

[0323] Exemplary checkpoint inhibitors of the TIGIT signaling pathway include, but are not limited to, anti-TIGIT antibodies such as BMS-986207, COM902 (CGEN-15137; Compugen), AB154 (Arcus Biosciences) or etigilimab (OMP-313M32; OncoMed Pharmaceuticals), or the antibodies disclosed in WO2017 / 059095, particularly "MAB10", US2018 / 0185482, WO2015 / 009856, and US2019 / 0077864.

[0324] Exemplary checkpoint inhibitors of B7-H3 include, but are not limited to, the Fc-optimized monoclonal antibody enoblituzumab (MGA271; Macrogenics; see US2012 / 0294796) and the anti-B7-H3 antibodies MGD009 (Macrogenics) and pidilizumab (see US7,332,582).

[0325] Exemplary B7-H4 inhibitors include, but are not limited to, antibodies described in Dangaj et al., 2013 (Cancer Research 73:4820-9) and Smith et al., 2014 (Gynecol Oncol, 134:181-189), antibodies described in WO2013 / 025779 (e.g., 2D1 encoded by SEQ ID NOs: 3 and 4, 2H9 encoded by SEQ ID NOs: 37 and 39, and 2E11 encoded by SEQ ID NOs: 41 and 43), and antibodies described in WO2013 / 067492 (e.g., antibodies having an amino acid sequence selected from SEQ ID NOs: 1 to 8), morpholino antisense oligonucleotides, e.g., antibodies described in Kryczek et al., 2006 (J Exp Med, 203:871-81), or soluble recombinant forms of B7-H4, such as those disclosed in US2012 / 0177645.

[0326] Exemplary BTLA inhibitors include, but are not limited to, anti-BTLA antibodies described in Crawford and Wherry, 2009 (J Leukocyte Biol 86:5-8), anti-BTLA antibodies described in WO2011 / 014438 (e.g., 4C7 or antibodies comprising heavy and light chains according to SEQ ID NOs: 8 and 15 and / or SEQ ID NOs: 11 and 18), anti-BTLA antibodies described in WO2014 / 183885 (e.g., the antibody deposited under number CNCM I-4752), and anti-BTLA antibodies described in US2018 / 155428.

[0327] Exemplary inhibitors of KIR signaling include, but are not limited to, the monoclonal antibodies lirilumab (1-7F9; IPH2102; see US 8,709,411), IPH4102 (Innate Pharma; Marie-Cardine et al., 2014, Cancer 74(21): 6060-70), such as the anti-KIR antibodies disclosed in US2018 / 208652, US2018 / 117147, US2015 / 344576, WO2005 / 003168, WO2005 / 009465, WO2006 / 072625, WO2006 / 072626, WO2007 / 042573, WO2008 / 084106 (e.g., antibodies comprising heavy and light chains according to SEQ ID NOs: 2 and 3), WO2010 / 065939, WO2012 / 071411, WO2012 / 160448, and WO2014 / 055648.

[0328] Exemplary LAG-3 inhibitors include, but are not limited to, the anti-LAG-3 antibodies BMS-986016 (Bristol-Myers Squibb; see WO2014 / 008218 and WO2015 / 116539), 25F7 (see US2011 / 0150892), IMP731 (see WO2008 / 132601), H5L7BW (see WO2014140180), MK-4280 (28G-10; Merck; see WO2016 / 028672), REGN3767 (Regneron / Sanofi), BAP050 (see WO2017 / 019894), IMP-701 (LAG-525; Novartis), Sym022 (Symphogen), TSR-033 (Tesaro), MGD013 (a bispecific DART antibody targeting LAG-3 and PD-1 developed by MacroGenics), BI754111 (Boehringer Ingelheim), FS118 (a bispecific antibody targeting LAG-3 and PD-1 developed by F-star), GSK2831781 (GSK), and WO2009 / 044273, WO2008 / 132601, WO2015 / 042246, EP2320940, US2019 / 169294, US2019 / 169292, WO2016 / 028672, WO2016 / 126858, WO2016 / 200782, WO2015 / 200119, WO 2017 / 220569, WO2017 / 087589, WO2017 / 219995, WO2017 / 019846, WO2017 / 106129, WO20 17 / 062888, WO2018 / 071500, WO2017 / 087901, US2017 / 0260271, WO2017 / 198741, WO201 7 / 220555, WO2017 / 015560, WO2017 / 025498, WO2017 / 149143, WO2018 / 069500, WO2018 / 083087, WO2018 / 034227, WO2014 / 140180, the LAG-3 antagonist protein AVA-017 (Avacta), the soluble LAG-3 fusion protein IMP321 (eftiragimode alfa; Immutep; EP2205257 and Brignone et al., 2007, J.Immunol., 179: 4202-4211), as well as the soluble LAG-3 protein disclosed in WO2018 / 222711.

[0329] Exemplary TIM-3 inhibitors include, but are not limited to, antibodies that target TIM-3, such as F38-2E2 (BioLegend), covolimab (TSR-022; Tesaro), LY3321367 (Eli Lilly), MBG453 (Novartis), and antibodies disclosed in, for example, WO2013 / 006490, WO2018 / 085469 (e.g., antibodies comprising heavy and light chain sequences encoded by nucleic acid sequences according to SEQ ID NOs: 3 and 4), WO2018 / 106588, WO2018 / 106529 (e.g., antibodies comprising heavy and light chain sequences according to SEQ ID NOs: 8-11).

[0330] Exemplary TIM-3 ligand inhibitors include, but are not limited to, CEACAM1 inhibitors, such as the anti-CEACAM1 antibody CM10 (cCAM Biotherapeutics; see WO2013 / 054331), antibodies disclosed in WO2015 / 075725 (e.g., CM-24, 26H7, 5F4, TEC-11, 12-140-4, 4 / 3 / 17, COL-4, F36-54, 34B1, YG-C28F2, D14HD11, M8.7.7, D11-AD11, HEA81, Bl.1, CLB-gran-10, F34-187, T84.1, B6.2, B1.13, YG-C94G7, 12-140-5, scFv DIATHIS1, TET-2; cCAM Biotherapeutics), Watt et al. al., 2001 (Blood, 98: 1469-1479) and the antibodies described in WO2010 / 12557, as well as PtdSer inhibitors such as bavituximab (Peregrine).

[0331] Exemplary CD94 / NKG2A inhibitors include, but are not limited to, monalizumab (IPH2201; Innate Pharma) and antibodies and methods for their production disclosed in US 9,422,368 (see, e.g., humanized Z199; EP 2628753), EP 3193929 and WO 2016 / 032334 (see, e.g., humanized Z270; EP 2628753).

[0332] Exemplary IDO inhibitors include, but are not limited to, exiguamine A, epacadostat (INCB024360; InCyte; see US 9,624,185), indoximod (Newlink Genetics; CAS No.: 110117-83-4), NLG919 (Newlink Genetics / Genentech; CAS No.: 1402836-58-1), GDC-0919 (Newlink Genetics / Genentech; CAS No.: 1402836-58-1), F001287 (Flexus Biosciences / BMS; CAS No.: 2221034-29-1), KHK2455 (Cheong et al., 2018, Expert Opin Ther Pat. 28(4):317-330), PF-06840003 (see WO2016 / 181348), navoximod (RG6078, GDC-0919, NLG919; CAS number: 1402837-78-8), linrodostat (BMS-986205; Bristol-Myers Suibb; CAS number: 1923833-60-6), small molecules such as 1-methyl-tryptophan, pyrrolidine-2,5-dione derivatives (see WO2015 / 173764), and the IDO inhibitors disclosed by Sheridan, 2015, Nat Biotechnol 33:321-322.

[0333] Exemplary CD39 inhibitors include, but are not limited to, A001485 (Arcus Biosciences), PSB069 (CAS number: 78510-31-3), and the anti-CD39 monoclonal antibody IPH5201 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8:2411-2425.E9).

[0334] Exemplary CD73 inhibitors include, but are not limited to, anti-CD73 antibodies such as CPI-006 (Corvus Pharmaceuticals), MEDI9447 (MedImmune; see WO2016075099), IPH5301 (Innate Pharma; see Perrot et al., 2019, Cell Reports 8:2411-2425.E9), the anti-CD73 antibodies described in WO2018 / 110555, and the small molecule inhibitors PBS12379 (Tocris Bioscience; CAS number: 1802226-78-3), A000830, A001190, and A001421 (Arcus Biosciences; Becker et al., 2018, Cancer Research 78(13 Supplement):3691-3691, doi: 10.1158 / 1538-7445.AM2018-3691), CB-708 (Calithera Biosciences), and the purine cytotoxic nucleoside analog-based diphosphonates described by Allard et al., 2018 (Immunol Rev., 276(1):121-144).

[0335] Exemplary A2AR inhibitors include, but are not limited to, istradefylline (KW-6002; CAS number: 155270-99-8), PBF-509 (Palobiopharma), ciforadenant (CPI-444: Corvus Pharma / Genentech; CAS number: 1202402-40-1), ST1535 ([2-butyl-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purine-6-xylamine]; CAS number: 496955-42-1), ST4206 (Stasi et al., 2015, Europ J Pharm 761:353-361; CAS No.: 1246018-36-9), tozadenant (SYN115; CAS No.: 870070-55-6), V81444 (see WO2002 / 055082), preladenant (SCH420814; Merck; CAS No.: 377727-87-2), bipadenant (BIIB014; CAS No.: 442908-10-3), ST1535 (CAS No.: 496955-42-1), SCH412348 (CAS No.: 377727-26-9), SCH442416 (Axon 2283; Axon Medchem; CAS No.: 316173-57-6), ZM241385 (4-(2-(7-amino-2-(2-furyl)-(1,2,4)triazolo(2,3-a)-(1,3,5)triazin-5-yl-amino)ethyl)phenol; CAS No.: 139180-30-6), AZD4635 (AstraZeneca), AB928 (dual A2AR / A2BR small molecule inhibitor; Arcus Biosciences), and SCH58261 (see Popoli et al., 2000, Neuropsychopharm 22:522-529; CAS No.: 160098-96-4).

[0336] Exemplary A2BR inhibitors include, but are not limited to, AB928 (dual A2AR / A2BR small molecule inhibitor; Arcus Biosciences), MRS1706 (CAS No.: 264622-53-9), GS6201 (CAS No.: 752222-83-6), and PBS1115 (CAS No.: 152529-79-8).

[0337] Exemplary VISTA inhibitors include, but are not limited to, anti-VISTA antibodies such as JNJ-61610588 (ombatilimab; Janssen Biotech), and the small molecule inhibitor CA-170 (anti-PD-L1 / L2 and anti-VISTA small molecule; CAS number: 1673534-76-3).

[0338] Exemplary Siglec inhibitors include, but are not limited to, the anti-Sigle-7 antibodies disclosed in US2019 / 023786 and WO2018 / 027203 (e.g., antibodies comprising a variable heavy chain region according to SEQ ID NO: 1 and a variable light chain region according to SEQ ID NO: 15), the anti-Siglec-2 antibody inotuzumab ozogamicin (Besponsa; see US8,153,768 and US9,642,918), the anti-Siglec-3 antibody gemtuzumab ozogamicin (Mylotarg; see US9,359,442), or the antibody disclosed in US2019 / 062427. , US2019 / 023786, WO2019 / 011855, WO2019 / 011852 (e.g., antibodies comprising CDRs according to SEQ ID NOs: 171-176, or 3 and 4, or 5 and 6, or 7 and 8, or 9 and 10, or 11 and 12, or 13 and 14, or 15 and 16, or 17 and 18, or 19 and 20, or 21 and 22, or 23 and 24, or 25 and 26), US2017 / 306014, and the anti-Siglec-9 antibodies disclosed in EP3146979.

[0339] Exemplary CD20 inhibitors include, but are not limited to, anti-CD20 antibodies such as rituximab (Rituxan; IDEC-102; IDEC-C2B8; see US 5,843,439), ABP798 (a biosimilar of rituximab), ofatumumab (2F2; see WO2004 / 035607), obinutuzumab, ocrelizumab (2h7; see WO2004 / 056312), ibritumomab tiuxetan (Zevalin), tositumomab, ublituximab (LFB-R603; LFB Biotechnologies), and antibodies disclosed in US2018 / 0036306 (e.g., antibodies comprising light and heavy chains according to SEQ ID NOS: 1-3 and 4-6, or 7 and 8, or 9 and 10).

[0340] Exemplary GARP inhibitors include, but are not limited to, anti-GARP antibodies such as ARGX-115 (arGEN-X), as well as the antibodies and methods for their production disclosed in US2019 / 127483, US2019 / 016811, US2018 / 327511, US2016 / 251438, EP3253796.

[0341] Exemplary CD47 inhibitors include, but are not limited to, HuF9-G4 (Stanford University / Forty Seven), CC-90002 / INBRX-103 (Celgene / Inhibrx), SRF231 (Surface Oncology), IBI188 (Innovent Biologics), AO-176 (Arch Oncology), bispecific antibodies targeting CD47 such as TG-1801 (NI-1701; a bispecific monoclonal antibody targeting CD47 and CD19; Novimmune / TG Therapeutics), and NI-1801 (a bispecific monoclonal antibody targeting CD47 and mesothelin; Novimmune), and CD47 fusion proteins, such as ALX148 (ALX Oncology; Kauder et al., 2019, PLoS One, doi: Examples of antibodies include anti-CD47 antibodies such as IgG1 (see 10.1371 / journal.pone.0201832).

[0342] Exemplary SIRPα inhibitors include, but are not limited to, anti-SIRPα antibodies such as OSE-172 (Boehringer Ingelheim / OSE), FSI-189 (Forty Seven), and anti-SIRPα fusion proteins such as TTI-621 and TTI-662 (Trillium Therapeutics; see WO2014 / 094122).

[0343] Exemplary PVRIG inhibitors include, but are not limited to, anti-PVRIG antibodies such as COM701 (CGEN-15029), and antibodies described, for example, in WO2018 / 033798 (e.g., CHA.7.518.1H4(S241P), CHA.7.538.1.2.H4(S241P), CPA.9.086H4(S241P), CPA.9.083H4(S241P), CHA.9.547.7.H4(S241P), CHA.9.547.13.H4(S241P), and antibodies comprising a variable heavy chain domain according to SEQ ID NO: 5 and a variable light chain domain according to SEQ ID NO: 10 of WO2018 / 033798, or an antibody comprising a heavy chain according to SEQ ID NO: 9 and a light chain according to SEQ ID NO: 14; WO2018 / 033798 98 further discloses anti-TIGIT antibodies and combination therapies using anti-TIGIT and anti-PVRIG antibodies), antibodies disclosed in WO2016134333, WO2018017864 and methods for producing the same (e.g., an antibody comprising a heavy chain according to SEQ ID NOS: 5 to 7 which has at least 90% sequence identity with SEQ ID NO: 11 and / or a light chain according to SEQ ID NOS: 8 to 10 which has at least 90% sequence identity with SEQ ID NO: 12, or an antibody encoded by SEQ ID NOS: 13 and / or 14 or SEQ ID NOS: 24 and / or 29, or another antibody disclosed in WO2018 / 017864), and anti-PVRIG antibodies and fusion peptides disclosed in WO2016 / 134335.

[0344] Exemplary CSF1R inhibitors include, but are not limited to, the anti-CSF1R antibody cabiralizumab (FPA008; FivePrime; see WO2011 / 140249, WO2013 / 169264, and WO2014 / 036357), IMC-CS4 (EiiLilly), emactuzumab (R05509554; Roche), RG7155 ( WO2011 / 70024, WO2011 / 107553, WO2011 / 131407, WO2013 / 87699, WO2013 / 119716, WO2013 / 132044), and the small molecule inhibitors BLZ945 (CAS number: 953769-46-5) and pexidartinib (PLX3397; Selleckchem; CAS number: 1029044-16-3).

[0345] Exemplary CSF1 inhibitors include, but are not limited to, anti-CSF1 antibodies disclosed in EP1223980 and Weir et al., 1996 (J Bone Mineral Res 11: 1474-1481), WO2014 / 132072, and antisense DNA and RNA disclosed in WO2001 / 030381.

[0346] Exemplary NOX inhibitors include, but are not limited to, NOX1 inhibitors such as the small molecule ML171 (Gianni et al., 2010, ACS Chem Biol 5(10):981-93, NOS31 (Yamamoto et al., 2018, Biol Pharm Bull. 41(3):419-426), NOX2 inhibitors such as the small molecule Ceplene (histamine dihydrochloride; CAS number: 56-92-8), BJ-1301 (Gautam et al., 2017, Mol Cancer Ther 16(10):2144-2156; CAS number: 1287234-48-3), and the inhibitors described by Lu et al., 2017, Biochem Pharmacol 143:25-38, the small molecule inhibitor VAS2870 (Altenhoefer et al., 2012, Cell Mol Life Sciences 69(14):2327-2343), diphenyleneiodonium (CAS number: 244-54-2), and NOX4 inhibitors such as GKT137831 (CAS number: 1218942-37-0; see Tang et al., 2018, 19(10):578-585).

[0347] Exemplary TDO inhibitors include, but are not limited to, 4-(indol-3-yl)-pyrazole derivatives (see US 9,126,984 and US 2016 / 0263087), 3-indole substituted derivatives (see WO 2015 / 140717, WO 2017 / 025868, WO 2016 / 147144), 3-(indol-3-yl)-pyridine derivatives (see US 2015 / 0225367 and WO2015 / 121812), dual IDO / TDO antagonists, such as the small molecule dual IDO / TDO inhibitors disclosed in WO2015 / 150097, WO2015 / 082499, WO2016 / 026772, WO2016 / 071283, WO2016 / 071293, WO2017 / 007700, and the small molecule inhibitor CB548 (Kim, C, et al., 2018, Annals Oncol 29 (suppl_8): viii400-viii441).

[0348] According to the present disclosure, immune checkpoint inhibitors are inhibitors of inhibitory checkpoint proteins, but preferably not inhibitors of stimulatory checkpoint proteins.

[0349] In a preferred embodiment, the immune checkpoint inhibitor inhibits one of the inhibitory immune checkpoint signaling pathways described herein, in particular the PD-1 pathway (the interaction of PD-1 with one or more of its ligands (e.g., PD-L1 and / or PD-L2)), the CTLA-4 pathway (the interaction of CTLA-4 with one or more of its ligands (e.g., CD80 or CD86)), the TIM-3 pathway (the interaction of TIM-3 with one or more of its ligands (e.g., galectin-9, PtdSer, HMGB1, and CEACAM1)), the KIR pathway (the interaction of KIR with The immune checkpoint inhibitor is an antibody, particularly an antagonist or blocking antibody, that disrupts or inhibits one of the inhibitory immune checkpoint signaling pathways selected from the group consisting of the PD-1 pathway (interaction of PD-1 with one or more of its ligands (e.g., PD-L1 and / or PD-L2)), the CTLA-4 pathway (interaction of CTLA-4 with one or more of its ligands (e.g., CD80 or CD86)), the LAG-3 pathway (interaction of LAG-3 with one or more of its ligands), the TIGIT pathway (interaction of TIGIT with one or more of its ligands (e.g., PVR, PVRL2, and PVRL3)), the VISTA pathway (interaction of VISTA with one or more of its ligands), and the GARP pathway (interaction of GARP with one or more of its ligands). In a preferred embodiment, the immune checkpoint inhibitor is an antibody, particularly an antagonist or blocking antibody, that disrupts or inhibits one of the inhibitory immune checkpoint signaling pathways selected from the group consisting of the PD-1 pathway (interaction of PD-1 with one or more of its ligands (e.g., PD-L1 and / or PD-L2)), the CTLA-4 pathway (interaction of CTLA-4 with one or more of its ligands (e.g., CD80 or CD86)). In a preferred embodiment, the immune checkpoint inhibitor is an antibody, particularly an antagonist or blocking antibody, that disrupts or inhibits the PD-1 pathway (the interaction of PD-1 with one or more of its ligands, e.g., PD-L1 and / or PD-L2). In a preferred embodiment, the immune checkpoint inhibitor is an antibody, particularly an antagonist or blocking antibody, that disrupts or inhibits the interaction of PD-1 with PD-L1.

[0350] Checkpoint inhibitors may be administered in the form of nucleic acids, such as DNA or RNA molecules, encoding immune checkpoint inhibitors, such as inhibitory nucleic acid molecules or antibodies or fragments thereof. For example, antibodies encoded by expression vectors can be delivered as described herein. Thus, nucleic acid molecules can be delivered, for example, in the form of plasmids or mRNA molecules, or complexed with delivery vehicles, such as liposomes, lipoplexes, or nucleic acid-lipid particles. Checkpoint inhibitors may also be administered via oncolytic viruses containing an expression cassette encoding the checkpoint inhibitor. Checkpoint inhibitors may also be administered by administering endogeneic or allogeneic cells capable of expressing the checkpoint inhibitor, for example, in the form of cell-based therapy.

[0351] In one embodiment, the cell-based therapy comprises genetically engineered cells. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor, such as an immune checkpoint inhibitor described herein. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor that is an inhibitory nucleic acid molecule, such as an siRNA, shRNA, an oligonucleotide, an antisense DNA or RNA, an aptamer, an antibody or fragment thereof, or a soluble immune checkpoint protein or fusion. The genetically engineered cells can also express additional agents that enhance T cell function. Such agents are known in the art. Cell-based therapies for use in inhibiting immune checkpoint signaling are disclosed, for example, in WO2018 / 222711, the entire contents of which are incorporated herein by reference.

[0352] Preferably, the checkpoint inhibitor is administered in a suitable amount, i.e., the amount of checkpoint inhibitor administered, for example, in each dose and / or treatment cycle, may be an amount that completely or partially reduces, inhibits, interferes with, or negatively modulates one or more checkpoint proteins, or may be an amount that completely or partially reduces, inhibits, interferes with, or negatively modulates the expression of one or more checkpoint proteins. Thus, a suitable amount of checkpoint inhibitor according to the present disclosure can completely or partially reduce, inhibit, interfere with, or negatively modulate one or more checkpoint proteins, or completely or partially reduces, inhibits, interferes with, or negatively modulates the expression of one or more checkpoint proteins. Therefore, the checkpoint inhibitor preferably prevents inhibitory signals associated with immune checkpoints, resulting in the prevention or reversal of immunosuppression and the establishment or enhancement of T cell immunity against cancer cells.

[0353] The amount of checkpoint inhibitor administered in each dose and / or treatment cycle may, inter alia, be within a range in which 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 said checkpoint inhibitor is bound to the checkpoint protein.

[0354] In a preferred embodiment, the amount of checkpoint inhibitor administered is, for example, in each dose and / or each treatment cycle: a) approximately 100-200 mg in total; and / or b) A total of approximately 0.20 × 10 -9 ~1350×10 -9 mol is.

[0355] The checkpoint inhibitor may be administered in any manner and by any route known in the art. The mode and route of administration will depend on the type of checkpoint inhibitor to be used. In a preferred embodiment, the checkpoint inhibitor is administered systemically, for example, parenterally, and particularly intravenously.

[0356] The checkpoint inhibitor may be administered in the form of any suitable pharmaceutical composition described herein. In a preferred embodiment, the checkpoint inhibitor is administered in the form of an infusion.

[0357] Additional therapeutic agents In addition to the binding agent and checkpoint inhibitor, the treatment regimen according to the first aspect of the present disclosure may further comprise administering to the subject one or more additional therapeutic agents.

[0358] In one embodiment, one or more additional therapeutic agents comprise one or more chemotherapeutic agents, particularly the chemotherapeutic agents commonly used in the treatment of tumor or cancer described herein.For example, one or more chemotherapeutic agents include platinum-based compounds (for example, cisplatin, oxaliplatin and carboplatin), taxane-based compounds (for example, paclitaxel and nab-paclitaxel), nucleoside analogues (for example, 5-fluorouracil and gemcitabine), and their combinations (for example, cisplatin / carboplatin+5-fluorouracil or nab-paclitaxel+gemcitabine).

[0359] Subject and tumor or cancer to be treated The subjects to be treated according to the present disclosure are preferably human subjects.

[0360] The tumor or cancer to be treated can be any tumor or cancer. Examples of tumors / cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, such as bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the genital and reproductive organs, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, kidney cancer, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumor, glioma, meningioma, and pituitary adenoma.

[0361] In one embodiment, the tumor or cancer to be treated is a non-central nervous system (CNS) tumor or cancer, eg, a non-CNS malignancy.

[0362] 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, 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. 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.

[0363] In a preferred embodiment, the tumor or cancer to be treated is a solid tumor or cancer. In one embodiment, the tumor or cancer to be treated is a non-CNS solid tumor or cancer, for example a non-CNS solid malignant tumor.

[0364] The tumor or cancer may be melanoma, specifically. Cutaneous melanoma is the 17th most common malignant tumor, with an estimated age-standardized incidence rate of 3.4 per 100,000 people. In 2020, there were an estimated 324,635 new cases of cutaneous melanoma worldwide, with 57,043 deaths (GLOBOCAN, 2020). The 5-year survival outcomes for patients with localized or distant disease are approximately 66% and 27%, respectively (SEER, 2018). In the first-line (1L) setting, targeted therapies and immune checkpoint (ICP) inhibitors, alone or in combination, are approved for the treatment of advanced or metastatic melanoma. Improved outcomes have been associated with combination therapy, for example, with programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors; however, patients experience a higher frequency of severe immune-related adverse events (irAEs) (NCCN, 2021c). Novel combination approaches aimed at enhancing efficacy and limiting toxicity offer opportunities to improve upon existing standard of care (SOC). Patients with advanced or metastatic melanoma who have progressed on targeted therapy or immunotherapy typically receive cytotoxic therapy with modest response rates; therefore, there remains a significant unmet medical need, even in the second-line (2L) and subsequent (2L+) settings (NCCN, 2021c).

[0365] In one embodiment, where the tumor or cancer is melanoma, the tumor or cancer is not ocular (uveal) or mucosal melanoma. In one embodiment, the tumor or cancer is cutaneous or acral melanoma.

[0366] In one embodiment where the tumor or cancer is melanoma, the melanoma is unresectable melanoma, particularly unresectable Stage III or Stage IV melanoma (preferably according to the American Joint Committee on Cancer staging system (AJCC; version 8)).

[0367] In one embodiment, where the tumor or cancer is melanoma, the subject has not received prior systemic anti-cancer treatment for unresectable or metastatic melanoma, i.e., prior to treatment according to the first aspect, the subject has not received systemic anti-cancer treatment for unresectable or metastatic melanoma.

[0368] In one embodiment, where the tumor or cancer is melanoma, the subject has a known tumor BRAF mutation status according to local standard test (preferably FDA approved test).For such subject, especially for subject with BRAF V600E mutation melanoma, preferably meet one or more (preferably all) of the following criteria: (i) lactate dehydrogenase < local upper limit of normal; (ii) no clinically significant tumor-related symptoms in the investigator's judgment; and (iii) no rapidly progressing metastatic melanoma in the investigator's judgment.

[0369] In one embodiment, where the tumor or cancer is melanoma, the subject has not received prior treatment with an immune checkpoint (ICP) inhibitor, i.e., prior to treatment according to the first aspect, the subject has not received treatment with an ICP inhibitor (in other words, the subject is ICP inhibitor naive (CPI-naive)).

[0370] The tumor or cancer may be, in particular, colorectal cancer. Colorectal cancer (CRC) is the third most commonly diagnosed cancer in men and the second most commonly diagnosed cancer in women. In 2020, approximately 1,931,590 new cases of CRC and 935,173 deaths are estimated worldwide (GLOBOCAN, 2020). The 5-year relative survival rate in the United States is 71% for patients with localized disease at diagnosis and 14% for patients with distant disease at diagnosis (SEER, 2018). Recommended initial therapy options for advanced or metastatic disease depend on whether the patient is a candidate for intensive therapy. More intensive initial therapy options include 5-fluorouracil (5-FU) / leucovorin and oxaliplatin (FOLFOX), 5-FU / leucovorin and irinotecan (FOLFIRI), capecitabine and oxaliplatin, and 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI). The addition of biologic agents (e.g., bevacizumab, cetuximab, panitumumab) is also an option in combination with some of these regimens (NCCN, 2021a). The approval of targeted agents such as bevacizumab and cetuximab has led to improved outcomes for patients with metastatic CRC, but all currently approved targeted agents target either the VEGF pathway or the EGFR pathway. Therefore, there remains a need for new agents with novel mechanisms of action (MoAs), particularly for patients whose tumors harbor RAS (KRAS, NRAS) or BRAF mutations and whose disease has progressed following available treatment options.

[0371] In one embodiment, where the tumor or cancer is CRC, the subject has not received prior treatment with an immune checkpoint (ICP) inhibitor, i.e., prior to treatment according to the first aspect, the subject has not received treatment with an ICP inhibitor.

[0372] The tumor or cancer may be, in particular, lung cancer. The lung cancer may be non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC. Lung cancer is the second most common malignant tumor, with an estimated age-standardized incidence rate of 22.4 per 100,000 people and the leading cause of cancer death for both men and women (Kantar, 2021). In 2020, there were an estimated 2,206,771 new cases of lung cancer worldwide, with 1,796,144 deaths (GLOBOCAN, 2020). NSCLC accounts for 85% to 90% of all cases, with a 5-year survival rate of approximately 18% across all disease stages and only 3.5% for metastatic disease (Jemal et al., 2011) (Kantar, 2021; SEER, 2018). In the first-level setting, treatment typically consists of platinum-based chemotherapy combined with immunotherapy or targeted therapy, depending on molecular and biomarker analysis and tumor histology (NCCN, 2021d). More recently, the advent of PD-1 and programmed death-ligand 1 (PD-L1) inhibitors has improved outcomes for patients without driver mutations (approximately 62% of non-squamous and 77% of squamous tumors (Kantar, 2021)). For patients whose tumors do not harbor specific oncogenic mutations or express biomarkers for checkpoint inhibitor (CPI) options, more treatment alternatives are needed. Novel combinations with complementary approaches to enhance response may further address unmet needs in this population. For patients in the second-level setting, SOC is limited to platinum-based chemotherapy, CPI monotherapy, or docetaxel with or without ramucirumab, depending on prior therapy. For patients in the third-line (3L) setting, chemotherapy monotherapy is standard. Novel therapies are needed to limit toxicity and potentially enhance efficacy in this population (NCCN, 2021d).

[0373] In one embodiment, where the tumor or cancer is lung cancer, the tumor or cancer is non-small cell lung cancer (NSCLC), eg, squamous or non-squamous NSCLC.

[0374] In one embodiment, where the tumor or cancer is lung cancer, particularly NSCLC, the tumor or cancer lacks an epidermal growth factor (EGFR)-sensitizing mutation and / or an anaplastic lymphoma (ALK) translocation / ROS1 rearrangement. For subjects known to have tumors with predominantly squamous histology, molecular testing for EGFR mutations and ALK translocations is not expected to be necessary, whether this is according to the local SOC.

[0375] In one embodiment, 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 can be determined by any means and method known to those skilled in the art, for example, by local SOC testing (preferably an FDA-approved test) or immunohistochemistry (IHC) determined by a central laboratory.

[0376] In one embodiment, where the tumor or cancer is lung cancer, the subject has a histologically confirmed diagnosis of stage IV metastatic or recurrent NSCLC (AJCC version 8) and has not received prior systemic anti-cancer therapy as primary therapy for advanced or metastatic disease.

[0377] In one embodiment, where the tumor or cancer is lung cancer, the subject has not received prior treatment with an immune checkpoint (ICP) inhibitor, i.e., prior to treatment according to the first aspect, the subject has not received treatment with an ICP inhibitor.

[0378] The tumor or cancer may be, in particular, head and neck cancer. More than 600,000 cases of head and neck squamous cell carcinoma (HNSCC) are diagnosed worldwide each year. In the United States, approximately 65,630 new cases of oral cavity, pharynx, and larynx cancer and an estimated 14,500 deaths are expected to occur in 2020 over the same period (NCCN, 2021b). Tobacco use, alcohol use, and human papillomavirus (HPV) infection increase the risk of developing HNSCC. Patients with locally HPV-positive HNSCC have improved treatment outcomes compared with patients with HPV-negative disease. For patients with recurrent or metastatic HNSCC, pembrolizumab / platinum (cisplatin or carboplatin) / 5-FU and pembrolizumab monotherapy (for patients with a PD-L1 combined positive score [CPS] ≥ 20 or ≥ 1) are recommended 1L regimens; however, median overall survival (mOS) is less than 15 months (NCCN, 2021b). Therefore, HNSCC remains an area of ​​significant unmet medical need, and further opportunities exist to improve outcomes using novel treatment approaches.

[0379] In one embodiment where the tumor or cancer is head and neck cancer, the tumor or cancer is head and neck squamous cell carcinoma (HNSCC).

[0380] In one embodiment, where the tumor or cancer is head and neck cancer, histologically or cytologically confirmed recurrent or metastatic HNSCC is considered untreatable by local therapy.

[0381] In one embodiment, where the tumor or cancer is head and neck cancer, the subject has not received prior systemic therapy in the recurrent or metastatic setting. Systemic therapy completed more than six months prior to signing the consent form is permitted if given as part of multimodal treatment for locally advanced disease.

[0382] In one embodiment where the tumor or cancer is head and neck cancer, eligible primary tumor locations are the oropharynx, oral cavity, hypopharynx, and larynx.

[0383] In one embodiment, where the tumor or cancer is head and neck cancer, the subject does not have a primary tumor site (any tissue structure) in the nasopharynx.

[0384] In one embodiment, where the tumor or cancer is head and neck cancer, the subject has a tumor PD-L1 IHC combined positive score (CPS) > 1 (which can be determined by local (preferably an FDA-approved test) or central laboratory testing (central testing is required in the expansion phase)).

[0385] In one embodiment, where the tumor or cancer is oropharyngeal cancer, the subject has a human papillomavirus (HPV) p16 test result (preferably available according to the local SOC). Oral cavity, hypopharyngeal, and laryngeal cancers do not necessarily need to be HPV tested by p16 IHC, as these tumor locations are traditionally assumed to be HPV negative.

[0386] In one embodiment, where the tumor or cancer is head and neck cancer, the subject has not been treated with an immune checkpoint (ICP) inhibitor, i.e., prior to treatment according to the first aspect, the subject has not been treated with an ICP inhibitor.

[0387] The tumor or cancer may be, in particular, pancreatic ductal adenocarcinoma. Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Approximately 60,430 new cases of pancreatic cancer and 48,220 deaths are estimated to occur in the United States in 2021 (Siegal, 2021). For patients with metastatic disease at the time of diagnosis, the prognosis is dire, with an mOS of less than one year. Forfloxacin and gemcitabine, alone or in combination with albumin-bound paclitaxel, are the predominant systemic treatment regimens used as 1L treatment in this setting, although other regimens containing agents such as irinotecan liposomal injection (combined with 5-FU and leucovorin), bevacizumab, or erlotinib, and Forfloxacin, may also be utilized as 2L+ treatments (NCCN, 2021e). Despite the increasing number of treatments available in this setting, significant toxicity and lack of survival benefit with current chemotherapy and combined modalities indicate that clinical trials are an important option for patients with newly diagnosed late-stage disease.

[0388] In one embodiment where the tumor or cancer is pancreatic cancer, the tumor or cancer is not pancreatic endocrine cancer.

[0389] In one embodiment where the tumor or cancer is pancreatic cancer, the tumor or cancer is pancreatic ductal adenocarcinoma (PDAC).

[0390] In one embodiment where the tumor or cancer is pancreatic cancer, the subject has not had prior treatment for metastatic disease with radiation therapy, surgery, chemotherapy, or experimental therapy, i.e., prior to treatment according to the first aspect, the subject has not had treatment for metastatic disease with radiation therapy, surgery, chemotherapy, or experimental therapy.

[0391] In one embodiment, where the tumor or cancer is pancreatic cancer, the subject has not received prior treatment with a checkpoint inhibitor, i.e., prior to treatment according to the first aspect, the subject has not received treatment with an ICP inhibitor.

[0392] In one embodiment, where the tumor or cancer is pancreatic cancer, the tumor or cancer does not have an actionable genetic alteration, such as a BRCA1 / 2 or PALB2 mutation.

[0393] Treatment regimen The binding agent and checkpoint inhibitor may be administered in any suitable manner, for example, intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, intranodally, or intratumorally.

[0394] In one embodiment of the first aspect, the binding agent is administered to the subject, particularly 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.

[0395] In one embodiment, the checkpoint inhibitor is administered to the subject, particularly by systemic administration.Preferably, the checkpoint inhibitor is administered to the subject by intravenous injection or infusion.In one embodiment, the checkpoint inhibitor is administered in at least one treatment cycle.

[0396] In one embodiment, the binding agent and checkpoint inhibitor are administered to the subject by systemic administration.Preferably, the binding agent and checkpoint inhibitor are administered to the subject by intravenous injection or infusion.In one embodiment, the binding agent and checkpoint inhibitor are administered in at least one treatment cycle.

[0397] In one embodiment, each treatment cycle is about 2 weeks (14 days), 3 weeks (21 days), or 4 weeks (28 days), preferably 3 weeks (21 days).

[0398] In certain embodiments, each dose is administered or infused every two weeks (1Q2W), every three weeks (1Q3W), or every four weeks (1Q4W), preferably every three weeks (1Q3W).

[0399] In some embodiments, one or each dose is administered or infused on day 1 of each treatment cycle. For example, one dose of the binding agent and one dose of the checkpoint inhibitor may be administered on day 1 of each treatment cycle.

[0400] 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.

[0401] The binding agent and the checkpoint inhibitor may be administered simultaneously. In an alternative preferred embodiment, the binding agent and the checkpoint inhibitor are administered separately.

[0402] In one embodiment, the method further comprises administering one or more additional therapeutic agents to the subject, and the one or more additional therapeutic agents preferably comprise one or more chemotherapeutic agents, such as platinum-based compounds (e.g., cisplatin, oxaliplatin, and carboplatin), taxane-based compounds (e.g., paclitaxel and nab-paclitaxel), nucleoside analogs (e.g., 5-fluorouracil and gemcitabine), and combinations thereof (e.g., cisplatin / carboplatin + 5-fluorouracil or nab-paclitaxel + gemcitabine).In this embodiment, the one or more additional therapeutic agents are preferably administered in at least one treatment cycle, and each treatment cycle is preferably 3 weeks (21 days).For example, one dose of the one or more additional therapeutic agents is administered at least every 3 weeks (1Q3W) during at least the first treatment cycle, for example, twice every 3 weeks (2Q3W) during at least the first treatment cycle. In one embodiment, one dose of the one or more additional therapeutic agents is administered on at least day 1 of at least the first treatment cycle, eg, on days 1 and 8 of at least the first treatment cycle.

[0403] The binding agent, checkpoint inhibitor, and, if present, one or more additional therapeutic agents can be administered in any suitable form (for example, naked).However, it is preferred that the binding agent, checkpoint inhibitor, and, if present, one or more additional therapeutic agents are administered in the form of any suitable pharmaceutical composition described herein.In one embodiment, at least the binding agent and the checkpoint inhibitor are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent, and one pharmaceutical composition for the checkpoint inhibitor), and preferably the binding agent, checkpoint inhibitor, and, if present, one or more additional therapeutic agents are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent, one pharmaceutical composition for the checkpoint inhibitor, and at least one pharmaceutical composition for one or more additional therapeutic agents).

[0404] The composition or pharmaceutical composition may be prepared as described in Remington: The Science and Practice of Pharmacy, 19 th The carrier, excipient, and / or diluent, as well as any other components suitable for pharmaceutical compositions, such as known adjuvants, may be formulated according to conventional techniques, such as those disclosed in "Therapeutic Agents for the Development of Novel Antigens," Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutically acceptable carrier or diluent, as well as any known adjuvants and excipients, should be suitable for the binding agent and / or checkpoint inhibitor, and / or one or more additional therapeutic agents, if present, and the selected mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined based on the lack of a significant negative effect on the desired biological properties of the selected compound or pharmaceutical composition (e.g., less than a substantial effect on antigen binding [e.g., 10% or less relative inhibition, 5% or less relative inhibition, etc.]).

[0405] The compositions, particularly the pharmaceutical compositions of the binding agent, the pharmaceutical composition of the checkpoint inhibitor, and, if present, at least one pharmaceutical composition of the one or more additional therapeutic agents, may include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugar- or protein-free amino acids), preservatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0406] 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. (A. R Gennaro edit. 1985).

[0407] Pharmaceutical carriers, excipients, or diluents can be selected according to the intended route of administration and standard pharmaceutical practice.

[0408] Pharmaceutically acceptable carriers include various suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, etc. that are physiologically compatible with the active compounds, particularly the binding agent, checkpoint inhibitor, and / or, if present, one or more additional therapeutic agents used herein.

[0409] Examples of suitable aqueous and non-aqueous carriers that may be employed in the (pharmaceutical) compositions include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical field.

[0410] 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 anticipated.

[0411] 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, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0412] The term "diluent" refers to diluting and / or thinning a drug. Furthermore, the term "diluent" includes any one or more of fluids, liquids or solids suspending and / or mixing media. Examples of suitable diluents include ethanol, glycerol, and water.

[0413] The (pharmaceutical) composition may also contain a pharmaceutically acceptable antioxidant, examples of which include, for example, (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0414] The (pharmaceutical) composition may also comprise isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the composition.

[0415] The (pharmaceutical) composition may also contain one or more adjuvants appropriate for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersing agents, preservatives, or buffers, which can enhance the shelf life or effectiveness of the composition. The compositions used herein may be prepared using carriers that are expected to protect the compound from 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, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with waxes, or other materials known in the art. Methods for preparing such formulations are generally known to those skilled in the art, see, for example, *Sustained and Controlled Release Drug Delivery Systems*, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0416] "Pharmaceutically acceptable salts" include, for example, acid addition salts, which can be formed by using pharmaceutically acceptable acids such as, for example, hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Further suitable pharmaceutically acceptable salts include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium (NH +); and salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations formed using counteranions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates, and arylsulfonates).Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentaneproline, cyclopentanediol ... Pionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, Hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothionate, 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 Examples of suitable salts include hydroxybenzoates, pamoates (embonates), palmitates, pantothenates, pectinates, persulfates, 3-phenylpropionates, phosphates / diphosphates, phthalates, picrates, pivalates, polygalacturonates, propionates, salicylates, stearates, sulfates, suberates, succinates, tannates, tartrates, teoclates, tosylates, triethyl iodides, undecanoates, valerates, 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 be used to prepare pharmaceutically acceptable salts, and are included in the disclosure of the present invention.

[0417] In one embodiment, the binding agent used herein, checkpoint inhibitor, and if present, one or more additional therapeutic agents 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 immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutical active substances is well known in the art.Unless any conventional media or agent is incompatible with the active compound, it is anticipated to use it in the composition.Other active compounds or therapeutic compounds can also be incorporated into the composition.

[0418] Pharmaceutical compositions for injections are typically sterile and must be 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 concentrations. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition substances that delay absorption, such as monostearate salts and gelatin. Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent, for example, with one or combination of the above-listed components as needed, and then sterilizing by microfiltration.Generally, dispersion is prepared by incorporating active compound into sterile medium that contains basic dispersion medium and other necessary components, for example, from the above-listed components.For the sterile powder that is used to prepare sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which can obtain the powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0419] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of above-listed components as needed, and then sterilized by microfiltration.Generally, dispersion is prepared by incorporating active compound into sterile medium that contains basic dispersion medium and other components required from above-listed.For the sterile powder that is used to prepare sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which can obtain the powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0420] In a second aspect, the present disclosure provides a kit comprising (i) a binding agent comprising a first binding region that binds to CD40 and a second binding region that binds to CD137, (ii) a checkpoint inhibitor, and optionally (iii) one or more additional therapeutic agents. The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, checkpoint inhibitor, and optionally one or more additional therapeutic agents) 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 (by itself or in the form of a (pharmaceutical) composition) and a second container contains the checkpoint inhibitor (by itself or in the form of a (pharmaceutical) composition). Where the kit also includes one or more additional therapeutic agents, it preferably comprises at least three containers, one containing the binding agent (by itself or in the form of a (pharmaceutical) composition), one containing the checkpoint inhibitor (by itself or in the form of a (pharmaceutical) composition), and at least a third container containing the one or more additional therapeutic agents (by itself or in the form of (a) (pharmaceutical) composition).

[0421] In a third aspect, the present disclosure provides a kit of the second aspect for use in a method for reducing or preventing tumor progression or for treating cancer in a subject. Embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, checkpoint inhibitor, optional one or more additional therapeutic agents, treatment regimens, specific tumors / cancers, and subjects) and / or the second aspect also apply to the kit for use of the third aspect.

[0422] In a fourth aspect, the present disclosure provides 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 after administration of a checkpoint inhibitor, the binding agent comprising a first binding region that binds CD40 and a second binding region that binds CD137. The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, checkpoint inhibitor, optional one or more additional therapeutic agents, treatment regimens, particular tumors / cancers, and subjects) also apply to the method of the fourth aspect.

[0423] In a further aspect, the present disclosure provides a checkpoint inhibitor for use in a method for reducing or preventing tumor progression or treating cancer in a subject, said method comprising administering a checkpoint inhibitor to said subject prior to, concurrently with, or after administration of a binding agent inhibitor, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137. The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, checkpoint inhibitor, optional one or more additional therapeutic agents, treatment regimens, particular tumors / cancers, and subjects) also apply to checkpoint inhibitors for use in this further aspect.

[0424] Citation of documents and works referenced herein is not intended as an admission that any of the foregoing is relevant prior art. All statements regarding the contents of these documents are based on information available to applicant and are not to be construed as any admission regarding the contents of these documents.

[0425] The description (including the following examples) is presented to enable those skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided merely as examples. Various modifications to the examples described herein will be readily apparent to those skilled 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. Accordingly, 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 claims.

[0426] Itemized claims 1. A binding agent for use in a method for reducing or preventing tumor progression or treating cancer in a subject, said method comprising administering a binding agent to said subject prior to, concurrently with, or after administration of a checkpoint inhibitor, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

[0427] 2. The binding agent for use according to item 1, wherein CD40 is human CD40, in particular human CD40 comprising the sequence set forth in SEQ ID NO: 36, and / or CD137 is human CD137, in particular human CD137 comprising the sequence set forth in SEQ ID NO: 38.

[0428] 3. The binding agent for use according to item 1 or 2, wherein the checkpoint inhibitor is at least one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a KIR inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a VISTA inhibitor, and a GARP inhibitor.

[0429] 4. The binding agent for use according to any one of items 1 to 3, wherein the checkpoint inhibitor is an antibody, such as a PD-1 blocking antibody, in particular pembrolizumab.

[0430] 4a. The binding agent for use of any one of paragraphs 1 to 4, wherein the checkpoint inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof comprising the complementarity-determining region (CDR) of one of the anti-PD-1 antibodies or antigen-binding fragments described herein, e.g., the CDR of one anti-PD-1 antibody or antigen-binding fragment selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0431] 4b. The binding agent for use of any one of paragraphs 1 to 4a, wherein the checkpoint inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof comprising the heavy chain variable region and the light chain variable region of one of the anti-PD-1 antibodies or antigen-binding fragments described herein, for example, the heavy chain variable region and the light chain variable region of one of the anti-PD-1 antibodies or antigen-binding fragments selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, and IgG1-PD1.

[0432] 4b1. The binding agent for use according to any one of paragraphs 1 to 4a, wherein the checkpoint inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region as defined in SEQ ID NO: 43 and a light chain variable region as defined in SEQ ID NO: 44.

[0433] 4c. The binding agent for use according to any one of items 1 to 4b, wherein the checkpoint inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, and IgG1-PD1.

[0434] 4c1. The binding agent for use according to any one of paragraphs 1 to 4b1, wherein the checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof, and the anti-PD-1 antibody comprises a VH sequence as defined in SEQ ID NO: 43, a VL sequence as defined in SEQ ID NO: 44, an Fc sequence as defined in SEQ ID NO: 61, and optionally a kappa sequence as defined in SEQ ID NO: 27.

[0435] 4d. The binding agent for use of any one of paragraphs 1 to 4, wherein the checkpoint inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a complementarity-determining region (CDR) of one of the anti-PD-L1 antibodies or antigen-binding fragments described her...

Claims

1. A binder for use in a method for reducing or preventing the progression of a tumor in a subject or for treating cancer, the method comprising administering the binder to the subject before, simultaneously with, or after administration of a PD-1 inhibitor, the binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137, 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: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; 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: 11, 12, and 13, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively, binder.

2. (i) CD40 is human CD40 or human CD40 comprising the sequence set forth in SEQ ID NO: 36, (ii) CD137 is human CD137 or human CD137 comprising the sequence set forth in SEQ ID NO: 38, (iii) the PD-1 inhibitor is a PD-1 blocking antibody, (iv) the PD-1 inhibitor is pembrolizumab, or (v) one or both of the binder and the PD-1 inhibitor are administered systemically or intravenously, the binder according to claim 1.

3. A) a) the first binding region 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 with SEQ ID NO: 7 or 9, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 8 or 10; and b) The second binding region 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 with SEQ ID NO: 17 or 19, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 18 or 20. B) a) The first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7 or 9, and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; and b) The second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19, and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 18 or 20, or C) a) The first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 10; and b) The second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO:

20. The binder according to claim 1.

4. (i) The binder is a multispecific antibody or a bispecific antibody, or (ii) The binder is in the form of a full-length antibody or an antibody fragment. The binder according to claim 1.

5. A) The binder is i) a polypeptide comprising, consisting of, or consisting essentially of the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and ii) a polypeptide comprising, consisting of, or consisting essentially of the second heavy chain variable region (VH) and the second heavy chain constant region (CH) comprising. B) The binder is i) a polypeptide comprising the first light chain variable region (VL) and further comprising the first light chain constant region (CL), and ii) a polypeptide comprising the second light chain variable region (VL) and further comprising the second light chain constant region (CL) comprising. C) The binder is an antibody comprising a first binding arm and a second binding arm, and 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) comprising; The second binding arm is 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) comprising,[[]] D) The binding agent is i) a first heavy chain and light chain comprising the antigen-binding region capable of binding to CD40, and ii) a second heavy chain and light chain comprising the antigen-binding region capable of binding to CD137 comprising, or E) The binding agent is i) a first heavy chain and light chain comprising the antigen-binding region capable of binding to CD40, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region, the first heavy chain and light chain; and ii) a second heavy chain and light chain comprising the antigen-binding region capable of binding to CD137, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region, the second heavy chain and light chain comprising,[[]] The binding agent according to claim 1. **Claim 6** (i) Each of the first and second heavy chain constant regions (CH) comprises 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, or at least a hinge region, a CH2 region, and a CH3 region, (ii) Each of the first and second heavy chain constant regions (CH) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations, (iii) In the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and the first and the second heavy chains are not substituted at the same position, or (iv) In the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted; in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted; the first and the second heavy chains are not substituted at the same position, and (a) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (b) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain. The binder according to claim 5. [

7. ] The binder according to claim 1, which induces Fc-mediated effector function to a lower degree compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CH) comprising the human IgG1 hinge, CH2, and CH3 regions. [

8. ] The first and second heavy chain constant regions (CH) are modified such that the antibody induces Fc-mediated effector function to a lower degree compared to an identical antibody except that the antibody comprises unmodified first and second heavy chain constant regions (CH). (i) Each of the unmodified first and second heavy chain constant regions (CH) comprises the amino acid sequence set forth in SEQ ID NO: 21 or 29. (ii) The Fc-mediated effector function is measured by binding to an Fcγ receptor, binding to C1q, or induction of cross-linking mediated by the Fc of an Fcγ receptor. (iii) The Fc-mediated effector function is measured by binding to C1q, or (iv) the first and second heavy chain constant regions are modified such that binding of said antibody to C1q is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody. The binding agent according to claim 7.

9. 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 the human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively, the binding agent according to claim 5.

10. (i) The positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, in said first and second heavy chains. (ii) The positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said first and second heavy chain constant regions (HC). (iii) The positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering of both the first and second heavy chain constant regions are F and E, respectively, (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, or (iv) The positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the 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 the 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 the EU numbering of the first heavy chain is R, and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L. The binder according to claim 9.

11. (i) The constant region of the first or second heavy chain is a) the sequence set forth in SEQ ID NO: 21 or 29; b) a subsequence of the sequence of a), starting from the N-terminus or C-terminus of the sequence defined in a), and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) a sequence having up to 10 substitutions, 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) selected from the group consisting of, or consisting essentially of, or consisting of, or (ii) The constant region of the first or second heavy chain is a) the sequence set forth in SEQ ID NO: 22 or 30; b) a subsequence of the sequence of a), starting from the N-terminus or C-terminus of the sequence defined in a), and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) a sequence having 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) selected from the group consisting of, or consisting essentially of, or consisting of, or (iii) The constant region of the first or second heavy chain is a) the sequence set forth in SEQ ID NO: 23 or 31; b) A sub - sequence of the array of a), starting from the N - terminus or C - terminus of the array defined in a), and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) An array having, compared with the amino acid sequence defined in a) or b), up to 10 substitutions, 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 comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of (iv) The constant region of the first or second heavy chain is a) The sequence set forth in SEQ ID NO: 24 or 32; b) A sub - sequence of the array of a), starting from the N - terminus or C - terminus of the array defined in a), and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) An array having, compared with the amino acid sequence defined in a) or b), up to 7 substitutions, e.g., up to 6 substitutions, up to 5, up to 4, up to 3, up to 2 substitutions or up to 1 substitution comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of (v) The constant region of the first or second heavy chain is a) The sequence set forth in SEQ ID NO: 25 or 33; b) A sub - sequence of the array of a), starting from the N - terminus or C - terminus of the array defined in a), and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) An array having, compared with the amino acid sequence defined in a) or b), up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of (vi) The constant region of the first or second heavy chain is a) The sequence set forth in SEQ ID NO: 26 or 34; b) A sub - sequence of the array of a), starting from the N - terminus or C - terminus of the array defined in a), and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) An array having, compared with the amino acid sequence defined in a) or b), up to 6 substitutions, up to 5 substitutions, up to 4, up to 3, up to 2 substitutions or up to 1 substitution comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of The binder according to claim 5. **Claim 12**: (i) The binder comprises a kappa (κ) light chain constant region, (ii) The binder comprises a lambda (λ) light chain constant region, (iii) The first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region, (iv) The second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region, or (v) 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, The binder according to claim 1. **Claim 13** The kappa (κ) light chain is a) the sequence set forth in SEQ ID NO: 27, b) a subsequence of the sequence of a) that starts from the N-terminus or C-terminus of the sequence defined in a) and has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) a sequence having at most 10 substitutions, 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 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b) comprises an amino acid sequence selected from the group consisting of, or The lambda (λ) light chain is a) the sequence set forth in SEQ ID NO: 28, b) a subsequence of the sequence of a) that starts from the N-terminus or C-terminus of the sequence defined in a) and has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and c) a sequence having at most 10 substitutions, 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 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b) comprises an amino acid sequence selected from the group consisting of, The binder according to claim 12. **Claim 14**: (i) The binder is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, (ii) The binder is a full-length IgG1 antibody, (iii) The binder is an antibody of the IgG1m(f) allotype, (iv) The subject is a human subject, (v) the tumor or cancer is a solid tumor or cancer, (vi) the tumor or cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney 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 lymphoma, non-Hodgkin lymphoma, Merkel cell carcinoma, and mesothelioma, or (vii) the tumor or cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, pancreatic cancer, and head and neck cancer, The binder according to claim 1.

15. (i) the tumor or cancer is melanoma, or cutaneous or nodal melanoma, or (ii) the tumor or cancer is melanoma, or cutaneous or nodal melanoma, and a. the melanoma is an unresectable melanoma or an unresectable stage III or stage IV melanoma, b. the subject has not received prior treatment with a checkpoint inhibitor, or c. the subject has not received prior systemic anti-cancer treatment for unresectable or metastatic melanoma, The binder according to claim 1.

16. (i) the tumor or cancer is lung cancer, or non-small cell lung cancer (NSCLC), or squamous or non-squamous NSCLC, (ii) the tumor or cancer is lung cancer, or non-small cell lung cancer (NSCLC), or squamous or non-squamous NSCLC, and a. the lung cancer or NSCLC has no epidermal growth factor (EGFR) sensitizing mutation or anaplastic lymphoma kinase (ALK) translocation / ROS1 rearrangement, b. the lung cancer or NSCLC contains cancer cells and PD-L1 is expressed in ≧1% of the cancer cells, or c. the subject has not received prior treatment with a checkpoint inhibitor, The binder according to claim 1.

17. (i) the tumor or cancer is head and neck cancer, or head and neck squamous cell carcinoma (HNSCC), or (ii) the tumor or cancer is head and neck cancer, or head and neck squamous cell carcinoma (HNSCC), and the subject has not received prior treatment with a checkpoint inhibitor, The binder according to claim 1.

18. (i) the tumor or cancer is pancreatic cancer, or pancreatic ductal adenocarcinoma (PDAC), or (ii) The tumor or cancer is pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC), and a. The subject has not received prior treatment for metastatic disease by radiotherapy, surgery, chemotherapy, or investigational therapy, or b. The subject has not received prior treatment with a checkpoint inhibitor, The binder according to claim 1.

19. (i) The tumor or cancer is colorectal cancer, or (ii) The tumor or cancer is colorectal cancer and the subject has not received prior treatment with a checkpoint inhibitor, The binder according to claim 1.

20. (i) The binder and the PD-1 inhibitor are administered in at least one treatment cycle, and each treatment cycle is 3 weeks (21 days), (ii) One dose of the binder and one dose of the PD-1 inhibitor are administered every 3 weeks (1Q3W), or (iii) One dose of the binder and one dose of the PD-1 inhibitor are administered on the first day of each treatment cycle, The binder according to claim 1.

21. (i) The method further comprises administering to the subject one or more additional therapeutic agents or chemotherapeutic agents, (ii) The method is Platinum-based compounds, Cisplatin, oxaliplatin, and carboplatin, Taxane-based compounds, Paclitaxel and nab-paclitaxel, Nucleoside analogs, 5-Fluorouracil and gemcitabine, Combinations thereof, Cisplatin / carboplatin + 5-fluorouracil or Nab-paclitaxel + gemcitabine The method further comprises administering to the subject one or more chemotherapeutic agents selected from The binder according to claim 1.

22. (i) One or more additional therapeutic agents or chemotherapeutic agents are administered in at least one treatment cycle, and each treatment cycle is 3 weeks (21 days), (ii) One dose of one or more additional therapeutic agents or chemotherapeutic agents is administered at least every 3 weeks (1Q3W) during at least the first treatment cycle, or is administered 2 times every 3 weeks (2Q3W) during at least the first treatment cycle, or (iii) One dose of one or more additional therapeutic agents or chemotherapeutic agents is administered on at least the first day of at least the first treatment cycle, or on days 1 and 8 of at least the first treatment cycle, The binder according to claim 21.

23. An agent comprising (i) a first binding region that binds to CD40 and a second binding region that binds to CD137, (ii) a PD-1 inhibitor, and optionally (iii) one or more additional therapeutic agents, The agent comprises a first binding region that binds to CD40 and a second binding region that binds to CD137, 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: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; 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: 11, 12, and 13, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively, Kit. **Claim 24** (i) The agent or one or more additional therapeutic agents are as defined in any one of claims 2 to 14 and 21, or (ii) The agent, the PD-1 inhibitor, and, if present, one or more additional therapeutic agents are for systemic administration or intravenous injection or infusion, The kit according to claim 23. **Claim 25** Use of the kit according to claim 23 for reducing or preventing tumor progression in a subject or for use in a method of treating cancer. **Claim 26** The kit according to claim 25, wherein the tumor or cancer or subject or method is as defined in any one of claims 14 to 22.

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