Multispecific binding agents against PD-L1 and CD137 in combination

Abstract

The present invention provides combination therapy using a binding agent that binds to human CD137 and human PD-L1 in combination with a PD-1 inhibitor to reduce or inhibit tumor progression or treat cancer. TIFF2024536383000051.tif59138

Description

[Technical Field]

[0001] Technical Field The present invention relates to combination therapy using a binding agent that binds to human CD137 and human PD-L1 in combination with a PD-1 inhibitor to reduce or inhibit tumor progression or treat cancer. [Background technology]

[0002] background CD137 (4-1BB) is a member of the TNFR family and is involved in the regulation of CD8 +CD137 is a costimulatory molecule on CD4+ T cells, regulatory T cells (Tregs), natural killer T cells (NK(T) cells), B cells, and neutrophils. On 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 agonistic antibodies leads to signal transduction using TRAF-2 and TRAF-1 as adaptors. Initial 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 prolonged 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 (Non-Patent Document 2)). 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 and include urelumab, a human IgG4 antibody (AU 2004279877 (Patent Document 1)), and utomilumab, a human IgG2 antibody (Fisher et al., 2012, Cancer Immunol. Immunother. 61: 1721-1733 (Non-Patent Document 3)).

[0003] Programmed death-ligand 1 (PD-L1, PDL1, CD274, B7H1) is a 33 kDa single-pass type I membrane protein. Based on alternative splicing, three PD-L1 isoforms have been described. PD-L1 belongs to the immunoglobulin (Ig) superfamily and contains one Ig-like C2-type domain and one Ig-like V-type domain. Freshly isolated T cells and B cells express only trace amounts of PD-L1 and are not CD14-like.+ A subset of monocytes (approximately 16%) constitutively expresses PD-L1, however, interferon-γ (IFNγ) is known to upregulate PD-L1 on tumor cells.

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

[0005] PD-L1 blocking antibodies have shown clinical activity in several types of cancer known to overexpress PD-L1 (including melanoma and NSCLC). For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. Atezolizumab is currently undergoing clinical trials as an immunotherapy for several indications, including various types of solid tumors (see, for example, Rittmeyer et al., 2017 Lancet 389:255-265), and has been approved for the indications of non-small cell lung cancer and bladder cancer. The PD-L1 antibody avelumab (Kaufman et al., Lancet Oncol. 2016;17(10):1374-1385) has been approved by the FDA for the treatment of adult and pediatric patients aged 12 years and older with metastatic Merkel cell carcinoma and is currently in clinical trials for several cancer indications, including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, NSCLC, ovarian cancer, and kidney cancer. The PD-L1 antibody durvalumab has been approved for the treatment of locally advanced or metastatic urothelial cancer and is in clinical development for multiple solid tumors and hematological cancers (see, e.g., Massard et al., 2016 J Clin Oncol. 34(26):3119-25). Additional anti-PD-L1 antibodies are described, for example, in WO 2004004771.

[0006] Horton et al. (J Immunother Cancer. 2015; 3(Suppl 2): ​​O10) (Non-Patent Document 8) discloses the combination of an agonist 4-1BB antibody with a neutralizing PD-L1 antibody. WO 2019 / 025545 (Patent Document 3) provides binding agents, such as bispecific antibodies, that bind to human PD-L1 and human CD137.

[0007] However, despite these advances in the art, there is a great need for improved therapies to inhibit tumor progression or treat cancer. [Prior art documents]

Patent Document

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Document

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

[0010] overview The present inventors have surprisingly found that the immune response is amplified by the combination of (i) stimulation with a binding agent that binds to human CD137 and human PD-L1, and (ii) a PD-1 inhibitor (particularly a PD-1 antibody).

[0011] Accordingly, in a first aspect, the present disclosure provides a binding agent for use in a method for reducing or inhibiting tumor progression or treating cancer in a subject, the method comprising administering a binding agent to the subject prior to, concurrently with, or after administration of a PD-1 inhibitor, wherein the binding agent comprises a first binding region that binds CD137 and a second binding region that binds PD-L1; and where: (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding domain that binds to PD-L1 comprises a heavy chain variable region (VH) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. case, The PD-1 inhibitor is not an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively.

[0012] In a second aspect, the present disclosure provides a kit comprising: (i) a binding agent comprising a first binding domain that binds to CD137 and a second domain that binds to PD-L1; and (ii) a PD-1 inhibitor; where: (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding domain that binds to PD-L1 comprises a heavy chain variable region (VH) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. case, The PD-1 inhibitor is not an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively.

[0013] In a third aspect, the present disclosure provides a kit of the second aspect for use in a method for reducing or inhibiting tumor progression or treating cancer in a subject.

[0014] In a fourth aspect, the present disclosure provides a method for reducing or inhibiting tumor progression or treating cancer in a subject, the method comprising administering to the subject, prior to, concurrently with, or after administration of a PD-1 inhibitor, a binding agent, the binding agent comprising a first binding region that binds CD137 and a second binding region that binds PD-L1; and where: (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding domain that binds to PD-L1 comprises a heavy chain variable region (VH) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. case, The PD-1 inhibitor is not an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively. [Brief explanation of the drawings]

[0015] [Figure 1] A schematic diagram of the predicted mechanism of action of the CD137×PD-L1 bispecific antibody is shown. (A) PD-L1 is expressed on both antigen-presenting cells (APCs) and tumor cells. Binding of PD-L1 to T cells expressing the negative regulatory molecule PD-1 effectively neutralizes T cell activation signals, ultimately leading to T cell inhibition. (B) Addition of the CD137×PD-L1 bispecific antibody blocks the inhibitory PD-1:PD-L1 interaction via the PD-L1-specific arm. At the same time, the bispecific antibody provides agonistic signaling to CD137 expressed on T cells through cell-cell interactions, resulting in potent T cell costimulation. [Figure 2]Figure 1 shows an MC38 syngeneic tumor model established by subcutaneous inoculation of 1x106 MC38 cells into C57BL / 6 mice. When tumors reached a mean volume of 64mm3, mice were randomized and treated with either mbsIgG2a-PD-L1x4-1BB (5mg / kg), anti-mouse PD-1 antibody (anti-mPD-1; 10mg / kg), alone or in combination, or PBS (all 2QWx3). A. Data shown are median tumor volumes per treatment group (n=10) and are carried forward for animals that met termination criteria. Growth curves were interrupted when <50% of animals within a treatment group remained alive (PBS, mbsIgG2a-PD-L1x4-1BB, anti-mPD-1) or by day 35 (mbsIgG2a-PD-L1x4-1BB in combination with anti-mPD-1). Arrows indicate days of treatment. B. Progression-free survival, defined as the percentage of mice with tumor volumes smaller than 500 mm3, is shown as a Kaplan-Meier curve. Mantel-Cox analysis was used to compare survival between treatment groups at day 45 (Table 8). [Figure 3] This figure shows the proliferative dose-response analysis of GEN1046 and the anti-PD-1 antibody nivolumab in an antigen-specific T cell assay using an active PD1 / PD-L1 axis. CFSE-labeled T cells electroporated with claudin-6-specific TCR- and PD-1-IVT-RNA were incubated with immature dendritic cells electroporated with claudin-6-IVT-RNA for 5 days in the presence of (A) GEN1046 (at 3-fold serial dilutions from 1 to 0.00015 μg / mL) or (B) nivolumab (at 4-fold serial dilutions from 0.8 to 0.00005 μg / mL). CD8+ T cell proliferation was measured by flow cytometry. Data shown are the expansion index as a function of antibody concentration. Error bars (SD) indicate intra-experimental variation (n = 3 replicates in (A); n = 2 duplicates in (B) using cells from one representative donor). Curves were fitted with a four parameter logarithmic fit using GraphPad Prism software v9.0 to determine EC50 values ​​and Hill slopes (shown in Tables 9 and 10). [Figure 4]This figure shows the release of PD-1 / PD-L1-mediated T cell inhibition by GEN1046 in the absence or presence of the anti-PD-1 antibody nivolumab, and the additional costimulation of CD8+ T cell proliferation. CFSE-labeled T cells electroporated with claudin-6-specific TCR- and PD-1-in vitro translated (IVT)-RNA were incubated with immature dendritic cells electroporated with claudin-6-IVT-RNA in combination with a fixed concentration of 1.6 μg / mL nivolumab or 0.8 μg / mL of the nonbinding control antibody IgG1-ctrl in the presence of 0.2 μg / mL, 0.0067 μg / mL, or 0.0022 μg / mL GEN1046 for 5 days (n=2 technical replicates / condition, using cells from n=3 individual donors). Baseline proliferation in the absence of GEN1046 was determined using medium alone, 0.8 μg / mL IgG1-ctrl alone, and 1.6 μg / mL nivolumab alone. CD8+ T cell proliferation was measured by flow cytometry. Bar graphs represent the mean ± SD of the expansion index per indicated condition, calculated using FlowJo software v10.7.1. The dashed line represents baseline proliferation in the presence of the anti-PD-1 antibody nivolumab. [Figure 5] FIG. 1 is a schematic diagram of a first-in-human, open-label, dose-escalation study with expansion cohorts to evaluate the safety of GEN1046 in subjects with malignant solid tumors. [Figure 6] FIG. 1 is a waterfall plot showing progression-free survival in subjects who had received prior checkpoint inhibitor therapy (gray line) and checkpoint inhibitor-naïve patients (black line). [Figure 7] Comparison of time since last upfront anti-PD-(L)1 in subjects among the CPI Experienced Expansion Cohort (GEN1046 monotherapy), comparing subjects with a clinical response (PR) with subjects with stable disease (SD) or progressive disease (PD). Response groups were compared using the Wilcoxon test. PR vs. PD: p=0.0017; PR vs. SD: p=0.034. [Figure 8-1]Figure 1 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 nonbinding 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 a negative control. (A-B) Data shown are the geometric mean fluorescence intensity (gMFI) ± SD of duplicate wells from one representative experiment of four. (C-D) Data shown are the gMFI ± SD of duplicate wells from one representative experiment of two. (E) Data shown are the geometric mean fluorescence intensity (gMFI) ± SD of duplicate wells from one representative experiment of four. Abbreviations: gMFI = geometric mean fluorescence intensity; PD-1 = programmed cell death protein 1; PE = R-phycoerythrin. [Figure 8-2] See description of Figure 8-1. [Figure 9A] Figure 1 shows the competitive binding of IgG1-PD1 to human PD-1 with PD-L1 and PD-L2. 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. The percentage of cells binding to biotinylated PD-L1 or PD-L2 was determined by staining cells with streptavidin-allophycocyanin and 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 the 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 9B] See legend to Figure 9A. [Figure 10] Figure 1 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 11] Figure 1 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 co-cultured with iDCs electroporated with CLDN6-encoding RNA 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 are shown for one representative donor (26268_B) out of four donors evaluated in three independent experiments. Error bars represent the standard deviation 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 12]Figure 1 shows IgG1-PD1-induced IFNγ secretion 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 was analyzed in supernatants using an IFNγ-specific immunoassay. Data shown are the mean ± standard error of the mean (SEM) concentrations for 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 of the mean. [Figure 13A] Figure 1 shows IgG1-PD1-induced cytokine secretion 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 was analyzed in the supernatant 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 13B] See legend to Figure 13A. [Figure 14]Figure 1 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 but with hexamerization-enhancing mutations), cells were incubated with human serum as a source of C1q. C1q binding was detected with a FITC-conjugated rabbit anti-C1q antibody. Data shown are the geometric mean fluorescence intensity (gMFI) ± standard deviation (SD) of duplicate wells from one representative donor of seven across three comparative experiments. Abbreviations: FITC = fluorescein isothiocyanate; gMFI = geometric mean fluorescence intensity; PE = R-phycoerythrocyanin. [Figure 15-1] Figure 1 shows FcγR binding of IgG1-PD1. Binding of IgG1-PD1 to immobilized human recombinant FcγR constructs was analyzed by SPR in a validated 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) is shown. 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 15-2] See description of Figure 15-1. [Figure 16-1]Figure 1 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). FcγRIa (A), FcγRIIa-H131 (B), FcγRIIa-R131 (C), FcγRIIb (D), FcγRIIIa-F158 (E), and FcγRIIIa-V158 (F) binding of the test antibodies was 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 16-2] See description of Figure 16-1. [Figure 17] Figure 1 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 18]Total human IgG in mouse plasma samples is shown. Mice were intravenously injected with 1 or 10 mg / kg IgG1-PD1 at t=0, and serial plasma samples were collected 10 minutes, 4 hours, 1 day, 2 days, 8 days, 14 days, and 21 days after injection. Total huIgG in plasma samples was determined by ECLIA for each mouse. Data are presented as the mean huIgG concentration ± SD for 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). 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 19A]This figure shows the antitumor activity of IgG1-PD1 in human PD-1 knock-in mice. An MC38 colon cancer syngeneic tumor model was established by subcutaneous implantation into hPD-1 KI mice. Mice were treated 2QW x 3 with 0.5, 2, or 10 mg / kg of IgG1-PD1 or pembrolizumab, or 10 mg / kg of IgG1-ctrl-FERR (nine mice per group). (A) Mean tumor volume ± SEM for each group until the last time point at which the group was completed. (B) Tumor volume for the different groups on the final day (day 11) at which all groups were completed. Data shown are tumor volumes for individual mice in each treatment group, as well as the mean tumor volume ± SEM per treatment group. Tumor volumes of treatment groups were compared to the IgG1-ctrl-FERR-treated group 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. One mouse in the 2 mg / kg IgG1-PD1 group died of unknown causes on day 16, before the tumor volume exceeded 500 mm3, and was excluded from the analysis. 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 the mean. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 20]IL-2 secretion induced by IgG1-PD1 in combination with GEN1046 in an allogeneic MLR assay is shown. Two unique donor pairs of allogeneic human mDCs and CD8+ T cells were co-cultured for 5 days in the presence of IgG1-PD1 (1 μg / mL), pembrolizumab (research grade, 1 μg / mL), GEN1046 (0.001–30 μg / mL), or a combination of either pembrolizumab or IgG1-PD1 with GEN1046. IgG1-ctrl-FERR (100 μg / mL), IgG4 (100 μg / mL), bsIgG1-PD-L1xctrl (30 μg / mL), bsIgG1-ctrlx4-1BB (30 μg / mL), and IgG1-ctrl-FEAL (30 μg / mL) were included as control antibodies. IL-2 secretion was analyzed in the supernatant by Luminex. Data shown are mean IL-2 levels ± SEM for two unique allogeneic donor pairs. Abbreviations: bsIgG1 = bispecific immunoglobulin G1; ctrl = control; FERR = mutation L234F / L235E / G236R, K409R; FEAL = mutation L234F / L235E / D265A, F405L; IL = interleukin; IgG = immunoglobulin G; mDC = mature dendritic cell; MLR = mixed lymphocyte reaction; PD1 = programmed cell death protein 1; PD-L1 = programmed cell death 1 ligand 1; SEM = standard error of the mean. [Figure 21]Figure 1 shows the enhancement of CD8+ T cell proliferation by IgG1-PD1 in combination with GEN1046 in an antigen-specific T cell stimulation assay. Human CD8+ T cells were electroporated with RNA encoding a CLDN6-specific TCR and RNA encoding PD1 and labeled with CFSE. T cells were then co-cultured with CLDN6-electroporated iDCs in the presence of 0.8 μg / mL of IgG1-PD1, pembrolizumab, or IgG1-ctrl-FERR, alone or in combination with the indicated concentrations of GEN1046. CFSE dilution in T cells was analyzed by flow cytometry after 4 days and used to calculate the expansion index. Data are shown for one representative donor out of four donors evaluated in two independent experiments. Error bars represent the SD of duplicate wells. The dotted line indicates the expansion index of CD8+ T cells co-cultured with mock-electroporated (i.e., non-CLDN6-expressing) iDCs. Abbreviations: CFSE = carboxyfluorescein succinimidyl ester; CLDN6 = claudin 6; ctrl = control; FERR = mutation L234F / L235E / G236R, K409R; iDC = immature dendritic cell; IgG1 = immunoglobulin G1; PD1 = programmed cell death protein 1; PD-L1 = programmed cell death 1 ligand 1; RNA = ribonucleic acid; SD = standard deviation; TCR = T-cell receptor. [Figure 22]Figure 2 shows the enhancement of cytokine secretion by IgG1-PD1 in combination with GEN1046 after antigen-specific CD8+ T cell stimulation. Human CD8+ T cells expressing CLDN6-specific TCR and PD1 were cocultured with CLDN6-expressing iDCs in the presence of 0.8 μg / mL of IgG1-PD1, pembrolizumab, or IgG1-ctrl-FERR alone or in combination with the indicated concentrations of GEN1046, as shown in Figure 21. Cytokine concentrations in the culture supernatants were determined by multiplex electrochemiluminescence immunoassay after 4 days. Data are shown for one representative donor out of four donors evaluated in two independent experiments. Error bars represent SD of duplicate wells. Abbreviations: CLDN6 = claudin 6; ctrl = control; FERR = mutation L234F / L235E / G236R, K409R; GM-CSF = granulocyte / macrophage colony-stimulating factor; iDC = immature dendritic cell; IgG1 = immunoglobulin G1; IFN = interferon; IL = interleukin; PD1 = programmed cell death protein 1; PD-L1 = programmed cell death 1 ligand 1; RNA = ribonucleic acid; SD = standard deviation; TCR = T-cell receptor. [Figure 23] The MC38 colon cancer model was established by SC inoculation of 1 x 10 MC38 cells into C57BL / 6 mice. When tumors reached a mean volume of 60 mm3, mice were randomized and treated with the indicated antibodies or combinations (all 2QW x 3). A. Data shown are median tumor volumes per treatment group (n = 10); data are carried forward for animals that met the termination criteria. Growth curves were interrupted when <50% of animals within a treatment group remained viable (mIgG2a-ctrl-AAKR, mbsIgG2a-PD-L1x4-1BB, anti-mouse PD-1 antibody [anti-mPD-1]) or by day 69 (mbsIgG2a-PD-L1x4-1BB in combination with anti-mPD-1). Downward-pointing triangles indicate the day of treatment. B. Progression-free survival, defined as the percentage of mice with tumor volumes smaller than 500 mm3, is shown as a Kaplan-Meier curve. [Figure 24]Shown are (re)challenges of treated mice that showed complete tumor regression and a control group of tumor-naive mice. Mice were (re)challenged with 1 x 10 MC38 tumor cells injected SC on day 121 after antibody treatment began. Data shown are mean tumor volumes ± SEM. [Figure 25] Figure 1 shows cytokine levels in the peripheral blood of MC38 tumor-bearing C57BL / 6 mice treated with mbsIgG2a-PD-L1x4-1BB, anti-mPD-1 antibodies either as single agents or in combination, or the non-binding control antibody IgG2a-ctrl-AAKR. Peripheral blood samples were collected at baseline (1 day before treatment [day -1], dotted line) and 2 days after each treatment (days 2 and 5). Cytokine analysis was performed by ECLIA. [Figure 26] Quantitative IHC and ISH data for cellular immune and tumor markers expressed in tumor tissue resected from the MC38 colon cancer model are shown. C57BL / 6 mice were inoculated with 1 x 106 MC38 cells. When tumors reached a mean volume of 50-70 mm3, mice were randomized and treated with mbsIgG2a-PD-L1 x 4-1BB, anti-mPD-1, or their combinations. Tumors were resected on day 7 (n = 5 per treatment group) or day 14 (n = 5 per treatment group) after the start of treatment. Because some resected tumor samples were too small to perform IHC analysis, four to five tumors per treatment group were analyzed. Resected tumor sections (4 μm) were stained by immunohistochemistry (IHC) using anti-CD3, anti-CD4, anti-CD8, or anti-PD-L1 antibodies, or by in situ hybridization (ISH) for 4-1BB or PD-L2. IHC data are presented as % of marker-positive cells relative to total cells counted on the slide and mean ± SEM per treatment group. ISH data are presented as RNAscope H-score per slide and mean ± SEM per treatment group. [Figure 27]Figure 1 shows GzmB and Ki67 expression in CD8 T-cell subsets from dissociated tumor tissue from an MC38 colon cancer model. C57BL / 6 mice were inoculated with 1 x 10 MC38 cells. When tumors reached a mean volume of 50-70 mm, mice were randomized and treated with mbsIgG2a-PD-L1x4-1BB, anti-mPD-1, or the combination. Seven days after treatment initiation (n = 5 per treatment group), tumors were excised, dissociated into single-cell suspensions, and analyzed by flow cytometry. Data shown are the percentage of GzmB+ cells (A) or Ki67+ cells (B) within the CD8+ T-cell population of individual mice and per treatment group, as well as the mean ± SEM. Mann-Whitney statistical analysis was performed to compare the percentage of GzmB+ or Ki67+ cells within the CD8+ T-cell population between treatment groups. *p < 0.05 and **p < 0.01.

[0016] Table 1: Sequences: Reference is made below to, among other things, the sequences and SEQ ID NOs set forth in the Sequence Listing. Reference is also made to specific examples of antibodies of the invention described herein, without the invention being limited thereto. These exemplary, but non-limiting, antibodies of the invention are designated herein by reference to the antibody names. The bold and underlined parts are F; E; G; A; L; R; and G, corresponding to positions 234; 235; 236; 265; 405; 409; and 430, respectively, according to EU numbering. In SEQ ID Nos: 83 and 84, the bolded amino acids represent the -AAKR or -AALT mutations required for controlled Fab arm exchange. In the variable regions, the CDR regions, annotated according to the IMGT definition (unless otherwise indicated or contradicted by context), are underlined.

[0017] TIFF2024536383000002.tif190150TIFF2024536383000003.tif234150TIFF2024536383000004.tif234150TIFF2024536383 000005.tif234150TIFF2024536383000006.tif231150TIFF2024536383000007.tif230150TIFF2024536383000008.tif23615 0TIFF2024536383000009.tif238150TIFF2024536383000010.tif232150TIFF2024536383000011.tif229150TIFF2024536383 000012.tif234150TIFF2024536383000013.tif237150TIFF2024536383000014.tif237150TIFF2024536383000015.tif39150 DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention The present disclosure will be further described in more detail below, but it should be understood that the present disclosure is not limited to the specific methodology, protocols and reagents described herein, as they can be modified.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0019] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The examples and preferred embodiments described in various ways should not be construed as limiting the disclosure to only those embodiments explicitly described. The description should be understood to support and encompass embodiments that combine an explicitly described embodiment with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered disclosed by the description of this application, unless the context dictates otherwise. For example, in a preferred embodiment of the binding agent used herein, the first heavy chain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR], and in another preferred embodiment of the binding agent used herein, the second heavy chain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL]; in a further preferred embodiment of the binding agent used herein, the first heavy chain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR] and the second heavy chain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL].

[0020] 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. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0021] The practice of this disclosure will be based, unless otherwise indicated, on the basis of references 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; Rompp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor). The methods employ conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques as described in (see, for example, J. Med. Chem. Soc., 1989).

[0022] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the disclosure and do not impose limitations on the scope of the otherwise claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0023] 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 indicated herein, each separate value is incorporated herein as if it were individually listed herein.

[0024] Several 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.

[0025] definition The following definitions apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined term has its art-recognized meaning.

[0026] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," as well as variants such as "comprises" and "comprising," will be understood to imply the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other member, integer, or step or group of members, integers, or steps. The term "consisting essentially of" means excluding any other member, integer, or step of essential significance. The term "comprising" encompasses the term "consisting essentially 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 "consisting essentially of" or "consisting of." Similarly, at each occurrence in this application, the term "consisting essentially of" can be replaced with the term "consisting of."

[0027] As used in the context of describing this disclosure (especially in the context of the claims), the terms "a," "an," and "the" and similar referents should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0028] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specific features or components, with or without the other features or components. For example, "X and / or Y" should be interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0029] In the context of the present disclosure, the term "about" refers to a degree of accuracy that a person skilled in the art would understand to still guarantee the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and, for example, ±0.01%. As will be appreciated by those skilled in the art, the specific deviation of a numerical value for a given technical effect depends on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater deviation than an artificial or engineered technical effect.

[0030] The term "binding agent" in the context of the present disclosure refers to any agent capable of binding to a desired antigen. In certain embodiments of the present disclosure, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also include synthetic, modified, or non-naturally occurring moieties, particularly non-peptide moieties. Such moieties may, for example, link a desired antigen-binding function or antigen-binding region, such as an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0031] As used herein, "immune checkpoint" refers to regulators of the immune system, particularly costimulatory and inhibitory signals that regulate the breadth and amount 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 between PD-1 and PD-L1 and / or PD-L2. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86, which replaces CD28 binding. In certain embodiments, the inhibitory signal is the interaction between LAG-3 and an MHC class II molecule. In certain embodiments, the inhibitory signal is the interaction between TIM-3 and one or more of its ligands, such as galectin-9, PtdSer, HMGB1, and CEACAM1. In certain embodiments, the inhibitory signal is the interaction between one or several KIRs and their ligands. In certain embodiments, the inhibitory signal is the interaction between TIGIT and one or more of its ligands, PVR, PVRL2, and PVRL3. In certain embodiments, the inhibitory signal is the interaction between CD94 / NKG2A and HLA-E. In certain embodiments, the inhibitory signal is the interaction between VISTA and its binding partner. In certain embodiments, the inhibitory signal is the interaction between one or more Siglecs and their ligands. In certain embodiments, the inhibitory signal is the interaction between GARP and one or more of its ligands. In certain embodiments, the inhibitory signal is the interaction between CD47 and SIRPα. In certain embodiments, the inhibitory signal is the interaction between PVRIG and PVRL2. In certain embodiments, the inhibitory signal is the interaction between CSF1R and CSF1. In certain embodiments, the inhibitory signal is the interaction between BTLA and HVEM. In certain embodiments, the inhibitory signal is the interaction between a part of the adenosinergic pathway, e.g., A2AR and / or A2BR, and adenosine produced by CD39 and CD73. In certain embodiments, the inhibitory signal is the interaction between B7-H3 and its receptor and / or B7-H4 and its receptor.In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX, or TDO.

[0032] The terms "checkpoint inhibitor" (CPI) and "immune checkpoint (ICP) inhibitor" are used interchangeably herein. The terms refer to a molecule, such as a binding agent, that wholly or partially reduces, inhibits, interferes with, or negatively modulates one or more checkpoint proteins, or that wholly or partially reduces, inhibits, interferes with, or negatively modulates the expression of one or more checkpoint proteins, e.g., a molecule, such as a binding agent, that inhibits an immune checkpoint, particularly an immune checkpoint inhibitory signal. In one aspect, an immune checkpoint inhibitor binds to one or more checkpoint proteins. In one aspect, an immune checkpoint inhibitor binds to one or more molecules that regulate a checkpoint protein. In one aspect, 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 according to the present disclosure can be used. 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% of the level of inhibition of a checkpoint protein, for example.

[0033] In one embodiment, a checkpoint inhibitor can be any compound, e.g., any binding agent, that inhibits the inhibitory signals of an immune checkpoint, where the inhibitory signals include interactions between PD-1 and PD-L1 and / or PD-L2; interactions between CTLA-4 and CD80 or CD86 that replace CD28 binding; interactions between LAG-3 and MHC class II molecules; interactions between TIM-3 and one or more of its ligands, such as galectin-9, PtdSer, HMGB1, and CEACAM1; interactions between one or several KIRs and their ligands; interactions between TIGIT and one or more of its ligands PVR, PVRL2, and PVRL3; interactions between CD94 / NKG2A and HLA-E; interactions between VISTA and its binding partners; interactions between one or more Siglecs and their ligands; interactions between GARP and one or more of its ligands; The checkpoint inhibitor is selected from the group consisting of: 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, 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 can be a blocking antibody, such as a PD-1 blocking antibody, a CTLA4 blocking antibody, a PD-L1 blocking antibody, a PD-L2 blocking antibody, a TIM-3 blocking antibody, a KIR blocking antibody, a LAG-3 blocking antibody, a TIGIT blocking antibody, a VISTA blocking antibody, or a GARP blocking antibody. Examples of PD-1 blocking antibodies include pembrolizumab, nivolumab, cemiplimab, and spartalizumab.Examples of CTLA4-blocking antibodies include ipilimumab and tremelimumab. Examples of PD-L1-blocking antibodies include atezolizumab, durvalumab, and avelumab.

[0034] 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.

[0035] 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 and 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.

[0036] 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.

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

[0038] The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2 nd ed. Raven Press, NY (1989). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains, linked via disulfide bonds. The chains are organized into immunoglobulin domains or regions, e.g., V L or VL (variable light chain) domain / region, C L or CL (constant light chain) domain / region, V H or a VH (variable heavy chain) domain / region, and C H or CH (constant heavy chain) domain / region C H 1(CH1), C H 2(CH2), C H 3(CH3), and C HThe heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically consists of a VL and a CL. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions that can be hypervariable in sequence and / or in the form of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise specified or contradicted by context, CDR sequences herein are identified according to the IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also the internet http address www.imgt.org). Unless otherwise stated or contradicted by context, references to amino acid positions in the constant region in this disclosure follow 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 immunoglobulin heavy chains in mammals: α, δ, ε, γ, and μ, which are responsible for the different antibody classes: 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 essentially conserved within 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 should not be understood as limiting. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups and a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids may be classified based on their structure and chemical properties. Thus, the classes of amino acids may be reflected in one or both of the following tables:

[0041] Table 2. Major classifications based on R group structure and general chemical characterization TIFF2024536383000016.tif33128

[0042] Table 3. Alternative physical and functional classifications of amino acid residues TIFF2024536383000017.tif55143

[0043] For purposes of this 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 that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0044] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of an amino acid sequence variant with an insertion, one or more amino acid residues are inserted into a specific site in the amino acid sequence, but random insertion with appropriate screening of the resulting product is 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 variants 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 variants containing deletions at the N-terminus and / or C-terminus of the protein are also called N-terminal and / or C-terminal truncated variants.

[0047] Amino acid substitution variants are characterized by the removal of at least one residue in a sequence and the insertion of another in its place. Substitution of one amino acid for another can be classified as a conservative or non-conservative substitution. It is preferred that the modification occurs at a position in the amino acid sequence that is not conserved among homologous proteins or peptides, and / or that 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 a similarly charged or uncharged amino acid. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in their side chains. In the context of the present disclosure, a "conservative substitution" is the substitution of one amino acid for another amino acid with similar structural and / or chemical properties, such as substitution of one amino acid residue for another amino acid residue of the same class, as defined in the two tables above: for example, leucine may be substituted with isoleucine, since both are aliphatic, branched, hydrophobic substances. Similarly, aspartic acid may be substituted with glutamic acid, since both are small, negatively charged residues. 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, refer to the position number of an amino acid in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence "corresponding to" an amino acid or segment in another sequence is one that, when aligned with the other amino acid or segment using a standard sequence alignment program, such as ALIGN, ClustalW, or equivalent, typically with default settings, has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain. Methods for aligning sequences or segments in a sequence, thereby determining positions in a sequence that correspond to amino acid positions according to the present disclosure, are believed to be well known in the art.

[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, that has the ability to specifically bind to an antigen (particularly an epitope on an antigen) under normal physiological conditions, preferably with a half-life of a substantial period, 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, or any other relevant, functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a period 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 connected between the chains 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 consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists 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, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), 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), where 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 can be an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, 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. As used herein, antibody includes 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 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 WO 2007 / 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)), consisting essentially of a VH domain, also called a domain antibody, a dAb fragment (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 an Fv fragment, VL and VH, are encoded by separate genes, the VL and VH may be linked by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods, and this VL and VH region pairs to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv), see, e.g., Bird et al., Science 2000, 103:141-142). 242 , 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. While such fragments are generally included within the meaning of antibody, they are a distinctive feature of the present disclosure, collectively and individually exhibiting distinct biological properties and utilities. These and other useful antibody fragments, as well as bispecific formats of such fragments, in the context of the present disclosure are further discussed herein. The term antibody, unless otherwise specified, should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, e.g., chimeric antibodies and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0052] The antibody produced can have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG (IgG1, IgG2, IgG3, IgG4, etc.), IgD, IgA (IgA1, IgA2, etc.), IgE, IgM, or IgY) encoded by the heavy chain constant region gene. When a particular isotype, such as IgG1, is mentioned herein, the term is not limited to a specific isotype sequence, such as a specific IgG1 sequence, but is used to indicate that the antibody sequence is closer to that isotype, such as IgG1, than to other isotypes. Thus, for example, the IgG1 antibody disclosed herein may be a sequence variant of a naturally occurring IgG1 antibody, including mutations 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 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 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, can be in any format, including any of the bispecific or multispecific antibody formats described herein below.

[0055] The term "full length," when used in reference to 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 nature for that particular isotype, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains for an IgG1 antibody.

[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 contain 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 originates from a non-human species (e.g., from a rodent) and whose constant region originates from a different species, such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term used to refer to various types of antibody modifications, and the process of antibody engineering is 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 human therapeutic applications are developed to reduce the expected antibody immunogenicity of non-human antibodies, such as rodent antibodies. These typically contain a non-human (e.g., murine or rabbit) variable region specific for an antigen of interest and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain," when used in the context of a chimeric antibody, refers 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 has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen-binding site, onto homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0 629 240). Substitution of framework residues from the parent antibody (i.e., non-human antibody) into the human framework regions (backmutation) 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, optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications may be applied to obtain humanized antibodies with favorable properties, such as affinity and biochemical properties, although the modifications do not necessarily have to 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 those 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 the 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, which 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 or similar to those of the binding arm or antigen-binding region of the parent antibody. However, as described elsewhere herein, amino acid modifications, such as mutations, can be made in the CDRs, constant regions, or elsewhere in the antibody, binding arm, antigen-binding region, etc., to introduce desired properties. 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 generated in a number of different species, for example, mouse, rabbit, chicken, guinea pig, llama, and goat.

[0061] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries, and 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 spleen cells from immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0063] As used herein, unless contradicted by context, the term "Fab arm" or "arm" refers to one heavy chain-light chain pair and is used interchangeably 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 can 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 an antibody region 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 a region comprising, from the N-terminus to the C-terminus of an antibody, 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 (such as 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 reference to antibodies, including multispecific antibodies, means that the antibody induces an Fc-mediated effector function, in particular such a function 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 comprising (i) the same CDR sequences, in particular CDR sequences comprising the same first and second antigen-binding regions as said antibody, 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 EU numbering as 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 be of any of the other subtypes as 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 EU numbering as set forth in Kabat (ibid.). However, the CH1 region may be of any of the other subtypes as 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 EU numbering as set forth in Kabat (ibid.). However, the CH2 region may be of any of the other subtypes as 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 EU numbering as set forth in Kabat (ibid.). However, the CH3 region may be of any of the other subtypes as 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 incapable of antigen cross-linking.

[0073] A "CD137 antibody" or "anti-CD137 antibody" is an antibody as described above that specifically binds to the antigen CD137.

[0074] A "CD137xPD-L1 antibody" or "anti-CD137xPD-L1 antibody" is a bispecific antibody that contains two different antigen-binding domains, one that specifically binds to the antigen CD137 and one that specifically binds to the antigen PD-L1.

[0075] The term "biosimilar" (e.g., of an approved reference product / biological drug), as used herein, refers to a biological product that is similar to the reference product based on data from: (a) analytical studies demonstrating that the biological product is highly similar to the reference product, with minor differences in clinically inactive components; (b) animal studies (including evaluation of toxicity); and / or (c) one or more clinical studies (including evaluation of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and efficacy (e.g., no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and efficacy) under one or more appropriate conditions of use for which the reference product is approved and intended for use and for which approval is sought. In some embodiments, the biosimilar biological product and the reference product utilize the same mechanism(s) of action for one or more conditions of use indicated, recommended, or suggested in the product labeling, but only to the extent that the mechanism(s) of action are known for the reference product. In some embodiments, one or more conditions of use indicated, recommended, or suggested in the labeling for the biologic have previously been approved for the reference product. In some embodiments, the route of administration, dosage form, and / or strength of the biologic are the same as those of the reference product. A biosimilar can be, for example, a currently known antibody that has the same primary amino acid sequence as a marketed antibody, but may be produced in a different cell type or by a different production, purification, or formulation method.

[0076] As used herein, the terms "bind" or "capable of binding" refer to an antibody binding to a given antigen or epitope, typically within about 10 s, as determined using biolayer interferometry (BLI) or, for example, as determined using surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10-10 M or less, or about 10 -11 M or even less K D An antibody binds to a given antigen with an affinity comparable to the K of binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D at least 10 times lower, for example at least 100 times lower, for example at least 1,000 times lower, for example at least 10,000 times lower, for example at least 100,000 times lower, D The amount at which the affinity is higher is the antibody's K D As a result, 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 nonspecific antigen.

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

[0078] "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 and to the same epitope. Whether a test antibody recognizes the same epitope as a particular antigen-binding antibody, i.e., whether the antibody binds to the same epitope, can be tested by various methods well known to those skilled in the art.

[0080] Competition between antibodies can be detected by a cross-blocking assay. For example, a competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the wells of a microtiter plate, and an antigen-binding antibody and a competing test antibody candidate can be added. The amount of antigen-binding antibody that binds to the antigen in the well positively correlates with the binding ability of the competing test antibody candidate that competes for binding to the same epitope. Specifically, as the affinity of the competing test antibody candidate for the same epitope increases, the amount of antigen-binding antibody that binds to the antigen-coated well decreases. The amount of antigen-binding antibody that binds to the well can be measured by labeling the antibody with a detectable or measurable label.

[0081] An antibody that competes for binding to an antigen with, or has specificity for, the antigen of another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, may be a variant of the heavy and / or light chain variable regions as described herein, e.g., an antibody that includes modifications and / or a degree of identity in the CDRs 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 CD137 and PD-L1 is substantially free of monospecific antibodies that specifically bind to CD137 or PD-L1).

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

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

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

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

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

[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 be reduced than oxidized.

[0089] The present disclosure also describes multispecific antibodies, such as bispecific antibodies, that comprise functional variants of the VL region, VH region, or one or more CDRs of exemplary bispecific antibodies. Functional variants of the VL, VH, or CDRs used in the context of a bispecific antibody enable each antigen-binding region of the bispecific antibody to still retain at least a significant 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 to 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), 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. In addition, 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 indicated, the following notation is used to describe mutations: i) a substitution of an amino acid at a given position is described, for example, as K409R, which means a 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 to denote any amino acid residue, including the codes Xaa and X. Thus, a substitution of lysine with arginine at position 409 is designated as K409R, and a substitution of lysine at position 409 with any amino acid residue is designated as K409X. In the case of a deletion of lysine at position 409, it is denoted 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, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions in the variant are conservative amino acid residue substitutions.

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

[0094] 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 1-23 of SEQ ID NO:37 correspond to the signal peptide of human CD137; while amino acids 24-186 of SEQ ID NO:37 correspond to the extracellular domain of human CD137; and the remainder of the protein, from amino acids 187-213 and 214-255 of SEQ ID NO:37, are the transmembrane and cytoplasmic domains, respectively.

[0095] The "programmed death-1 (PD-1)" receptor refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 (also known as CD279 or SLEB2) is preferentially expressed on previously activated 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). As used herein, the term "PD-1" 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, particularly a protein having the amino acid sequence as set forth in SEQ ID NO: 113 of the Sequence Listing (NCBI Reference Sequence: NP_005009.2), or preferably a protein encoded by the nucleic acid sequence as set forth in SEQ ID NO: 115 (NCBI Reference Sequence: NM_005018.2). "Programmed death-ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1.

[0096] 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 Nos. NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), and analogs that share at least one epitope with hPD-L1. The sequence of human PD-L1 is also shown in SEQ ID NO:40 (mature sequence), and SEQ ID NO:39, in which amino acids 1-18 are predicted to be a signal peptide. The term "PD-L2," as used herein, includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, as well as analogs that share at least one common epitope with hPD-L2. PD-1 ligands (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 between PD-1 and its ligand results in a reduction in tumor-infiltrating lymphocytes, a decrease in 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 the effect is additive when the interaction of PD-1 with PD-L2 is similarly blocked.

[0097] The term "dysfunction" as used herein refers to immune cells in a state of reduced immune responsiveness to antigen stimulation. Dysfunction includes 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.

[0098] The term "anergy," as used herein, refers to a state of non-responsiveness to antigenic stimulation resulting from defective or insufficient signals delivered through the T cell receptor (TCR). T cell anergy can also occur upon stimulation by antigen in the absence of costimulation, resulting in cells becoming refractory to subsequent activation by antigen, even in the presence of costimulation. The non-responsive state can often be reversed by the presence of IL-2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0099] The term "exhaustion," as used herein, refers to immune cell exhaustion, e.g., T cell exhaustion, as a state of T cell dysfunction due to persistent TCR signaling, which occurs during many chronic infections and cancers. It is distinguished from anergy in that it results from persistent signaling rather than through defective or insufficient signaling. Exhaustion is defined by defective effector function, persistent expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector or memory T cells. Exhaustion prevents optimal control of disease (e.g., infection and tumors). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) and cell-intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways, e.g., as described herein).

[0100] "Enhancing T cell function" refers to inducing, eliciting, 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 secretion of gamma interferon from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor clearance) compared to pre-intervention levels. 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.

[0101] The terms "inhibitory nucleic acid" or "inhibitory nucleic acid molecule," as used herein, refer to a nucleic acid molecule, e.g., DNA or RNA, that reduces, inhibits, interferes with, or negatively modulates one or more PD-1 proteins, in whole or in part. 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).

[0102] The term "oligonucleotide," as used herein, refers to a nucleic acid molecule capable of reducing protein expression, particularly the expression of a PD-1 protein, such as the PD-1 protein described herein. Oligonucleotides are short DNA or RNA molecules typically containing 2 to 50 nucleotides. Oligonucleotides may be single-stranded or double-stranded. PD-1 inhibitory oligonucleotides may be antisense oligonucleotides.

[0103] Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, particularly the nucleic acid sequence of the PD-1 protein (or a fragment thereof). Antisense RNA is typically used to bind to mRNA, thereby interfering with the protein translation of mRNA, for example, the mRNA encoding the PD-1 protein. Antisense DNA is typically used to target specific and 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 with tumor-associated antigen (TAA)-specific T cells.

[0104] The terms "siRNA," "small 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 a specific gene with a complementary nucleotide sequence, such as the gene encoding the PD-1 protein. In one embodiment, the siRNA interferes with mRNA, thus blocking translation, e.g., of the PD-1 protein. Transfection of exogenous siRNA can be used for gene knockdown, but the effect is likely to 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. The siRNA sequence can also be modified to introduce a short loop between the two strands to generate "small hairpin RNA" or "shRNA." shRNA can be processed into functional siRNA by Dicer. shRNA has a relatively low degradation and turnover rate. Thus, the PD-1 inhibitor can be an shRNA.

[0105] The term "aptamer," as used herein, refers to a single-stranded nucleic acid molecule, e.g., DNA or RNA, typically 25-70 nucleotides in length, capable of binding to a target molecule, e.g., a polypeptide. In one embodiment, the aptamer binds to an immune PD-1 protein, such as the PD-1 checkpoint protein described herein. For example, an aptamer according to the present disclosure can specifically bind to a PD-1 protein or polypeptide, or to a molecule in a signaling pathway that modulates the expression of a PD-1 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).

[0106] 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 reduces, inhibits, interferes with, or negatively modulates one or more PD-1 proteins, as described above, in whole or in part.Such small molecule inhibitors are usually synthesized by organic chemistry, but can also be isolated from natural sources such as plants, fungi, and bacteria.The low molecular weight allows small molecule inhibitors to rapidly pass through cell membranes.For example, various A2AR antagonists known in the art are organic compounds with molecular weights of less than 500 daltons.

[0107] The term "cell-based therapy" refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells, or stem cells) that express an immune PD-1 inhibitor into a subject for the purpose of treating a disease or disorder (e.g., a cancer disease).

[0108] The term "oncolytic virus," as used herein, refers to a virus that can selectively replicate in cancerous or hyperproliferative cells, slowing their growth, or inducing their death, either in vitro or in vivo, while having no or minimal effect on normal cells. Oncolytic viruses for delivering PD-1 inhibitors contain an expression cassette that can encode a PD-1 inhibitor that is an inhibitory nucleic acid molecule, such as an siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or soluble PD-1 protein or fusion. The oncolytic virus is preferably replication-competent, and the expression cassette is under the control of a viral promoter, such as a synthetic early / late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picornaviruses, 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, Sinbis virus, vaccinia virus (including Copenhagen, Western Reserve, and Wyeth strains), as exemplarily described in WO 2017 / 209053, and adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF). The production and use of recombinant oncolytic viruses containing soluble forms of PD-1 inhibitors are disclosed in WO 2018 / 022831, which is incorporated herein by reference in its entirety. The oncolytic virus can be used as an attenuated virus.

[0109] A "treatment cycle" is defined herein as the period of time within which the pharmacodynamics of the binding agent result in the increased effectiveness of separate doses of the binding agent, or in other words, the period after the administered binding agent has essentially been eliminated from the subject's body. Multiple smaller doses over a short time frame, e.g., a short 2-24 hour period, e.g., 2-12 hour period, or within the same day, would be equivalent to a larger single dose.

[0110] In the present context, the terms "treatment," "treating," or "therapeutic intervention" refer to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition afflicting a subject, e.g., the administration of therapeutically effective compounds to alleviate symptoms or complications, slow the progression of the disease, disorder, or condition, relieve or reduce symptoms and complications, and / or prevent the disease, disorder, or condition, as well as cure or eliminate the condition, where prevention is to be understood as the management and care of an individual with the aim of combating the disease, condition, or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications. In one aspect, "treatment" refers to the administration of an effective amount of a therapeutically active binding agent, e.g., a therapeutically active antibody, of the present disclosure with the aim of alleviating, ameliorating, preventing, or eradicating (curing) the symptoms or disease state.

[0111] The response to treatment with the binding agent of the present disclosure, as well as resistance to treatment, failure to respond to treatment, and / or recurrence from treatment, can be assessed according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST Criteria v1.1). The RECIST Criteria are shown in the table below (LD: widest range).

[0112] Table 4. Definition of response (RECIST Criteria v1.1) TIFF2024536383000018.tif84151

[0113] "Best overall response" is the best response recorded from the start of treatment to disease progression / relapse (in PD, the minimum measurement recorded since the start of treatment is used as the criterion). Subjects with CR or PR are considered to have an objective response. Subjects with CR, PR, or SD are considered to have disease control. Subjects with NE are counted as non-responders. "Best overall response" is the best response recorded from the start of treatment to disease progression / relapse (in PD, the minimum measurement recorded since the start of treatment is used as the criterion). Subjects with CR, PR, or SD are considered to have disease control. Subjects with NE are counted as non-responders.

[0114] "Duration of response (DOR)" applies only to subjects with a confirmed best overall response of CR or PR and is defined as the time from the first documented objective tumor response (CR or PR) to the date of first PD or death from underlying cancer.

[0115] "Progression-free survival (PFS)" is defined as the number of days from Day 1 of Cycle 1 to the date of first progression or death from any cause.

[0116] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 to death from any cause. If the subject died or was unknown, OS was censored at the last date the subject was known to be alive (on or before the cutoff date).

[0117] In the context of this disclosure, the term "treatment regimen" refers to a structured treatment plan designed to improve and maintain health.

[0118] The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result at the dosage or time required. A therapeutically effective amount of a binding agent, e.g., an antibody such as a multispecific antibody or a monoclonal antibody, can vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the binding agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the binding agent or fragment thereof are outweighed by the therapeutically beneficial effects. If the initial dose is insufficient in the patient, a higher dose (or an effective higher dose achieved by a different, more localized route of administration) can be used. If a patient experiences undesired side effects at a certain dose, a lower dose (or an effective lower dose achieved by a different, more localized route of administration) can be used.

[0119] As used herein, the term "cancer" includes diseases characterized by dysregulated 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 stimuli that initiated the new growth have ceased.

[0120] The term "cancer" in the present disclosure also includes cancer metastasis. "Metastasis" refers to the spread of cancer cells from their original location 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, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and subsequent infiltration of target organs after transport by the blood. Finally, the growth of new tumors, i.e., secondary or metastatic tumors, at target sites depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one aspect, the term "metastasis" in the present disclosure relates to "distant metastasis," which refers to metastasis away from the primary tumor and the regional lymph node system.

[0121] 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, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0122] 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%.

[0123] "Physiological pH," as used herein, refers to a pH of 7.5 or about 7.5.

[0124] As used in this disclosure, "wt. %" refers to weight percent, which is a unit of concentration that measures the amount in grams (g) of a substance, expressed as a percentage of the total weight in grams (g) of the total composition.

[0125] The term "freezing" refers to the solidification of a liquid, usually by removal of heat.

[0126] The term "lyophilizing" or "freeze-drying" refers to the freeze-drying of 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 freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used interchangeably herein.

[0127] The term "recombinant" in the context of this disclosure means "produced through genetic engineering." In one aspect, a "recombinant entity" in the context of this disclosure is not naturally occurring.

[0128] The term "naturally occurring" as used herein refers to an entity that can be found in nature. For example, 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 is naturally occurring. The term "naturally occurring" refers to "naturally occurring" and includes not only known entities, but also entities that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0129] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance containing 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 large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are generally used synonymously herein.

[0130] A "therapeutic protein" has a positive or beneficial effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one aspect, a therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides and can also refer to therapeutically active fragments thereof. It can 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.

[0131] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" can refer to a contiguous or discontinuous portion of the structure.

[0132] The terms "portion" and "fragment" are used interchangeably herein and refer to a single continuous element. For example, a portion of a structure such as an amino acid sequence or a protein refers to a single 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%).

[0133] A "fragment" of 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. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) 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 contains, 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 contains at least 6, particularly 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.

[0134] 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 of the pharmacological activities of the peptide or protein from which it is derived. A portion or fragment of a peptide or protein preferably comprises a sequence of at least 6, 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 at most 8, particularly at most 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.

[0135] As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid modification. The parent amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence, or can be a modified version of a wild-type amino acid sequence. Preferably, the variant 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 compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0136] As used herein, "wild-type" or "WT" or "native" 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.

[0137] 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 at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given 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 given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed with 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.

[0138] "Sequence similarity" refers to the percentage of amino acids that are either identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0139] The terms "% identical" and "% identity" or similar terms are intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences to be compared. The percentage is entirely statistical, and the differences between the two sequences can be, 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 over a segment or "window of comparison" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs using such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA, 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 on 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 on the NCBI website include (i) an Expect Threshold set to 10; (ii) a word size set to 28; (iii) maximum matches in the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) gap costs set to linear; and (vi) use of a filter for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include (i) an Expect Threshold set to 10; (ii) a word size set to 3; (iii) maximum matches in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to Existence: 11 Extension: 1; and (vi) a conditional compositional score matrix adjustment.

[0140] The percentage of identity is obtained by determining the number of identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this by 100.

[0141] In some embodiments, the degree of similarity or identity is given over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is given over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acid residues, in some embodiments, consecutive amino acid residues. In some embodiments, the degree of similarity or identity is given over the entire length of the reference sequence.

[0142] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of 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% of the amino acid residues.

[0143] The amino acid sequence variants described herein can be easily 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 easily prepared with the aid of known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0144] 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., it is functionally equivalent. With respect to an antigen or antigen sequence, one specific 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 the parent molecule or sequence, and that still performs one or more functions of the parent molecule or sequence, for example, is capable of inducing an immune response. In one embodiment, the alteration in the amino acid sequence of the parent molecule or sequence does not significantly affect or alter the properties 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 that of the parent molecule or sequence, however, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0145] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence, or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence, or a fragment thereof. For example, one of skill in the art will understand that antigens suitable for use herein may be altered in sequence to differ from the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

[0146] "Isolated" means altered or removed from the 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 can exist in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell. In preferred aspects, the binding agents used in the present disclosure are in a substantially purified form.

[0147] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein can be present in vitro or in vivo; for example, the cells can form part of an organ, tissue, and / or organism of a patient. According to this disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during the transfection process are not typically integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If transfected nucleic acid is desired to remain in the genome of the cell and its daughter cells, stable transfection must occur.Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding the antigen is transiently transfected into cells.RNA can be transfected into cells to transiently express its encoded protein.

[0148] According to the present disclosure, a peptide or protein analog is a modified form of the peptide or protein from which it is derived that retains 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 molecules 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 extends to all functional chemical equivalents of proteins and peptides.

[0149] " Activation " or " stimulation " as used herein refers to the state of immune effector cells, such as T cells, that are stimulated sufficiently to induce detectable cell proliferation. Activation can 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 to immune effector cells that are undergoing cell division.

[0150] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first comes into contact with its specific antigen, triggering differentiation into an effector cell, such as an effector T cell.

[0151] 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 immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize the antigen are amplified. Preferably, clonal expansion leads to differentiation of the immune effector cells.

[0152] "Antigen" in accordance with the present disclosure encompasses any substance that elicits an immune response and / or any substance against which an immune response or immune mechanism, such as a cellular response, is directed. This also includes situations in which an antigen is processed into antigenic peptides and an immune response or immune mechanism is directed against one or more antigenic peptides, particularly when presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). According to the present disclosure, the term "antigen" includes any molecule that contains at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that induces an immune response, preferably specific to the antigen (including cells expressing the antigen), optionally after processing. In one aspect, 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.

[0153] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a portion or fragment thereof that is recognized by the immune system, e.g., by antibodies in 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 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, the amino acid residues are within the footprint of the specifically antigen-binding peptide).

[0154] An epitope of a protein preferably comprises a contiguous or discontinuous portion of the 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, an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present disclosure is a T cell epitope.

[0155] The terms "optional" or "optionally," as used herein, mean that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes instances where the event, circumstance, or condition occurs and instances where it does not occur.

[0156] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining together of two or more elements, components, or domains.

[0157] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition that adversely affects an individual's body. Disease is often understood as a medical condition accompanied by specific symptoms and signs. Disease can be caused by factors resulting from external sources, such as infection, or by internal dysfunction, such as autoimmune disease. In humans, "disease" is often used more broadly to refer to any condition that causes pain, impairment, suffering, social problems, or death in the afflicted individual, or similar problems for those who come into contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical structural and functional changes, although in other contexts and for other purposes, these may be considered distinct categories. Because living with many illnesses can alter one's outlook on life and personality, illnesses usually adversely affect individuals not only physically but also emotionally.

[0158] The term "therapeutic treatment" refers to any treatment that improves the health status and / or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in an individual, halt or delay the onset of the disease in an individual, prevent or delay 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 has previously had the disease.

[0159] The term "preventive treatment" or "prophylactic treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "preventive treatment" or "prophylactic treatment" are used interchangeably herein. Similarly, the term "method for preventing" relates to any method intended to prevent the progression of a disease in an individual in the context of disease progression, for example tumor or cancer progression.

[0160] 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, including a bird (chicken), fish, or any other animal species, that can suffer from or is susceptible to a disease or disorder (e.g., cancer). Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly individuals, children, and newborns. In aspects of the present disclosure, an "individual" or "subject" is a "patient."

[0161] The term "patient" means an individual or subject for treatment, in particular an individual or subject suffering from a disease.

[0162] Aspects and Embodiments of the Disclosure In a first aspect, the present disclosure provides a binding agent for use in a method for reducing or inhibiting tumor progression or treating cancer in a subject, the method comprising administering a binding agent to the subject prior to, concurrently with, or after administration of a PD-1 inhibitor, wherein the binding agent comprises a first binding region that binds CD137 and a second binding region that binds PD-L1; and where: (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding domain that binds to PD-L1 comprises a heavy chain variable region (VH) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. case, The PD-1 inhibitor is not an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively.

[0163] It should be understood that neither the combination nor use of a binding agent comprising a first binding region that binds CD137 and a second binding region that binds PD-L1 with an antibody comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively, is part of the invention provided herein. It should also be understood that combinations or use of a binding agent comprising a first binding region that binds CD137 and a second binding region that binds PD-L1 with an antibody comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:146, 147, and 148, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:149, 150, and 151, respectively (CDRs are defined by Kabat numbering). Although this application refers to pembrolizumab and experimental data regarding pembrolizumab is presented herein, combinations or use with pembrolizumab are not intended to be included in any aspect or embodiment of the invention.

[0164] Binding agents that bind to CD137 and PD-L1 In one aspect, the CD137 is human CD137, particularly human CD137 comprising the sequence set forth in SEQ ID NO:38. In one aspect, the PD-L1 is human PD-L1, particularly human PD-L1 comprising the sequence set forth in SEQ ID NO:40. In one aspect, the CD137 is human CD137 and the PD-L1 is human PD-L1. In one aspect, the CD137 is human CD137 comprising the sequence set forth in SEQ ID NO:38 and the PD-L1 is human PD-L comprising the sequence set forth in SEQ ID NO:40.

[0165] In one embodiment of the binder according to the first aspect, (a) a first binding region that binds to human CD137 comprises a heavy chain variable region (VH) of SEQ ID NO: 1 or 9 comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) of SEQ ID NO: 5 or 10 comprising CDR1, CDR2, and CDR3 sequences; and (b) The second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) of SEQ ID NO:11, which comprises the CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) of SEQ ID NO:15, which comprises the CDR1, CDR2, and CDR3 sequences.

[0166] In one embodiment of the binder according to the first aspect, (a) a first binding region that binds to human CD137, comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) The second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:16, 17, and 18, respectively.

[0167] In one embodiment of the binding agent according to the first aspect, the first binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:1 or 9, and a light chain variable region (VL) 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:5 or 10.

[0168] In a further embodiment of the binding agent according to the first aspect, the second binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:11, and a light chain variable region (VL) 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:15.

[0169] In one embodiment of the binder according to the first aspect, (a) a first binding region that binds to human CD137, comprising a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 9, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 5 or 10; and (b) The second binding domain that binds to human PD-L1 comprises a heavy chain variable domain (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:11, and a light chain variable domain (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:15.

[0170] In one embodiment of the binding agent according to the first aspect, the first binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:1 or 9, and a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:5 or 10.

[0171] In a further embodiment of the binding agent according to the first aspect, the second binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:15.

[0172] In one embodiment of the binder according to the first aspect, (a) the first binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 or 9 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 5 or 10; and (b) The second binding domain that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:15.

[0173] In one embodiment of the binder according to the first aspect, (a) a first binding region that binds to human CD137, comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and (b) The second binding domain that binds to human PD-L1 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:15.

[0174] The binding agent may in particular be an antibody, such as a multispecific antibody, e.g., a bispecific antibody, and may be in the format of a full-length antibody or an antibody fragment.

[0175] It is further preferred that the binding agent is a human or humanized antibody.

[0176] Each variable region may comprise three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).

[0177] 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.

[0178] 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); (ii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH); Includes.

[0179] 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); (ii) a polypeptide comprising the second light chain variable region (VL) and further comprising a second light chain constant region (CL); Includes.

[0180] 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); (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); (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL); Includes.

[0181] In one embodiment of the first aspect, the binder is (i) a first heavy chain and a light chain comprising an antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; (ii) a second heavy chain and a light chain comprising an antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region; and Includes.

[0182] 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.

[0183] Each of the first and second heavy chain constant regions (CHs) can comprise a CH3 region, wherein the two CH3 regions comprise asymmetric mutations. Asymmetric mutations mean that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions. For example, one of the first and second CH3 regions contains a mutation at a position corresponding to EU numbering position 405 in the human IgG1 heavy chain, and the other of the first and second CH3 regions contains a mutation at a position corresponding to EU numbering position 409 in the human IgG1 heavy chain.

[0184] 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).

[0185] In one embodiment of the binding agent according to the first aspect, (i) the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said first heavy chain and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said second heavy chain.

[0186] 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.

[0187] 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 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) can comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 19 or 25.

[0188] 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.

[0189] 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%, wherein C1q binding is preferably determined by ELISA.

[0190] 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.

[0191] 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.

[0192] In particular, the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to EU numbering may be F, E and A in said first and second heavy chain constant regions, respectively.

[0193] In one embodiment of the binding agent according to the first aspect, the positions corresponding to 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 wherein (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.

[0194] 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, wherein (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.

[0195] In one embodiment of the binding agent according to the first aspect, the constant region of the first and / or second heavy chain comprises: (a) the sequence shown in SEQ ID NO:19 or SEQ ID NO:25 [IgG1-FC]; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0196] 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 second heavy chain, comprises: (a) the sequence shown in SEQ ID NO:20 or SEQ ID NO:26 [IgG1-F405L]; (b) a subsequence of the sequence in (a), e.g., 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:

[0197] 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 shown in SEQ ID NO: 21 or 27 [IgG1-F409R]; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0198] In one embodiment of the binding agent according to the first aspect, the constant region of the first and / or second heavy chain comprises: (a) the sequence shown in SEQ ID NO:22 or SEQ ID NO:28 [IgG1-Fc_FEA]; (b) a subsequence of the sequence in (a), e.g., 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 seven substitutions, e.g., at most six substitutions, at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions, or at most one 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:

[0199] In one embodiment of the binding agent according to the first aspect, the constant region of the first and / or second heavy chain, e.g. the second heavy chain, comprises: (a) the sequence shown in SEQ ID NO:24 or SEQ ID NO:30 [IgG1-Fc_FEAL]; (b) a subsequence of the sequence in (a), e.g., 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 six substitutions, e.g., at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions, or at most one 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:

[0200] In one embodiment of the binding agent according to the first aspect, the constant region of the first and / or second heavy chain, e.g. the first heavy chain, comprises: (a) the sequence shown in SEQ ID NO:23 or SEQ ID NO:29 [IgG1-Fc_FEAR]; (b) a subsequence of the sequence in (a), e.g., 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 six substitutions, e.g., at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions, or at most one 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:

[0201] In one embodiment of the first aspect, the binding agent comprises a kappa (κ) light chain constant region.

[0202] In one embodiment of the first aspect, the binding agent comprises a lambda (λ) light chain constant region.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] In one embodiment of the binding agent according to the first aspect, the kappa (κ) light chain is (a) the sequence shown in SEQ ID NO:35; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0207] In one embodiment of the binding agent according to the first aspect, the lambda (λ) light chain is (a) the sequence shown in SEQ ID NO:36; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0208] 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.

[0209] In a preferred embodiment of the binder according to the first aspect, the binder comprises: (i) a first heavy chain and a light chain comprising an antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises the sequence set forth in SEQ ID NO:31 and the first light chain comprises the sequence set forth in SEQ ID NO:32; (ii) a second heavy chain and a light chain comprising an antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises the sequence set forth in SEQ ID NO:33 and the second light chain comprises the sequence set forth in SEQ ID NO:34. Includes.

[0210] The binding agent for use according to the first aspect may in particular be acasanlimab or a biosimilar thereof.

[0211] In a currently preferred embodiment, the amount of binding agent administered at each dose and / or in each treatment cycle is: (a) about 0.3 to 5 mg / kg body weight or about 25 to 400 mg in total; and / or (b) Approximately 2.1×10 -9 ~3.4×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~2.7×10 -6 mol is.

[0212] According to these embodiments, a dose defined in mg / kg can be converted to a fixed dose based on the median body weight of subjects to whom the binding agent is administered being 80 kg, and vice versa.

[0213] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 0.3 to 4.0 mg / kg body weight or about 25 to 320 mg in total; and / or about 2.1 x 10 -9 ~2.7×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~2.2×10 -6 It can be mol.

[0214] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 0.38-4.0 mg / kg body weight or about 30-320 mg in total; and / or about 2.6 x 10 -9 ~2.7×10 -8 mol / kg body weight or a total of approximately 2.4 × 10 -7 ~2.2×10 -6 It can be mol.

[0215] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 0.5 to 3.3 mg / kg body weight or about 40 to 260 mg in total; and / or about 3.4 x 10 -9 ~2.2×10 -8 mol / kg body weight or a total of approximately 2.7 × 10 -7 ~1.8×10 -6 It can be mol.

[0216] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 0.6-2.5 mg / kg body weight or about 50-200 mg in total; and / or about 4.3 x 10 -9 ~1.7×10 -8 mol / kg body weight or a total of approximately 3.4 × 10 -7 ~1.4×10 -6 It can be mol.

[0217] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 0.8-1.8 mg / kg body weight or about 60-140 mg in total; and / or about 5.1 x 10 -9 ~1.2×10-8 mol / kg body weight or a total of approximately 4.1 × 10 -7 ~9.5×10 -7 It can be mol.

[0218] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 0.9-1.8 mg / kg body weight or about 70-140 mg in total; and / or about 6.0 x 10 -9 ~1.2×10 -8 mol / kg body weight or a total of approximately 4.8 × 10 -7 ~9.5×10 -7 It can be mol.

[0219] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 1-1.5 mg / kg body weight or about 80-120 mg in total; and / or about 6.8 x 10 -9 ~1.0×10 -8 mol / kg body weight or a total of approximately 5.5 × 10 -7 ~8.2×10 -7 It can be mol.

[0220] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 1.1-1.4 mg / kg body weight or about 90-110 mg in total; and / or about 7.7 x 10 -9 ~9.4×10 -9 mol / kg body weight or a total of approximately 6.1 × 10 -7 ~7.5×10 -7 It can be mol.

[0221] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 1.2-1.3 mg / kg body weight or about 95-105 mg in total; and / or about 6.8 x 10 -9 ~8.9×10 -9 mol / kg body weight or a total of approximately 6.5 × 10 -7 ~7.2×10 -7 It can be mol.

[0222] The amount of binding agent administered in each dose and / or in each treatment cycle is, in particular, about 0.8-1.5 mg / kg body weight or about 65-120 mg in total; and / or about 5.5 x 10 -9 ~1.0×10 -8 mol / kg body weight or a total of approximately 4.4 × 10 -7 ~8.2×10 -7 It can be mol.

[0223] The amount of binding agent administered in each dose and / or in each treatment cycle is, inter alia, about 0.9-1.3 mg / kg body weight or about 70-100 mg in total; and / or about 6.0 x 10 -9 ~8.5×10 -9 mol / kg body weight or a total of approximately 4.8 × 10 -7 ~6.8×10 -7 It can be mol. about 0.9 to 1.1 mg / kg body weight or about 75 to 90 mg in total; and / or Approximately 6.4×10 -9 ~7.7×10 -9 mol / kg body weight or a total of approximately 5.1 × 10 -7 ~6.1×10 -7 mol.

[0224] Furthermore, the amount of binder administered in each dose and / or in each treatment cycle may be, in particular, 0.3 to 4.0 mg / kg body weight or 25 to 320 mg in total; and / or 2.1 x 10 -9 ~2.7×10 -8 mol / kg body weight or a total of 1.7 × 10 -7 ~2.2×10 -6 It can be mol.

[0225] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.38 to 4.0 mg / kg body weight or 30 to 320 mg in total; and / or 2.6 x 10 -9 ~2.7×10 -8 mol / kg body weight or a total of 2.4 × 10 -7 ~2.2×10 -6 It can be mol.

[0226] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.5 to 3.3 mg / kg body weight or 40 to 260 mg in total; and / or 3.4 x 10 -9 ~2.2×10 -8 mol / kg body weight or a total of 2.7 × 10 -7 ~1.8×10 -6 It can be mol.

[0227] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.6 to 2.5 mg / kg body weight or 50 to 200 mg in total; and / or 4.3 x 10 -9 ~1.7×10 -8 mol / kg body weight or a total of 3.4 × 10 -7 ~1.4×10 -6 It can be mol.

[0228] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.8 to 1.8 mg / kg body weight or a total of 60 to 140 mg; and / or 5.1 x 10 -9 ~1.2×10 -8 mol / kg body weight or a total of 4.1 x 10 -7 ~9.5×10 -7 It can be mol.

[0229] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.9 to 1.8 mg / kg body weight or 70 to 140 mg in total; and / or 6.0 x 10 -9 ~1.2×10 -8 mol / kg body weight or a total of 4.8 × 10 -7 ~9.5×10 -7 It can be mol.

[0230] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 1 to 1.5 mg / kg body weight or a total of 80 to 120 mg; and / or 6.8 x 10 -9 ~1.0×10 -8mol / kg body weight or a total of 5.5 × 10 -7 ~8.2×10 -7 It can be mol.

[0231] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 1.1 to 1.4 mg / kg body weight or 90 to 110 mg in total; and / or 7.7 x 10 -9 ~9.4×10 -9 mol / kg body weight or a total of 6.1 x 10 -7 ~7.5×10 -7 It can be mol.

[0232] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 1.2 to 1.3 mg / kg body weight or 95 to 105 mg in total; and / or 6.8 x 10 -9 ~8.9×10 -9 mol / kg body weight or a total of 6.5 × 10 -7 ~7.2×10 -7 It can be mol.

[0233] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.8 to 1.5 mg / kg body weight or a total of 65 to 120 mg; and / or 5.5 x 10 -9 ~1.0×10 -8 mol / kg body weight or a total of 4.4 × 10 -7 ~8.2×10 -7 It can be mol.

[0234] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.9 to 1.3 mg / kg body weight or 70 to 100 mg in total; and / or 6.0 x 10 -9 ~8.5×10 -9 mol / kg body weight or a total of 4.8 × 10 -7 ~6.8×10 -7 It can be mol.

[0235] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.9 to 1.1 mg / kg body weight or 75 to 90 mg in total; and / or 6.4 x 10 -9 ~7.7×10 -9 mol / kg body weight or a total of 5.1 x 10 -7 ~6.1×10 -7 It can be mol.

[0236] The binding agent administered at each dose and / or in each treatment cycle is (a) about 1.1 mg / kg body weight or about 80 mg in total; and / or (b) Approximately 6.8×10 -9 mol / kg body weight or a total of approximately 5.5 × 10 -7 mol It could be.

[0237] The binding agent administered at each dose and / or in each treatment cycle is (a) 1.1 mg / kg body weight or 80 mg in total; and / or (b) 6.8 × 10 -9 mol / kg body weight or a total of 5.5 × 10 -7 mol It could be.

[0238] The amount of binding agent administered at each dose and / or in each treatment cycle is (a) about 1.25 mg / kg body weight or about 100 mg in total; and / or (b) Approximately 8.5×10 -9 mol / kg body weight or a total of approximately 6.8 × 10 -7 mol It is currently preferred that

[0239] The amount of binding agent administered at each dose and / or in each treatment cycle is (a) 1.25 mg / kg body weight or 100 mg in total; and / or (b) 8.5 × 10 -9 mol / kg body weight or a total of 6.8 × 10 -7mol It is equally preferable that

[0240] The binding agent may be administered in any manner and by any route known in the art. In a preferred embodiment, the binding agent is administered systemically, e.g., parenterally, particularly intravenously.

[0241] The binding agent may 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 solution.

[0242] The binding agent for use according to the invention may be administered by using intravenous (IV) infusion, for example by intravenous infusion over a period of at least 30 minutes, for example by intravenous infusion over a period of at least 60 minutes, for example by intravenous infusion over a period of 30 to 120 minutes. Preferably, the binding agent for use according to the invention is administered by using intravenous (IV) infusion over a period of 30 minutes.

[0243] The binding agent can be administered before, simultaneously with, or after administration of the PD-1 inhibitor.

[0244] In one embodiment, the binding agent is administered prior to administration of the PD-1 inhibitor. For example, the gap between the end of administration of the binding agent and the start of administration of the PD-1 inhibitor can be at least about 10 minutes, e.g., at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, and up to about 14 days (up to about 2 weeks), e.g., up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days (up to about 1 week), up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day (up to about 24 hours), up to about 18 hours, up to about 12 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2.5 hours, or up to about 2 hours.

[0245] In one embodiment, the binding agent is administered after administration of the PD-1 inhibitor. For example, the gap between the end of administration of the PD-1 inhibitor and the start of administration of the binding agent can be at least about 10 minutes, e.g., at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, and can be up to about 14 days (up to about 2 weeks), e.g., up to about 13 days, up to about 12 days, up to about 11 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days (up to about 1 week), up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, up to about 1 day (up to about 24 hours), up to about 18 hours, up to about 12 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2.5 hours, or up to about 2 hours.

[0246] In one embodiment, the binding agent is administered simultaneously with the PD-1 inhibitor. For example, the binding agent and the PD-1 inhibitor may be administered using a composition containing both drugs. Alternatively, the binding agent may be administered to one limb of the subject, and the PD-1 inhibitor may be administered to another limb of the subject.

[0247] PD-1 inhibitors In one embodiment, the PD-1 inhibitor blocks inhibitory signals associated with PD-1. In one embodiment, the PD-1 inhibitor is an antibody or fragment thereof that disrupts or inhibits inhibitory signaling associated with PD-1. In one embodiment, the PD-1 inhibitor is a small molecule inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the PD-1 inhibitor is a peptide-based inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the PD-1 inhibitor is an inhibitory nucleic acid molecule that disrupts or inhibits inhibitory signaling.

[0248] Inhibiting or blocking PD-1 signaling as described herein results in preventing or reversing immunosuppression and establishing or enhancing T cell-mediated immunity against cancer cells. In one aspect, inhibiting PD-1 signaling as described herein reduces or inhibits immune system dysfunction. In one aspect, inhibiting PD-1 signaling as described herein reduces dysfunction of dysfunctional immune cells. In one aspect, inhibiting PD-1 signaling as described herein reduces dysfunction of dysfunctional T cells.

[0249] In one embodiment, the PD-1 inhibitor blocks the interaction between PD-1 and PD-L1.

[0250] The PD-1 inhibitor may be an antibody, an antigen-binding fragment thereof, or a construct thereof, comprising an antibody portion having an antigen-binding fragment of the required specificity. The antibody or antigen-binding fragment thereof is as described herein. Antibodies or antigen-binding fragments thereof that are PD-1 inhibitors include antibodies or antigen-binding fragments thereof that specifically bind to PD-1. The antibodies or antigen-binding fragments may also be conjugated to further moieties described herein. In particular, the antibodies or antigen-binding fragments thereof are chimeric, humanized, or human antibodies.

[0251] In a preferred embodiment, the antibody that is a PD-1 inhibitor is an isolated antibody.

[0252] In one embodiment, the PD-1 inhibitor is an antibody, fragment thereof, or construct that blocks the interaction of PD-1 with PD-L1.

[0253] PD-1 inhibitors can 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, PD-1 checkpoint inhibitors that are siRNAs interfere with mRNA and thus block translation, e.g., translation of PD-1 protein.

[0254] In one embodiment, the PD-1 inhibitor 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 certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-1.

[0255] In a further preferred embodiment, the PD-1 inhibitor is an antibody that binds to PD-1, such as a PD-1-blocking antibody. Without being bound by theory, it is believed that the combination of a binding agent comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1 with an antibody that binds to PD-1 increases the response rate and improves the duration of response in subjects receiving the combination therapy, as the combination therapy results in complete blockade of the PD-1 pathway with simultaneous conditional activation of 4-1BB. PD-1-blocking antibodies block the interaction with PD-L1 and PD-L2. It is also believed that combination therapy with an antibody that binds to PD-1 increases the amount of PD-L1 that can be bound by the binding agent.

[0256] Exemplary PD-1 inhibitors include, but are not limited to, anti-PD-1 antibodies, such as BGB-A317 (BeiGene; see U.S. Patent No. 8,735,553, WO2015 / 35606 and US2015 / 0079109), lambrolizumab (disclosed, for example, in WO2008 / 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 nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb). Squibb; U.S. Patent No. 8,008,449; WO2013 / 173223; see 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 US 2015 / 0203579 and WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; 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. Pat. No. 9,834,606, and US 2015 / 0079109), BI 754091, SHR-1210 (see WO2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, INCSHR1210 (Jiangsu Hengrui), which are 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), genolimuzumab (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):Abstract), toripalimab (JS-001; see US 2016 / 0272708), camrelizumab (SHR-1210; INCSHR-1210; see US 2016 / 376367; Huang et al., Clin. Cancer Res. 2018; 24(6):1296-1304), spartalizumab (PDR001; see WO2017 / 106656; 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 ), ezabenlimab (BI-754091; US ​​2017 / 334995 ; Johnson et al., J. Clin. Oncol. 36, no.5_suppl (2018) 212-212), zimberelimab (GLS-010; see WO2017 / 025051), LZM-009 (US 2017 / 210806), AK-103 (see WO2017 / 071625, WO2017 / 166804, and WO2018 / 036472), retifanlimab (MGA-012; see WO2017 / 019846), Sym-021 (see WO2017 / 055547), CS1003 (see CN107840887), anti-PD-1 antibodies, e.g., those described in U.S. Patent Nos. 7,488,802, 8,008,449, 8,168,757, WO2018 / 04 2402, WO2010 / 089411 (which further discloses anti-PD-L1 antibodies), WO2010 / 036959, WO2011 / 159877 (which further discloses antibodies against TIM-3), WO2011 / 082400, WO2011 / 161699, WO2009 / 014708, WO03 / 099196, WO2009 / 114335, WO2012 / 145493 (which further discloses antibodies against PD-L1), WO2015 / 035606, WO2014 / 055648 (which further discloses anti-KIR antibodies), US 2018 / 0185482 (which further discloses anti-PD-L1 antibodies and anti-TIGIT antibodies), U.S. Pat. No. 8,008,449, U.S. Pat. No. 8,779,105, U.S. Pat. No. 6,808,710, U.S. Pat. No. 8,168,757, US 2016 / 0272708, and U.S. Pat. No. 8,354,509; small molecule antagonists against the PD-1 signaling pathway, e.g., those described in Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, siRNA against PD-1, such as those disclosed in WO2019 / 000146 and WO2018 / 103501, soluble PD-1 protein disclosed in WO2018 / 222711, and oncolytic viruses containing soluble forms of PD-1, such as those described in WO2018 / 022831.

[0257] In certain embodiments, the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558) or a biosimilar thereof, pembrolizumab (KEYTRUDA; MK-3475) or a biosimilar thereof, pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514) or a biosimilar thereof, TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI 754091, or SHR-1210.

[0258] The PD-1 inhibitor may in particular be pembrolizumab or a biosimilar thereof. Alternatively, the antibody may be nivolumab or a biosimilar thereof.

[0259] In certain embodiments, the PD-1 inhibitor immunomodulatory agent is an anti-PD-1 antibody or antigen-binding fragment thereof that comprises the complementarity-determining region (CDR) of one of the above-mentioned anti-PD-1 antibodies or antigen-binding fragments, 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.

[0260] In some embodiments, the CDRs of an anti-PD-1 antibody are described 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).

[0261] In certain embodiments, the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof comprising the heavy and light chain variable regions of one of the above-described anti-PD-1 antibodies or antigen-binding fragments, such as the heavy and light chain variable regions 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.

[0262] In certain embodiments, the PD-1 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, and CS1003.

[0263] The CDR sequences of pembrolizumab are identified herein by SEQ ID NOs:59-61 (VH CDRs 1, 2, and 3, respectively) and SEQ ID NOs:62-64 (VL CDRs 1, 2, and 3, respectively). The VH and VL sequences are identified by SEQ ID NOs:65 and 66, respectively, and the heavy and light chain sequences are identified by SEQ ID NOs:67 and 68, respectively. Thus, in one embodiment, the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively.

[0264] In a further embodiment, the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) comprising, consisting of, or consisting essentially of the sequence set forth in SEQ ID NO:65, and a light chain variable region (VL) comprising, consisting of, or consisting essentially of the sequence set forth in SEQ ID NO:66. The PD-1 inhibitor may particularly be an antibody comprising a heavy chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:67, and a light chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:68.

[0265] The CDR sequences of nivolumab are identified herein by SEQ ID NOs:69-71 (VH CDRs 1, 2, and 3, respectively) and SEQ ID NOs:72-74 (VL CDRs 1, 2, and 3, respectively). The VH and VL sequences are identified by SEQ ID NOs:75 and 76, respectively, and the heavy and light chain sequences are identified by SEQ ID NOs:77 and 78, respectively. Thus, in one embodiment, the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:69, 70, and 71, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:72, 73, and 74, respectively.

[0266] In a further embodiment, the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) comprising, consisting of, or consisting essentially of the sequence set forth in SEQ ID NO:75, and a light chain variable region (VL) comprising, consisting of, or consisting essentially of the sequence set forth in SEQ ID NO:76. The PD-1 inhibitor may particularly be an antibody comprising a heavy chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:77, and a light chain comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:78.

[0267] The anti-PD-1 antibodies of the present disclosure are preferably monoclonal and can 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 can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0268] 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 antibody, disulfide-linked Fv (sdFv), and V L or V H Antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with all or a portion of the following: hinge region, CH1, CH2, CH3, and CL domains. Antigen-binding fragments comprising any combination of the variable region(s) and the hinge region, CH1, CH2, CH3, and CL domains are also included in the present disclosure. 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.

[0269] 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; 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547 1553.

[0270] The anti-PD-1 antibodies disclosed herein may be described or specified in terms of the particular CDRs they contain.The precise boundaries of a given CDR or FR amino acid sequence can be determined using the Kabat numbering scheme (Kabat et al., 1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD; 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 specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., its variable region) should be understood to encompass the CDRs (or specific CDRs) as defined by any of the above schemes. For example, a specific CDR (e.g., CDR-H3) may be included in a given V. H or V L When a region's amino acid sequence is described as including the amino acid sequence of the corresponding CDR, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region as defined by any of the schemes described above. Schemes for identifying a particular CDR or CDRs, such as CDRs as defined by the Kabat, Chothia, AbM, or IMGT methods, may be specified.

[0271] 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.

[0272] 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 described 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 from the monoclonal antibody nivolumab, and (b) a set of four framework regions that differ from the set of framework regions in the monoclonal antibody nivolumab, wherein the anti-PD-1 antibody or derivative thereof binds to PD-1. In certain embodiments, the anti-PD-1 antibody is nivolumab.

[0273] 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 are 5×10 -2 M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M, or 10 -15 These include those with a dissociation constant or Kd less than M.

[0274] Anti-PD-1 antibodies also include derivatives and constructs that are modified, i.e., by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from binding to PD-1. For example, but not limited to, anti-PD-1 antibody derivatives include antibodies modified by, e.g., 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 can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, derivatives or constructs may contain one or more non-classical amino acids.

[0275] In a preferred embodiment, the PD-1 inhibitor is an antibody, particularly an antagonistic or blocking antibody, that disrupts or inhibits the PD-1 pathway (the interaction between PD-1 and one or more of its ligands, such as PD-L1 and / or PD-L2). In a preferred embodiment, the PD-1 inhibitor is an antibody, particularly an antagonistic or blocking antibody, that disrupts or inhibits the interaction between PD-1 and PD-L1.

[0276] The PD-1 inhibitor can be administered in the form of a nucleic acid, such as a DNA or RNA molecule, encoding the PD-1 inhibitor, e.g., an inhibitory nucleic acid molecule or an antibody or fragment thereof. For example, an antibody can be delivered encoded in an expression vector described herein. In such cases, the nucleic acid molecule can be delivered, for example, in the form of a plasmid or mRNA molecule, or complexed with a delivery vehicle, e.g., a liposome, lipoplex, or nucleic acid-lipid particle. The PD-1t inhibitor can also be administered via an oncolytic virus containing an expression cassette encoding the PD-1 inhibitor. PD-1 can also be administered by administering endogenous or allogeneic cells capable of expressing the PD-1 inhibitor, for example, in the form of cell-based therapy.

[0277] Preferably, the PD-1 inhibitor is administered in an appropriate amount. The amount of PD-1 inhibitor administered in each dose and / or treatment cycle may be, inter alia, 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 the PD-1 inhibitor is bound to PD-1.

[0278] In certain embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, e.g., the amount of PD-1 inhibitor administered in each dose and / or each treatment cycle is about 10 to about 1000 mg total, e.g., about 100 to about 600 mg total, e.g., about 150 to about 600 mg total, about 150 to about 500 mg total, about 175 to about 500 mg total, about 175 to about 450 mg total, about 200 to about 450 mg total, or e.g., about 200 to about 400 mg total.

[0279] In certain embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the amount of PD-1 inhibitor administered is, e.g., 10 to 1000 mg total, e.g., 100 to 600 mg total, e.g., 150 to 600 mg total, 150 to 500 mg total, 175 to 500 mg total, 175 to 450 mg total, 200 to 450 mg total, or e.g., 200 to 400 mg total, for each dose and / or each treatment cycle.

[0280] In certain embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the amount of PD-1 inhibitor administered is, e.g., about 100-600 mg total at each dose and / or each treatment cycle; and / or about 6.84 x 10 -7 ~4.11×10 -7 mol.

[0281] In certain embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the amount of PD-1 inhibitor administered is, for example, about 100-400 mg total at each dose and / or each treatment cycle; and / or about 6.84 x 10 -7 ~2.73×10 -6 mol, e.g., 100–400 mg total; and / or 6.84 × 10 total -7 ~2.73×10 -6 mol.

[0282] In certain embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the amount of PD-1 inhibitor administered is, for example, about 200-400 mg total at each dose and / or each treatment cycle; and / or about 6.84 x 10 -7 ~2.73×10 -6 mol, e.g., 200–400 mg total; and / or 6.84 × 10 total -7 ~2.73×10 -6 mol.

[0283] In certain embodiments, the amount of PD-1 inhibitor administered is, for example, about 200 mg total or about 1.37×10 at each dose and / or each treatment cycle. -6 mol, e.g., a total of 200 mg or 1.37 x 10 -6 mol.

[0284] In certain embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the amount of PD-1 inhibitor administered is, for example, about 200 mg total or about 1.37×10 for each dose and / or each treatment cycle. -6 mol, e.g., a total of 200 mg or 1.37 x 10 -6 mol.

[0285] In certain embodiments, the amount of PD-1 inhibitor administered is, for example, about 400 mg total or about 2.73 x 10 total at each dose and / or each treatment cycle. -6 , e.g., a total of 400 mg or a total of 2.73 × 10 -6 is.

[0286] In certain embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the amount of PD-1 inhibitor administered is, for example, about 400 mg total or about 2.73 x 10 total at each dose and / or each treatment cycle. -6 , e.g., a total of 400 mg or a total of 2.73 × 10 -6 is.

[0287] The PD-1 inhibitor can be administered by any method and route known in the art. The mode and route of administration depend on the type of PD-1 inhibitor to be used. In a preferred embodiment, the PD-1 inhibitor is administered systemically, for example, parenterally, particularly intravenously.

[0288] The PD-1 inhibitor may be administered in the form of any suitable pharmaceutical composition described herein. In a preferred embodiment, the PD-1 inhibitor is administered in the form of an infusion, for example, an intravenous infusion.

[0289] An antibody that binds PD-1 may comprise a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise or have the sequences set forth in SEQ ID NO:104, SEQ ID NO:101, and SEQ ID NO:100, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise or have the sequences set forth in SEQ ID NO:107, QAS, and SEQ ID NO:105, respectively. A specific, but non-limiting, example of such an antibody is MAB-19-0202.

[0290] The terms "heavy chain variable region" (also referred to as "VH") and "light chain variable region" (also referred to as "VL") are used herein in their most general sense and include any sequences that can include complementarity-determining regions (CDRs) interspersed with other regions, also termed framework regions (FRs). The framework regions, among other things, space the CDRs so that they can form an antigen-binding site, particularly after folding and pairing of the VH and VL. Preferably, each VH and VL is composed of three CDRs and four FRs arranged, from amino-terminus to carboxy-terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, the terms "heavy chain variable region" and "light chain variable region" should not be construed as limited to sequences that can be found in native antibodies or in the VH and VL sequences exemplified herein (SEQ ID NOs: 109-112 in the Sequence Listing). These terms include any sequence that contains and can accurately map CDRs, such as sequences derived from the VL and VH regions of a native antibody or from the sequences set forth in SEQ ID NOs: 109-112 in the Sequence Listing. In particular, those skilled in the art will recognize that the sequences of the framework regions can be modified without losing the characteristics of the VH and VL regions, respectively (including both variants involving amino acid substitutions and variants involving sequence length, i.e., insertion or deletion variants). In preferred embodiments, any modifications are limited to the framework regions. However, those skilled in the art will also appreciate that CDR, hypervariable, and variable regions can also be modified without losing the ability to bind to PD-1. For example, the CDR regions may be identical to or highly homologous to the regions specified herein. By "highly homologous," it is contemplated that one to five, preferably one to four, e.g., one to three, or one or two substitutions may be made in the CDRs. In addition, the hypervariable and variable regions may be modified so as to exhibit substantial homology with the regions specifically disclosed herein.

[0291] In the antibodies that bind to PD-1, the CDRs as specified herein have been identified using two different CDR identification methods. The first numbering scheme used herein is according to Kabat (Wu and Kabat, 1970; Kabat et al., 1991), and the second scheme is the IMGT numbering (Lefranc, 1997; Lefranc et al., 2005). In the third approach, the intersection of both identification schemes is used.

[0292] Antibodies that bind to PD-1 may comprise one or more CDRs, sets of CDRs, or combinations of sets of CDRs described herein, together with their intervening framework regions (also referred to herein as framing regions or FRs), or portions of the framework regions. Preferably, the portions include at least about 50% of one or both of the first and fourth framework regions, the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of antibodies produced by recombinant DNA techniques may result in the introduction of residues N- or C-terminal to the variable region encoded by linkers introduced to facilitate cloning or other engineering steps, including the introduction of linkers for joining the variable regions of the present disclosure to additional protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or protein labels.

[0293] An antibody that binds to PD-1 may comprise a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VH sequence set forth in any one of SEQ ID NOs: 111. In one embodiment, the antibody comprises a heavy chain variable region (VH), wherein the VH comprises a sequence set forth in any one of SEQ ID NOs: 111. In one embodiment, the antibody comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence set forth in any one of SEQ ID NOs: 112. In one embodiment, the antibody comprises a light chain variable region (VL), wherein the VL comprises a sequence set forth in any one of SEQ ID NO:112.

[0294] An antibody that binds to PD-1 may comprise a heavy chain variable region (VH) and a light chain variable region (VL), where the VH comprises or has the sequence set forth in SEQ ID NO:111, and the VL comprises or has the sequence set forth in SEQ ID NO:112, or a variant of each of these sequences. Another example of an antibody that binds to PD-1 may comprise a VH that comprises or has the sequence set forth in SEQ ID NO:111, or a variant thereof, and a VL that comprises or has the sequence set forth in SEQ ID NO:112, or a variant thereof. A specific, but non-limiting, example of such an antibody is MAB-19-0618. Antibody MAB-19-0618 is derived from MAB-19-0202. Variants of the heavy chain variable region (VH) and the light chain variable region (VL), as well as respective combinations of these variant VHs and VLs, are also encompassed by the present disclosure.

[0295] An antibody that binds to PD-1 may comprise a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region that comprises or has the sequence set forth in SEQ ID NO:93 or 90, and a heavy chain variable region (VH) that comprises or has the sequence set forth in SEQ ID NO:111, and the light chain comprises a light chain constant region that comprises or has the sequence set forth in SEQ ID NO:97, and a light chain variable region (VL) that comprises or has the sequence set forth in SEQ ID NO:112.

[0296] An antibody that binds to PD-1 can comprise a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region comprising or having the sequence set forth in SEQ ID NO:93 or 90, and a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of the sequence set forth in SEQ ID NO:111, and the light chain comprises a light chain constant region comprising or having the sequence set forth in SEQ ID NO:97, and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences of the sequence set forth in SEQ ID NO:112. For example, the CDR1, CDR2, and CDR3 sequences are as specified herein.

[0297] Antibodies that bind to PD-1 can be monoclonal, chimeric, or monoclonal humanized antibodies, or fragments of such antibodies. The antibodies can be whole antibodies or antigen-binding fragments thereof, including, for example, bispecific antibodies.

[0298] In antibodies that bind to PD-1, one or more, preferably both, heavy chain constant regions may be modified such that binding of C1q to the 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%. In one embodiment, C1q binding can be determined by ELISA.

[0299] By "wild-type" or "WT" or "native" herein is meant 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.

[0300] In an antibody that binds to PD-1, one or more, preferably both, heavy chain constant regions may be modified such that binding of the antibody to one or more IgG Fc-gamma receptors is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, compared to a wild-type antibody. In one embodiment, the one or more IgG Fc-gamma receptors are selected from at least one of Fc-gamma RI, Fc-gamma RII, and Fc-gamma RIII. In one embodiment, the IgG Fc-gamma receptor is Fc-gamma RI.

[0301] In one embodiment, the antibody that binds PD-1 is unable to induce Fc-gammaRI-mediated effector function, or wherein the induced Fc-gammaRI-mediated effector function is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, compared to the wild-type antibody.

[0302] In one aspect, an antibody that binds PD-1 is unable to induce at least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular lysis (ADCC)-mediated lysis, apoptosis, homotypic adhesion, and / or phagocytosis, or at least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular lysis (ADCC)-mediated lysis, apoptosis, homotypic adhesion, and / or phagocytosis is induced to a low degree, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%.

[0303] Antibody-dependent cell-mediated cytotoxicity is also referred to herein as "ADCC." ADCC describes the cell-killing ability of effector cells, particularly lymphocytes, described herein, which preferably requires that the target cell be marked by an antibody.

[0304] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the Fc domain of the antibody engages with the Fc receptor (FcR) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express specific Fc receptors. ADCC can be viewed as a mechanism for directly inducing various degrees of immediate tumor destruction, leading to antigen presentation and the induction of anti-tumor T cell responses. Preferably, in vivo induction of ADCC leads to anti-tumor T cell responses and host-derived antibody responses.

[0305] Complement-dependent cytotoxicity is also referred to herein as "CDC." CDC is another cell killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also both highly effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex is initiated by the CDC of the participating antibody molecules, such as IgG molecules. H This results in the uncloaking of multiple, closely spaced C1q-binding sites on the two domains (C1q is one of three subcomponents of complement C1). Preferably, these exposed C1q-binding sites convert the previously low-affinity C1q-IgG interaction into a high-avidity interaction, which triggers a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of the effector cell chemotactic / activating agents C3a and C5a. Preferably, the complement cascade terminates with the formation of a membrane attack complex, which opens a pore in the cell membrane that facilitates the free passage of water and solutes into and out of the cell, which may lead to apoptosis.

[0306] In one embodiment, the antibody that binds PD-1 has reduced or depleted effector function, hi one embodiment, the antibody does not mediate ADCC or CDC, or both.

[0307] In one embodiment, one or more, preferably both, heavy chain constant regions of an antibody that binds PD-1 are modified such that binding of the antibody to the neonatal Fc receptor (FcRn) is unaffected compared to the wild-type antibody.

[0308] In one embodiment, the PD-1 that the antibody can bind is human PD-1. In one embodiment, the PD-1 has or comprises the amino acid sequence set forth in SEQ ID NO:113 or SEQ ID NO:114, or the amino acid sequence of PD-1 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:113 or SEQ ID NO:114, or an immunogenic fragment thereof. In one embodiment, the antibody is capable of binding to a native epitope of PD-1 present on the surface of a living cell.

[0309] In one embodiment, an antibody that binds PD-1 comprises a heavy chain constant region, wherein the heavy chain constant region comprises an aromatic or nonpolar amino acid at a position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at a position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering.

[0310] The term "amino acid corresponding to position ..." and similar expressions, as used herein, refers to the number of the amino acid position in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain when aligned with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW, or the like, typically with default settings. Methods for aligning sequences or segments in sequences, and thereby determining positions in sequences that correspond to amino acid positions according to the present disclosure, are believed to be well known in the art.

[0311] For example, with reference to the amino acid sequence according to SEQ ID NO:93 in the Sequence Listing of the present disclosure, the amino acid positions corresponding to positions 234-236 in the human IgG1 heavy chain according to EU numbering are amino acid positions 117-119 of SEQ ID NO:93, with F located at position 117 (corresponding to position 234 in the human IgG1 heavy chain according to EU numbering), E located at position 118 (corresponding to position 235 in the human IgG1 heavy chain according to EU numbering), and R located at position 119 (corresponding to position 236 in the human IgG1 heavy chain according to EU numbering). In the sequence below, the FER amino acid sequence is underlined and shown in bold.

[0312] TIFF2024536383000019.tif37128

[0313] Unless otherwise indicated herein or clearly contradicted by context, all references throughout this disclosure to amino acid positions in antibody heavy chain constant regions refer to the positions corresponding to the respective positions in the human IgG1 heavy chain according to EU numbering as set forth in Kabat (as described in 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)).

[0314] In one embodiment, an antibody that binds PD-1 comprises a heavy chain constant region that has reduced or depleted Fc-mediated effector function, or that induces Fc-mediated effector function to a lesser extent, compared to another antibody that comprises the same antigen-binding region and a heavy chain constant region (CH) comprising a human IgG1 hinge, CH2, and CH3 regions.

[0315] In one particular embodiment, the heavy chain constant region (CH) in an antibody that binds PD-1 is modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an identical antibody except for comprising an unmodified heavy chain constant region (CH).

[0316] The term "Fc-mediated effector function" as used herein refers in particular to a function selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC, CDC and any combination thereof.

[0317] In the context of the present disclosure, the term "having reduced or depleted Fc-mediated effector function," as used in relation to antibodies, including multispecific antibodies, means that the antibody causes an overall reduction in Fc-mediated effector function, particularly a function selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC, or CDC, preferably at a level of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, compared to a human IgG1 antibody (i) comprising the same CDR sequences as the antibody, particularly the same first and second antigen-binding regions, and (ii) comprising two heavy chains comprising the human IgG1 hinge, CH2, and CH3 regions. "Depleted Fc-mediated effector function" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0318] In the context of the present disclosure, the term "induce Fc-mediated effector functions to a lesser extent" when used in relation to antibodies, including multispecific antibodies, means that the antibody induces Fc-mediated effector functions, in particular 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) comprises the same CDR sequences as the antibody, in particular the same first and second antigen-binding regions, and (ii) comprises two heavy chains comprising a human IgG1 hinge, CH2 and CH3 region.

[0319] Fc-mediated effector function can be determined by measuring 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 binding of a binding agent to C1q and / or IgG Fc-gamma RI.

[0320] In one embodiment relating to the use of an antibody that binds PD-1, the amino acid at the position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering is a basic amino acid.

[0321] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not 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 may be classified based on their structure and chemical characteristics.

[0322] In this disclosure, amino acid residues are represented by using the following abbreviations: Also, unless expressly indicated otherwise, amino acid sequences of peptides and proteins are identified from N-terminus to C-terminus (left to right), with the N-terminus identified as the first residue. Amino acids are referred to by their three-letter abbreviations, one-letter abbreviations, or full names as follows: Ala: A: alanine; Asp: D: aspartic acid; Glu: E: glutamic acid; Phe: F: phenylalanine; Gly: G: glycine; His: H: histidine; Ile: I: isoleucine; Lys: K: lysine; Leu: L: leucine; Met: M: methionine; Asn: N: asparagine; Pro: P: proline; Gln: Q: glutamine; Arg: R: arginine; Ser: S: serine; Thr: T: threonine; Val: V: valine; Trp: W: tryptophan; Tyr: Y: tyrosine; Cys: C: cysteine.

[0323] Naturally occurring amino acids can also generally be categorized 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 jointly as aromatic amino acids.

[0324] In one embodiment relating to the use of an antibody that binds to PD-1, the basic amino acid at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is selected from the group consisting of lysine, arginine, and histidine. In one embodiment, the basic amino acid at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is arginine (G236R). Such an amino acid substitution is also referred to herein as G236R. The term "G236R" indicates a substitution of the amino acid glycine (G) with arginine (R) at position 236 in the human IgG1 heavy chain according to EU numbering. Similar terms are used for other amino acid positions and amino acids within the scope of this disclosure. Unless indicated to the contrary, the amino acid positions referred to by these terms are amino acid positions in the human IgG1 heavy chain according to EU numbering.

[0325] In one embodiment of the present invention, the amino acid at a position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering is an aromatic amino acid. In one embodiment, the aromatic amino acid at this position is selected from the group consisting of phenylalanine, tryptophan, and tyrosine.

[0326] In one embodiment relating to the use of an antibody that binds PD-1, the amino acid at a position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering is a nonpolar amino acid. In one embodiment, the nonpolar amino acid at this position is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. In one embodiment, the nonpolar amino acid at this position is selected from the group consisting of isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0327] In one embodiment relating to the use of an antibody that binds PD-1, the amino acid at the position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering is phenylalanine (L234F).

[0328] Exemplary combinations of possible amino acids at positions corresponding to positions 234 and 236 in the human IgG1 heavy chain according to EU numbering are shown in the table below.

[0329] (Table 5) TIFF2024536383000020.tif128128

[0330] For example, at positions corresponding to positions 234 and 236 in a human IgG1 heavy chain according to EU numbering, the following amino acids, among others, can be present in the heavy chain constant region of an antibody that binds PD-1: 234F / 236R, 234W / 236R, 234Y / 236R, 234A / 236R, 234L / 236R, 234F / 236K, 234W / 236K, 234Y / 236K, 234A / 236K, 234L / 236K, 234F / 236H, 234W / 236H, 234Y / 236H, 234A / 236H, or 234L / 236H.

[0331] The above-described amino acids or amino acid substitutions at positions 234 and 236 can be present in only one heavy chain of the antibody that binds PD-1 or in both heavy chains of the antibody that binds PD-1. Each amino acid present in the first and second heavy chains of the antibody can be selected independently of each other.

[0332] For example, at least one heavy chain of an antibody that binds to PD-1 can comprise the following sequence (SEQ ID NO: 93): TIFF2024536383000021.tif37128

[0333] In one embodiment relating to an antibody that binds PD-1, the heavy chain comprises a human IgG1 heavy chain, wherein the amino acids at positions corresponding to positions 234 and 236 according to EU numbering are as specified above, and further wherein the amino acid at position corresponding to position 235 according to EU numbering in a human IgG1 heavy chain is an acidic amino acid. In one embodiment, the acidic amino acid at this position is selected from aspartic acid or glutamic acid. In one embodiment, the amino acid at the position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering is glutamic acid (L235E).

[0334] In one embodiment relating to an antibody that binds PD-1, in the heavy chain constant region, the amino acids at positions corresponding to positions 234, 235, and 236 in a human IgG1 heavy chain according to EU numbering are a nonpolar or aromatic amino acid at position 234, an acidic amino acid at position 235, and a basic amino acid at position 236.

[0335] Exemplary combinations of possible amino acids at positions corresponding to positions 234, 235 and 236 in the human IgG1 heavy chain according to EU numbering are shown in the table below.

[0336] (Table 6) TIFF2024536383000022.tif128154

[0337] For example, the following amino acids may be present in the heavy chain constant region of an antibody that binds PD-1, particularly at positions corresponding to positions 234, 235, and 236 in a human IgG1 heavy chain according to EU numbering: 234F / 235E / 236R, 234W / 235E / 236R, 234Y / 235E / 236R, 234A / 235E / 236R, 234L / 235E / 236R, 234F / 235D / 236R, 234W / 235D / 236R, 234Y / 235D / 236R, 234A / 235D / 236R, 234L / 235E / 236R, 234F / 235L / 236 R, 234W / 235L / 236R, 234Y / 235L / 236R, 234A / 235L / 236R, 234L / 235L / 236R , 234F / 235A / 236R, 234W / 235A / 236R, 234Y / 235A / 236R, 234A / 235A / 236R, 2 34L / 235A / 236R, 234F / 235E / 236K, 234W / 235E / 236K, 234Y / 235E / 236K, 234 A / 235E / 236K, 234L / 235E / 236K, 234F / 235D / 236K, 234W / 235D / 236K, 234Y / 235D / 236K, 234A / 235D / 236K, 234L / 235D / 236K, 234F / 235L / 236K, 234W / 2 35L / 236K, 234Y / 235L / 236K, 234A / 235L / 236K, 234L / 235L / 236K, 234F / 235 A / 236K, 234W / 235A / 236K, 234Y / 235A / 236K, 234A / 235A / 236K, 234L / 235A / 236K, 234F / 235E / 236H, 234W / 235E / 236H, 234Y / 235E / 236H, 234A / 235E / 2 36H, 234L / 235E / 236H, 234F / 235D / 236H, 234W / 235D / 236H, 234Y / 235D / 23 6H, 234A / 235D / 236H, 234L / 235D / 236H, 234F / 235L / 236H, 234W / 235L / 236H , 234Y / 235L / 236H, 234A / 235L / 236H, 234L / 235L / 236H, 234F / 235A / 236H, 234W / 235A / 236H, 234Y / 235A / 236H, 234A / 235A / 236H, or 234L / 235A / 236H.

[0338] The above-mentioned amino acids or amino acid substitutions at positions 234, 235, and 236 can be present in only one heavy chain of the antibody or in both heavy chains of the antibody. Each amino acid present in the first and second heavy chains of the antibody can be selected independently of each other.

[0339] For example, at least one heavy chain of an antibody that binds to PD-1 may have the following sequence (SEQ ID NO:90 or 93): May contain TIFF2024536383000023.tif37128.

[0340] For example, as shown in Tables 5 and 6, any permutation and combination of all described amino acid substitutions at positions 234, 236, and 235 in the present application should be considered to be disclosed by the description of the present application, unless the context indicates otherwise, where applicable. For example, in one embodiment of the antibody, the first heavy chain comprises the amino acids FER at positions corresponding to positions 234-236 in a human IgG1 heavy chain according to EU numbering, or the first heavy chain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 93, and the second heavy chain of the antibody comprises other amino acids, e.g., the amino acids AAG or LLG, at positions corresponding to positions 234-236 in a human IgG1 heavy chain according to EU numbering, or the second heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 92 or 98. In another embodiment of the antibody, the first and second heavy chains comprise the same amino acids at positions corresponding to positions 234-236 in a human IgG1 heavy chain according to EU numbering, i.e., the same aromatic or non-polar amino acid, e.g., F, at the position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering, and the same amino acid other than glycine, e.g., R, at the position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering, e.g., in a specific combination of FER or FLR.

[0341] In one embodiment, an antibody that binds PD-1 comprises at least one or two heavy chain constant regions, wherein the amino acid corresponding to position 234 is phenylalanine, the amino acid corresponding to position 235 is glutamic acid, and the amino acid corresponding to position 236 is arginine (L234F / L235E / G236R=FER).

[0342] In one embodiment, an antibody that binds PD-1 comprises one or more heavy chain constant regions (CH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the heavy chain constant region sequence set forth in SEQ ID NO:93.

[0343] In one embodiment, the antibody that binds to PD-1 comprises one or more, e.g., two, heavy chain constant regions (CH), wherein the heavy chain constant region comprises the sequence set forth in SEQ ID NO:93.

[0344] The antibody is preferably of the IgG1 isotype.

[0345] As used herein, the term "isotype" refers to the immunoglobulin class encoded by heavy chain constant region genes. When referring to an IgG1 isotype 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 mutations in the constant region.

[0346] IgG1 antibodies can exist as 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 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 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.

[0347] Mammals have two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved among different immunoglobulin isotypes, while the variable region is highly diverse and accounts for antigen recognition.

[0348] For example or in an embodiment, the antibody, preferably a monoclonal antibody, used in accordance with the present invention is of the IgG1, κ or λ isotype, preferably comprising a human IgG1 / κ or human IgG1 / λ constant region, or the antibody, preferably a monoclonal antibody, is derived from an IgG1, λ (lambda) or IgG1, κ (kappa) antibody, preferably a human IgG1, λ (lambda) or human IgG1, κ (kappa) antibody.

[0349] In one embodiment, an antibody that binds PD-1 comprises a light chain having a light chain constant region (LC) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the LC sequence set forth in SEQ ID NO: 97. In one embodiment, the antibody comprises a light chain having a light chain constant region (LC) comprising the sequence set forth in SEQ ID NO: 97.

[0350] In one embodiment of the invention, the antibody that binds to PD-1 is a full-length IgG1 antibody, e.g., IgG1,κ. In one embodiment of the invention, the binding agent is a full-length human IgG1 antibody, e.g., IgG1,κ.

[0351] In one embodiment, an antibody that binds PD-1 can be derivatized, linked, or co-expressed with another binding specificity. In another embodiment, the antibody can be derivatized, linked, or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., a Fab' fragment). For example, the antibody can be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, e.g., another antibody (e.g., to generate a bispecific or multispecific antibody).

[0352] Antibodies that bind to PD-1 can be human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies that bind to human PD-1 can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0353] The present disclosure includes the use of bispecific and multispecific molecules comprising at least one first binding specificity for PD-1 and a second binding specificity (or additional binding specificity) for a second target epitope (or additional target epitope).

[0354] In one embodiment, the first antigen-binding region of the multispecific antibody that binds to PD-1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) shown herein.

[0355] In one embodiment relating to the use of a multispecific antibody that binds to PD-1, the antibody comprises first and second binding arms derived from a full-length antibody, such as the full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibodies described above. In one embodiment, the first and second binding arms are derived from a monoclonal antibody. For example, or in a preferred embodiment, the first and / or second binding arms are derived from an IgG1, κ or λ isotype, preferably comprising a human IgG1 / κ or human IgG1 / λ constant region.

[0356] The first antigen-binding region of a multispecific or bispecific antibody used in accordance with the invention that binds PD-1 can comprise heavy and light chain variable regions of an antibody that competes with PD-L1 and / or PD-L2 for binding to PD-1. In one embodiment relating to the use of a multispecific or bispecific antibody, the first antigen-binding region that binds PD-1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) shown herein.

[0357] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the recognition and activation phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytotoxic T cells (CTLs), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils).

[0358] "Target cell" is intended to mean any unwanted cell in a subject (e.g., a human or animal) that can be targeted by an antibody. In a preferred embodiment, the target cell is a tumor cell.

[0359] Subjects and tumors or cancers to be treated The subjects to be treated according to the present disclosure are preferably human subjects.

[0360] In a preferred embodiment, the tumor or cancer to be treated is a solid tumor or cancer. The tumor or cancer may be a metastatic tumor or cancer.

[0361] 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, renal cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma. 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.

[0362] In certain embodiments, the tumor or cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urothelial cancer (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g., cancer of the oral cavity, pharynx, or larynx), and cervical cancer.

[0363] Preferably, the tumor is a PD-L1 positive tumor. In certain embodiments, PD-L1 is preferably expressed on ≥ 1% of cancer or tumor cells. PD-L1 expression may be determined using techniques known to those skilled in the art, and may be assessed, for example, by immunohistochemistry (IHC).

[0364] The tumor or cancer may be, in particular, lung cancer. The lung cancer may be non-small cell lung cancer (NSCLC), e.g., squamous or non-squamous NSCLC. Lung cancer is the second most common malignant tumor with an estimated age-adjusted incidence rate of 22.4 per 100,000 people and is the leading cause of cancer death in both men and women (Kantar, 2021). Worldwide, approximately 2,206,771 new cases and 1,796,144 deaths from lung cancer are estimated in 2020 (GLOBOCAN, 2020). NSCLC accounts for 85% to 90% of all cases, with a 5-year survival rate of approximately 18% across all stages of the disease and only 3.5% for metastatic disease (Jemal et al., 2011) (Kantar, 2021; SEER, 2018). In the 1L setting, treatment typically consists of platinum-based chemotherapy combined with immunotherapy or targeted therapy, depending on molecular and biomarker analysis and tumor histology (NCCN, 2021d). Recently, the advent of PD-1 and programmed cell death ligand 1 (PD-L1) inhibitors has improved outcomes for patients without driver mutations (approximately 62% of the non-squamous population and 77% of the squamous population (Kantar, 2021)). Patients whose tumors do not harbor certain oncogenic mutations or do not express biomarkers for checkpoint inhibitor (CPI) options require more treatment options. Novel combinations with complementary approaches to enhance response may further address unmet needs in this population. For patients in the 2L setting, SOC is limited to platinum-based chemotherapy, CPI monotherapy, or docetaxel with or without ramucirumab, depending on prior therapy received. For patients in the third-line (3L) setting, chemotherapy monotherapy is standard. Novel treatments are needed to limit toxicity and potentially increase efficacy in this population (NCCN, 2021d).

[0365] In one embodiment, where the tumor or cancer is lung cancer, the tumor or cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC. The tumor or cancer may be, in particular, a metastatic cancer, such as metastatic NSCLC.

[0366] In one embodiment, where the tumor or cancer is lung cancer, particularly NSCLC, the tumor or cancer does not have an epidermal growth factor receptor (EGFR) sensitizing mutation and / or an anaplastic lymphoma (ALK) rearrangement / ROS1 rearrangement. An EGFR sensitizing mutation is one that is amenable to treatment with an approved tyrosine kinase inhibitor (TKI).

[0367] In one embodiment, where the tumor or cancer is lung cancer, particularly NSCLC, the tumor or cancer comprises cancer cells and PD-L1 is expressed in >1% of the cancer cells. Such expression may be determined by any means and method known to those skilled in the art, for example, by immunohistochemistry (IHC), for example, by a local SOC test (preferably an FDA-approved test) or at a central laboratory.

[0368] In one embodiment, the subject has not received prior systemic treatment for metastatic disease, i.e., the subject has not received any systemic treatment for metastatic disease prior to receiving treatment according to the present invention. According to this embodiment, the tumor or cancer is preferably lung cancer, e.g., NSCLC.

[0369] In one embodiment, the subject has not received prior treatment with a checkpoint inhibitor / 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 a further embodiment, the subject has not received prior treatment with a PD-1 or PD-L1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody. In these embodiments, the tumor or cancer is preferably lung cancer, e.g., NSCLC.

[0370] In a further embodiment, the subject has not been previously treated with a 4-1BB (CD137) targeting agent, an anti-tumor vaccine, or an autologous cellular immunotherapy. In one embodiment, the subject has not been previously treated with an anti-4-1BB (CD137) antibody. In these embodiments, the tumor or cancer is preferably lung cancer, e.g., NSCLC.

[0371] In other embodiments, the tumor or cancer has relapsed and / or is refractory after treatment, e.g., systemic treatment with a checkpoint inhibitor. The subject may have received at least one prior systemic treatment, e.g., systemic treatment including a PD-1 or PD-L1 inhibitor, e.g., an anti-PD-1 or anti-PD-L1 antibody. The cancer or tumor may particularly relapse and / or become refractory, or the subject may have progressed after treatment with a PD-1 or PD-L1 inhibitor, e.g., an anti-PD-1 or anti-PD-L1 antibody, where the PD-1 or PD-L1 inhibitor is administered as a monotherapy or as part of a combination therapy.

[0372] In certain embodiments, treatment according to the present invention is provided to a subject who has received a prior therapy, such as those defined above, wherein the last prior therapy was with a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, where the PD-1 inhibitor or PD-L1 inhibitor is administered as monotherapy or as part of a combination therapy. The last prior therapy may be with a PD1 inhibitor or PD-L1 inhibitor as defined above.

[0373] Preferably, treatment according to the present invention is provided to subjects who have received last treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, and where the time since progression is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0374] By analogy, it may be preferable to administer a treatment method according to the present invention to a subject where the time since the last dose of a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, as part of the final conditioning treatment is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0375] In a further embodiment, the cancer or tumor is recurrent and / or refractory, or the subject is (i) platinum-based chemotherapy followed by treatment with an anti-PD-1 antibody or anti-PD-L1 antibody; or (ii) Treatment with anti-PD-1 antibody or anti-PD-L1 antibody after platinum-based chemotherapy Also in these embodiments, the tumor or cancer is preferably lung cancer, e.g., NSCLC.

[0376] A subject undergoing treatment according to the present invention may in particular be a subject who has not undergone prior treatment with a taxane chemotherapy drug; such as docetaxel or paclitaxel, for example, prior treatment of NSCLC with a taxane chemotherapy drug, such as docetaxel.

[0377] Treatment regimen The binding agent and PD-1 inhibitor can be administered by any suitable method, for example, intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, intranodally, or intratumorally.

[0378] In one embodiment of the first aspect, the binding agent is administered to the subject by systemic administration. Preferably, the binding agent is administered to the subject by intravenous injection or infusion. In one embodiment, the binding agent is administered in at least one treatment cycle.

[0379] In one embodiment, the PD-1 inhibitor is administered to the subject systemically. Preferably, the PD-1 inhibitor is administered to the subject by intravenous injection or infusion. In one embodiment, the PD-1 inhibitor is administered in at least one treatment cycle.

[0380] In one embodiment, the binding agent and the PD-1 inhibitor are administered to the subject systemically. Preferably, the binding agent and the PD-1 inhibitor are administered to the subject by intravenous injection or infusion. In one embodiment, the binding agent and the PD-1 inhibitor are administered in at least one treatment cycle.

[0381] In one embodiment, each treatment cycle is about 2 weeks (14 days), 3 weeks (21 days), or 4 weeks (28 days), 5 weeks (35 days), or 6 weeks (48 days). In a preferred embodiment, each treatment cycle is 3 weeks (21 days). In another preferred embodiment, each treatment cycle is 6 weeks (48 days).

[0382] In certain embodiments, one dose of the binder and one dose of the PD-1 inhibitor are administered or infused every 2 weeks (1Q2W), every 3 weeks (1Q3W), or every 4 weeks (1Q4W), every 5 weeks (1Q5W), preferably every 3 weeks (1Q3W). In other embodiments, one dose of the binder and one dose of the PD-1 inhibitor are administered every 6 weeks (1Q6W). The amounts of the binder and the PD-1 inhibitor are preferably as defined above.

[0383] In some embodiments, one and 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 PD-1 inhibitor can be administered on day 1 of each treatment cycle.

[0384] In some embodiments, a 100 mg dose of the binding agent and a 200 mg dose of the PD-1 inhibitor are administered every three weeks (1Q3W).

[0385] In other embodiments, a 100 mg dose of the binding agent and a 400 mg dose of the PD-1 inhibitor are administered every six weeks (1Q6W).

[0386] In certain embodiments, a 100 mg dose of the binding agent that is acasunlimab or a biosimilar thereof and a 200 mg dose of the PD-1 inhibitor that is nivolumab or a biosimilar thereof are administered every three weeks (1Q3W), e.g., on day 1 of each three-week treatment cycle.

[0387] In certain embodiments, the tumor or cancer is NSCLC; a 100 mg dose of the binding agent that is acasunlimab or a biosimilar thereof, and a 200 mg dose of the PD-1 inhibitor that is nivolumab or a biosimilar thereof, are administered every three weeks (1Q3W), e.g., on day 1 of each three-week treatment cycle.

[0388] In other embodiments, a 100 mg dose of the binding agent that is acasunlimab or a biosimilar thereof and a 400 mg dose of the PD-1 inhibitor that is nivolumab or a biosimilar thereof are administered every six weeks (1Q6W), e.g., on day 1 of each six-week treatment cycle.

[0389] In still other embodiments, the tumor or cancer is NSCLC; a 100 mg dose of the binding agent that is acasunlimab or a biosimilar thereof, and a 400 mg dose of the PD-1 inhibitor that is nivolumab are administered every six weeks (1Q6W), e.g., on day 1 of each six-week treatment cycle.

[0390] The PD-1 inhibitor may be administered first, followed by the binding agent, or the binding agent may be administered first, followed by the PD-1 inhibitor.

[0391] Each dose may be administered or infused over at least 30 minutes, for example, at least 60 minutes, at least 90 minutes, at least 120 minutes or at least 240 minutes.

[0392] The binding agent may in particular be administered over 30 minutes, such as at least 40 minutes, at least 50 minutes, or such as at least 60 minutes, by using intravenous (IV) infusion.

[0393] The PD-1 inhibitor may in particular be administered as an intravenous infusion over 30 minutes, such as over at least 40 minutes, at least 50 minutes, or such as over at least 60 minutes.

[0394] The binding agent and the PD-1 inhibitor can be administered simultaneously. In an alternative preferred embodiment, the binding agent and the PD-1 inhibitor are administered separately.

[0395] The binding agent and the PD-1 inhibitor may be administered in any suitable form (e.g., naked per se). However, the binding agent and the PD-1 inhibitor are preferably administered in the form of any suitable pharmaceutical composition described herein. In one embodiment, at least the binding agent and the PD-1 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 PD-1 inhibitor), and preferably the binding agent and the PD-1 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 PD-1 inhibitor).

[0396] The composition or pharmaceutical composition may be prepared by mixing the composition with any of the ingredients listed in Remington: The Science and Practice of Pharmacy, 1999, together with carriers, excipients and / or diluents as well as any other ingredients suitable for pharmaceutical compositions, including known adjuvants. th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutically acceptable carriers or diluents, as well as any known adjuvants and excipients, should be suitable for the binding agent and / or PD-1 inhibitor and the chosen mode of administration. Compatibility of carriers and other components of pharmaceutical compositions is determined based on not having 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 [10% or less relative inhibition, 5% or less relative inhibition, etc.]).

[0397] The compositions, particularly pharmaceutical compositions of the binding agents and pharmaceutical compositions of the PD-1 inhibitors, may include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or non-protein amino acids), preservatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0398] 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).

[0399] Pharmaceutical carriers, excipients or diluents can be selected having regard to the intended route of administration and standard pharmaceutical practice.

[0400] Pharmaceutically acceptable carriers include any and all 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 and PD-1 inhibitor.

[0401] Examples of suitable aqueous and non-aqueous carriers that can be utilized 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 solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical arts.

[0402] 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 pharmaceutically 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 (pharmaceutical) compositions is contemplated.

[0403] The term "excipient" as used herein refers to a substance that may be present in the (pharmaceutical) composition of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0404] The term "diluent" refers to a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension and / or mixture. Examples of suitable diluents include ethanol, glycerol, and water.

[0405] The (pharmaceutical) compositions may also include pharmaceutically acceptable antioxidants, such as: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0406] The (pharmaceutical) composition may also comprise isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the composition.

[0407] The (pharmaceutical) composition may also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersing agents, preservatives, or buffers, which can increase the shelf life or effectiveness of the composition. Sustained-release formulations, including compositions such as implants, transdermal patches, and microencapsulated delivery systems, as used herein, can be prepared using carriers that protect the compound from rapid release. Such carriers can 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 wax, 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.

[0408] "Pharmaceutically acceptable salts" include, for example, acid addition salts, which may 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. Additionally, 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 appropriate organic ligands (e.g., quaternary ammonium and amine cations formed with counteranions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates, and aryl sulfonates).Illustrative examples of pharmaceutically acceptable salts are acetate, adipate, alginate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate , edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolyl arsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy- Ethanesulfonate, 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, pamoate Pharmaceutically acceptable salts include, but are not limited to, embonate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, suberate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and others (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 present disclosure.

[0409] In one embodiment, the binding agents and PD-1 inhibitors used herein can be formulated to ensure proper in vivo distribution. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Other active or therapeutic compounds may also be incorporated into the compositions.

[0410] Injectable pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug 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. It is often 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 agents 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 a combination of components listed above as necessary, and then carrying out microfiltration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile medium that contains a basic dispersion medium and other components required, for example, from those listed above.For sterile powder for preparing sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which produces the powder of active ingredient and any additional desired component from its solution that has been previously sterile filtered.

[0411] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in a suitable solvent, and optionally one or a combination of the above-listed components, followed by microfiltration sterilization.Generally, dispersion is prepared by incorporating the active compound into a sterile medium that contains the basic dispersion medium and other necessary components from the above-listed components.For the preparation of sterile injectable solution, the example of the method of preparation is vacuum drying and freeze-drying (lyophilization), which produces the powder of the active ingredient and any additional desired components from its solution that has been previously sterile-filtered.

[0412] In certain embodiments, the binder for use according to the present invention comprises histidine, sucrose, and polysorbate 80 and is formulated into a composition or formulation having a pH of about 5 to about 6, e.g., a pH of 5 to 6. In particular, the binder for use according to the present invention may be in a composition or formulation comprising about 20 mM histidine, about 250 mM sucrose, and about 0.02% polysorbate 80 and having a pH of about 5.5, e.g., a composition or formulation comprising 20 mM histidine, 250 mM sucrose, and 0.02% polysorbate 80 and having a pH of 5.5. In certain embodiments, the formulation may comprise about 10 to about 30 mg / mL of binder, e.g., 10 to 30 mg / mL of binder, particularly about 20 mg / mL of binder, e.g., 20 mg / mL of binder.

[0413] The binding agent for use according to the invention may be provided in a composition as defined above, which may then be diluted in 0.9% NaCl (saline) prior to administration.

[0414] In a second aspect, the present disclosure provides a kit comprising: (i) a binding agent comprising a first binding region that binds CD137 and a second binding region that binds PD-L1; and (ii) a PD-1 inhibitor; where: (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding region that binds to PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; case, The PD-1 inhibitor is not an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively.

[0415] Embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent and the PD-1 inhibitor) also apply to the kit of the second aspect. In one embodiment, the kit comprises at least two containers, one of which contains the binding agent (per se or in a (pharmaceutical) composition) and the second of which contains the PD-1 inhibitor (per se or in a (pharmaceutical) composition).

[0416] 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 treating cancer in a subject. Embodiments disclosed herein with respect to the first aspect (particularly with respect to binding agents, PD-1 inhibitors, treatment regimens, specific tumors / cancers, and subjects) and / or the second aspect also apply to the kit for use of the third aspect.

[0417] 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 before, simultaneously with, or after administration of a PD-1 inhibitor, wherein the binding agent comprises a first binding region that binds CD137 and a second binding region that binds PD-L1; and where: (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding domain that binds to PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; case, The PD-1 inhibitor is not an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively.

[0418] Embodiments disclosed herein with respect to the first aspect (particularly with respect to binding agents, PD-1 inhibitors, treatment regimens, specific tumors / cancers, and subjects) also apply to the methods of the fourth aspect.

[0419] In a further aspect, the present disclosure provides a PD-1 inhibitor for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject a PD-1 inhibitor prior to, concurrently with, or following administration of a binding agent, wherein the binding agent comprises a first binding region that binds CD137 and a second binding region that binds PD-L1; and where: (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding region that binds to PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; case, The PD-1 inhibitor is not an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:62, 63, and 64, respectively.

[0420] Embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, the PD-1 inhibitor, the treatment regimen, the particular tumor / cancer, and the subject) also apply to the PD-1 inhibitor for use in this further aspect.

[0421] A further aspect of the invention relates to a binding agent comprising a first binding domain that binds CD137 and a second binding domain that binds PD-L1 for use in reducing or preventing tumor progression or treating cancer in a subject, wherein the subject's last prior treatment was with a PD1 or PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0422] The time from progression on the subject's last treatment with a PD-1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, is preferably 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0423] The time from the last dose of a PD-1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, as part of the final conditioning treatment is preferably 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0424] It is understood that the binding agent may have any of the characteristics defined above in relation to the first aspect of the invention. Similarly, the tumor or cancer and / or subject to which the binding agent is administered may be as defined above. The route and frequency of administration and the amount of binding agent administered may be as defined in relation to the first aspect of the invention above.

[0425] A still further aspect of the invention provides a method of reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject a binding agent comprising a first binding domain that binds CD137 and a second binding domain that binds PD-L1, wherein the subject's last prior treatment was with a PD1 or PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0426] The time from progression on the subject's last treatment with a PD-1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, is preferably 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0427] The time from the last dose of a PD-1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, as part of the final conditioning treatment is preferably 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0428] It is understood that the binding agent may have any of the characteristics defined above in relation to the first aspect of the invention. Similarly, the tumor or cancer and / or subject to which the binding agent is administered may be as defined above. The route and frequency of administration and the amount of binding agent administered may be as defined above in relation to the first aspect of the invention.

[0429] Citation of documents and works referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to the applicant and do not constitute an admission that the contents of these documents are accurate.

[0430] This specification (including the following examples) is presented to enable those skilled in the art to make and use the 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 embodiments 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 appended claims.

[0431] Items of this disclosure 1. A binding agent for use in a method for reducing or inhibiting 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 PD-1 inhibitor, the binding agent comprising a first binding region that binds CD137 and a second binding region that binds PD-L1; and where (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second binding domain that binds to PD-L1 comprises a heavy chain variable region (VH) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) that includes the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. case, the PD-1 inhibitor is not an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively; Binder. 2. 2. The binding agent for use in item 1, wherein the PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence shown in SEQ ID NO:40, and / or the CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO:38. 3. 4. The binding agent for use according to any one of items 1 to 3, wherein the PD-1 inhibitor is a PD-1 antibody. 4. 5. The binding agent for use according to any one of items 1 to 4, wherein the PD-1 inhibitor is a PD-1 blocking antibody. 5. The conjugate for use in any one of the preceding items, wherein the PD-1 inhibitor is pembrolizumab or a biosimilar thereof. 6. The binding agent for use in any one of the preceding items, wherein the PD-1 inhibitor is nivolumab or a biosimilar thereof. 7. (a) the first binding region comprises a heavy chain variable region (VH) of SEQ ID NO: 1 or 9 comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) of SEQ ID NO: 5 or 10 comprising CDR1, CDR2, and CDR3 sequences; and (b) the second antigen-binding region comprises a heavy chain variable region (VH) of SEQ ID NO: 11 comprising the CDR1, CDR2 and CDR3 sequences, and a light chain variable region (VL) of SEQ ID NO: 15 comprising the CDR1, CDR2 and CDR3 sequences; A binder for use in any one of the preceding items. 8. (a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; A binder for use in any one of the preceding items. 9. 8. The binding agent for use in any one of the preceding items, wherein 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 to SEQ ID NO:1 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:5 or 10. 10. 8. The binding agent for use in any one of the preceding items, wherein 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 to SEQ ID NO:11, 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:15. 11. 8. The binding agent for use in any one of the preceding items, wherein the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 or 9 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5 or 10. 12. 8. The binding agent for use in any one of the preceding items, wherein the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:15. 13. (a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and (b) the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 15; A binder for use in any one of the preceding items. 14. 8. The binding agent for use in any one of the preceding items, wherein said binding agent is a multispecific antibody, such as a bispecific antibody. 15. 8. A binding agent for use in any one of the preceding items, wherein said binding agent is in the format of a full length antibody or antibody fragment. 16. 13. The binding agent for use according to any one of items 6 to 12, wherein each variable region comprises three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4). 17. 14. The binding agent for use in item 13, wherein the complementarity determining regions and the framework regions are arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. 18. (i) a polypeptide comprising, consisting of, or consisting essentially of said first heavy chain variable region (VH) and first heavy chain constant region (CH); (ii) a polypeptide comprising, consisting of, or consisting essentially of the second heavy chain variable region (VH) and second heavy chain constant region (CH); 18. A binder for use in any one of items 7 to 17, comprising 19. (i) a polypeptide comprising the first light chain variable region (VL) and further comprising a first light chain constant region (CL); (ii) a polypeptide comprising the second light chain variable region (VL) and further comprising a second light chain constant region (CL); 19. A binder for use in any one of items 7 to 18, comprising 20. the binding agent is an antibody comprising a first binding arm and a second binding arm; The first binding arm is (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH); (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL); and The second binding arm is (iii) a polypeptide comprising the second heavy chain variable region (VH) and a second heavy chain constant region (CH); (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL); Including, A binder for use in any one of items 7 to 19. twenty one. (i) a first heavy chain and a light chain comprising the antigen-binding region capable of binding to CD137; (ii) a second heavy chain and a light chain comprising the antigen-binding region capable of binding to PD-L1; 10. A binder for use in any one of the preceding items, comprising twenty two. (i) a first heavy chain and a light chain comprising the antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; (ii) a second heavy chain and a light chain comprising the antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region; and 10. A binder for use in any one of the preceding items, comprising twenty three. 23. The binding agent for use in any one of items 18 to 22, wherein 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, preferably at least a hinge region, a CH2 region, and a CH3 region. twenty four. 24. The binding agent for use according to any one of items 18 to 23, wherein each of the first and second heavy chain constant regions (CHs) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations. twenty five. 24. The binding agent for use according to any one of items 18 to 23, wherein in the first heavy chain constant region (CH), at least one amino acid is substituted 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, and in the second heavy chain constant region (CH), at least one amino acid is substituted 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, and the first and second heavy chains do not have substitutions at the same positions. 26. (i) the amino acid at the position corresponding to F405 in a 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 a human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH); or (ii) the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in the second heavy chain; Binding agent for use in item 25. 27. 10. The binding agent for use in any one of the preceding items, which 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 a human IgG1 hinge, CH2 and CH3 regions. 28. 28. The binding agent for use of item 27, wherein the first and second heavy chain constant regions (CHs) have been modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an otherwise identical antibody comprising unmodified first and second heavy chain constant regions (CHs). 29. 29. The binding agent for use according to item 28, wherein each of the unmodified first and second heavy chain constant regions (CHs) comprises the amino acid sequence shown in SEQ ID NO: 19 or 25. 30. 30. The binding agent for use in item 28 or 29, wherein the Fc-mediated effector function is measured by binding to an Fcγ receptor, binding to C1q, or induction of Fe-mediated cross-linking of an Fcγ receptor. 31. 31. The binding agent for use of item 30, wherein said Fc-mediated effector function is measured by binding to C1q. 32. 32. The binding agent for use according to any one of items 27 to 31, wherein the first and second heavy chain constant regions are modified such that binding of C1q to the antibody is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%; and C1q binding is preferably determined by ELISA. 33. 33. The binding agent for use according to any one of items 18 to 32, wherein in at least one of the first and second heavy chain constant regions (CH), one or more amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively. 34. 34. The binding agent for use in item 33, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E in the first and second heavy chains, respectively. 35. 35. The binding agent for use according to item 33 or 34, wherein the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering are F, E and A in the first and second heavy chain constant regions, respectively. 36. the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively; (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in a 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; A binder for use in any one of items 33 to 35. 37. the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F, E, and A, respectively; (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 of the first heavy chain is R, and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L; A binder for use in any one of items 33 to 36. 38. the constant region of the first and / or second heavy chain comprises: (a) the sequence shown in SEQ ID NO: 19 or 25 [IgG1-FC]; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: A binder for use in any one of items 18 to 37. 39. the constant region of the first or second heavy chain, e.g., the second heavy chain, comprises: (a) the sequence shown in SEQ ID NO: 20 or 26 [IgG1-F405L]; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: A binder for use in any one of items 18 to 38. 40. the constant region of the first or second heavy chain, e.g., the first heavy chain, comprises: (a) the sequence shown in SEQ ID NO: 21 or 27 [IgG1-K409R]; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: A binder for use in any one of items 18 to 38. 41. the constant region of the first and / or second heavy chain comprises: (a) the sequence shown in SEQ ID NO: 22 or 28 [IgG1-Fc_FEA]; (b) a subsequence of the sequence in (a), e.g., 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 seven substitutions, e.g., at most six substitutions, at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions, or at most one substitution, compared to the amino acid sequence defined in (a) or (b). comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: A binder for use in any one of items 18 to 37. 42. the constant region of the first and / or second heavy chain, e.g., the second heavy chain, comprises: (a) the sequence shown in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL]; (b) a subsequence of the sequence in (a), e.g., 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 six substitutions, e.g., at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions, or at most one substitution, compared to the amino acid sequence defined in (a) or (b); comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: A binder for use in any one of items 18 to 41. 43. the constant region of the first and / or second heavy chain, e.g., the first heavy chain, comprises: (a) the sequence shown in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR]; (b) a subsequence of the sequence in (a), e.g., 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 six substitutions, e.g., at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions, or at most one substitution, compared to the amino acid sequence defined in (a) or (b); comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: A binder for use in any one of items 18 to 42. 44. Item 11. A binding agent for use in any one of the preceding items, wherein said binding agent comprises a kappa (κ) light chain constant region. 45. Item 11. A binding agent for use in any one of the preceding items, wherein said binding agent comprises a lambda (λ) light chain constant region. 46. Item 11. The binding agent for use in any one of the preceding items, wherein said first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region. 47. Item 11. The binding agent for use in any one of the preceding items, wherein said second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region. 48. 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; A binder for use in any one of the preceding items. 49. Kappa (κ) light chains (a) the sequence shown in SEQ ID NO:35; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). comprising an amino acid sequence selected from the group consisting of: A binder for use in any one of items 44 to 48. 50. Lambda (λ) light chains (a) the sequence shown in SEQ ID NO:36; (b) a subsequence of the sequence in (a), e.g., 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 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution, compared to the amino acid sequence defined in (a) or (b). comprising an amino acid sequence selected from the group consisting of: A binder for use in any one of items 45 to 49. 51. Item 11. The binding agent for use in any one of the preceding items, wherein said binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. 52. Item 11. The binding agent for use in any one of the preceding items, wherein said binding agent is a full-length IgG1 antibody. 53. Item 11. The binding agent for use in any one of the preceding items, wherein said binding agent is an antibody of the IgG1m(f) allotype. 54. The binder is (i) a first heavy chain and a light chain comprising an antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises the sequence set forth in SEQ ID NO:31 and the first light chain comprises the sequence set forth in SEQ ID NO:32; (ii) a second heavy chain and a light chain comprising an antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises the sequence set forth in SEQ ID NO:33, and the second light chain comprises the sequence set forth in SEQ ID NO:34. 10. A binder for use in any one of the preceding items, comprising 55. 8. The binder for use in any one of the preceding items, wherein said binder is acasanlimab or a biosimilar thereof. 56. 8. The binder for use in any one of the preceding items, wherein the binder is in a composition or formulation comprising histidine, sucrose, and polysorbate-80, and has a pH of 5 to 6. 57. 8. The binder for use in any one of the preceding items, wherein the binder is in a composition or formulation comprising about 20 mM histidine, about 250 mM sucrose, about 0.02% polysorbate-80, and has a pH of about 5.5. 58. 8. The binder for use in any one of the preceding items, wherein the binder is in a composition or formulation comprising 10-30 mg binder / mL, for example, 20 mg binder / mL. 59. 59. A binder for use in any one of the preceding items, wherein the binder is in a composition as defined in any one of items 56 to 58 and is diluted in 0.9% NaCl (saline) before administration. 60. 8. The binding agent for use in any one of the preceding items, wherein the PD-1 inhibitor is an antibody that binds to PD-1, and the antibody that binds to PD-1 comprises VH regions CDR1, CDR2, and CDR3 comprising the sequences as set forth in SEQ ID NOs:104, 101, and 100, respectively, and VL regions CDR1, CDR2, and CDR3 comprising the sequences as set forth in SEQ ID NOs:107, QAS, and SEQ ID NO:105, respectively. 61. 61. The binding agent for use of item 60, wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:111. 62. 62. The binding agent for use of item 60 or 61, wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH), wherein the VH comprises a sequence as set forth in SEQ ID NO:111. 63. 63. The binding agent for use according to any one of paragraphs 60 to 62, wherein the antibody that binds to PD-1 comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence as set forth in SEQ ID NO:112. 64. 64. The binding agent for use of item 63, wherein the antibody that binds to PD-1 comprises a light chain variable region (VL), and the VL comprises a sequence as set forth in SEQ ID NO:112. 65. 65. The binding agent for use according to any one of items 60 to 64, wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or has the sequence as set forth in SEQ ID NO:111 and the VL comprises or has the sequence as set forth in SEQ ID NO:112. 66. 66. The binding agent for use according to any one of items 60 to 65, wherein the antibody that binds to PD-1 comprises a heavy chain constant region, wherein the heavy chain constant region comprises an aromatic or nonpolar amino acid at a position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at a position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering. 67. 67. The binding agent for use according to item 66, wherein the amino acid at the position corresponding to position 236 is a basic amino acid. 68. 68. The binding agent for use according to item 67, wherein the basic amino acid is selected from the group consisting of lysine, arginine, and histidine. 69. 69. The binding agent for use according to item 67 or 68, wherein the basic amino acid is arginine (G236R). 70. 69. The binding agent for use in any one of items 66 to 69, wherein the amino acid at the position corresponding to position 234 is an aromatic amino acid. 71. 71. The binder for use according to item 70, wherein the aromatic amino acid is selected from the group consisting of phenylalanine, tryptophan, and tyrosine. 72. 69. The binding agent for use according to any one of items 66 to 69, wherein the amino acid at the position corresponding to position 234 is a non-polar amino acid. 73. 73. The binder for use according to item 72, wherein the nonpolar amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. 74. 74. The binder for use according to item 72 or 73, wherein the nonpolar amino acid is selected from the group consisting of isoleucine, proline, phenylalanine, methionine, and tryptophan. 75. 75. The binding agent for use in any one of items 66 to 74, wherein the amino acid corresponding to position 234 is phenylalanine (L234F). 76. 76. The binding agent for use according to any one of items 66 to 75, wherein the amino acid at a position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering in the heavy chain constant region of the antibody that binds to PD-1 is an acidic amino acid. 77. 77. The binder for use according to item 76, wherein the acidic amino acid is aspartic acid or glutamic acid. 78. 78. The binding agent for use according to any one of items 66 to 77, wherein the amino acid at a position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering in the heavy chain constant region of the antibody that binds to PD-1 is glutamic acid (L235E). 79. 79. The binding agent for use according to any one of items 66 to 78, wherein the amino acids at positions corresponding to positions 234, 235, and 236 in the heavy chain constant region of an antibody that binds to PD-1 are a nonpolar or aromatic amino acid at position 234, an acidic amino acid at position 235, and a basic amino acid at position 236. 80. 80. The binding agent for use according to any one of items 66 to 79, wherein in the heavy chain constant region of the antibody that binds to PD-1, the amino acid corresponding to position 234 is phenylalanine, the amino acid corresponding to position 235 is glutamic acid, and the amino acid corresponding to position 236 is arginine (L234F / L235E / G236R). 81. 81. The binding agent for use according to any one of items 60 to 80, wherein the heavy chain constant region of the antibody that binds to PD-1 comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the heavy chain constant region sequence as set forth in SEQ ID NO:93. 82. 82. The binding agent for use in any one of items 60 to 81, wherein the heavy chain constant region of the antibody that binds to PD-1 comprises a sequence as set forth in SEQ ID NO:93. 83. 83. The binding agent for use in any one of items 60 to 82, wherein the heavy chain constant region of the antibody that binds to PD-1 is of the isotype IgG1. 84. 84. The binding agent for use according to any one of items 60 to 83, wherein the antibody that binds to PD-1 is a monoclonal, chimeric or humanized antibody or a fragment of such an antibody. 85. 85. The binding agent for use according to any one of items 60 to 84, wherein the antibody that binds to PD-1 has reduced or depleted Fc-mediated effector function. 86. 86. The binding agent for use according to any one of items 60 to 85, wherein binding of the complement protein C1q to the constant region of the antibody that binds to PD-1 is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, compared to the wild-type antibody. 87. 87. The binding agent for use according to any one of items 60 to 86, wherein binding of one or more IgG Fc-gamma receptors to the antibody that binds to PD-1 is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, compared to the wild-type antibody. 88. 88. The binding agent for use according to item 87, wherein the one or more IgG Fc-gamma receptors are selected from at least one of Fc-gamma RI, Fc-gamma RII and Fc-gamma RIII. 89. 89. The binding agent for use according to item 87 or 88, wherein the IgG Fc-gamma receptor is Fc-gamma RI. 90. 89. The binding agent for use according to any one of items 60 to 89, wherein the antibody that binds to PD-1 is unable to induce Fc-gammaRI-mediated effector function, or the induced Fc-gammaRI-mediated effector function is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, compared to a wild-type antibody. 91. an antibody that binds to PD-1 fails to induce at least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion, and / or phagocytosis; or At least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion, and / or phagocytosis is induced to a reduced extent, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%; A binder for use in any one of items 60 to 90. 92. 92. The binding agent for use according to any one of items 60 to 91, wherein binding of the neonatal Fc receptor (FcRn) to the antibody that binds to PD-1 is not affected compared to the wild-type antibody. 93. 93. The binding agent for use in any one of items 60 to 92, wherein the PD-1 is human PD-1. 94. PD-1 has or comprises an amino acid sequence as set forth in SEQ ID NO:113 or SEQ ID NO:114, or the amino acid sequence of PD-1 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence as set forth in SEQ ID NO:113 or SEQ ID NO:114, or an immunogenic fragment thereof; Binders for use in item 93. 95. 95. The binding agent for use according to any one of items 60 to 94, wherein the antibody that binds to PD-1 binds to a native epitope of PD-1 present on the surface of a living cell. 96. 96. The binding agent for use according to any one of items 60 to 95, wherein the antibody that binds to PD-1 is a multispecific antibody comprising a first antigen-binding regi...

Claims

1. A medicament comprising a bispecific antibody for use in a method for reducing or inhibiting tumor progression or treating cancer in a subject, the method comprising administering the bispecific antibody to the subject prior to, simultaneously with, or after administration of a PD-1 inhibitor, the bispecific antibody comprising a first binding region that binds CD137 and a second binding region that binds PD-L1; and where (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, GAS, and SEQ ID NO: 8, respectively; and (b) the second binding region that binds to PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, DDN, and SEQ ID NO: 18, respectively; case, the PD-1 inhibitor is not an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively; Medicine.

2. The pharmaceutical composition of claim 1, wherein the PD-1 inhibitor is a PD-1 antibody or a PD-1 blocking antibody.

3. 2. The pharmaceutical composition of claim 1, wherein the PD-1 inhibitor is an anti-PD-1 antibody or an 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, and CS1003.

4. (a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 5; and (b) the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 15; The pharmaceutical composition according to claim 1.

5. The pharmaceutical according to claim 1, wherein the bispecific antibody is in the format of a full-length antibody or an antibody fragment.

6. the bispecific antibody is an antibody comprising a first binding arm and a second binding arm; The first binding arm comprises: (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH); (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL); and The second binding arm is (iii) a polypeptide comprising the second heavy chain variable region (VH) and a second heavy chain constant region (CH); (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL); Including, The pharmaceutical composition according to claim 1.

7. (i) the amino acid at the position corresponding to F405 in a 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 a human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH); or (ii) the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in the first heavy chain constant region, and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in the second heavy chain constant region; The pharmaceutical composition according to claim 6.

8. The pharmaceutical agent according to claim 6, wherein the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in the first and second heavy chain constant regions.

9. the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F, E, and A, respectively; (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in a 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 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 constant region is L; The pharmaceutical composition according to claim 6.

10. 2. The pharmaceutical according to claim 1, wherein the bispecific antibody is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and is preferably a full-length IgG1 antibody.

11. the bispecific antibody (i) a first heavy chain and a light chain comprising the first binding region capable of binding to CD137, wherein the first heavy chain comprises the sequence set forth in SEQ ID NO: 31 and the first light chain comprises the sequence set forth in SEQ ID NO: 32; (ii) a second heavy chain and a light chain comprising a second binding region capable of binding to PD-L1, wherein the second heavy chain comprises the sequence set forth in SEQ ID NO: 33, and the second light chain comprises the sequence set forth in SEQ ID NO:

34. The pharmaceutical composition of claim 1, comprising:

12. The pharmaceutical composition of claim 1, wherein the bispecific antibody is acasanlimab.

13. the bispecific antibody in a composition or formulation comprising histidine, sucrose, and polysorbate-80 and having a pH of 5 to 6; or in a composition or formulation comprising about 20 mM histidine, about 250 mM sucrose, about 0.02% polysorbate-80, and having a pH of about 5.5; The pharmaceutical composition according to claim 1.

14. the PD-1 inhibitor is an antibody that binds to PD-1, an antibody that binds to PD-1 comprising a VH region, CDR1, CDR2, and CDR3, comprising the sequences as set forth in SEQ ID NOs: 104, 101, and 100, respectively, and a VL region, CDR1, CDR2, and CDR3, comprising the sequences as set forth in SEQ ID NOs: 107, QAS, and SEQ ID NO: 105, respectively; or an antibody that binds to PD-1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or has a sequence as set forth in SEQ ID NO: 111, and the VL comprises or has a sequence as set forth in SEQ ID NO: 112; The pharmaceutical composition according to claim 1.

15. The pharmaceutical agent of claim 14, wherein the antibody that binds to PD-1 comprises a heavy chain constant region, and in the heavy chain constant region of the antibody that binds to PD-1, the amino acid corresponding to position 234 in the human IgG1 heavy chain according to EU numbering is phenylalanine, the amino acid corresponding to position 235 in the human IgG1 heavy chain according to EU numbering is glutamic acid, and the amino acid corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is arginine (L234F / L235E / G236R).

16. the antibody that binds to PD-1 is a multispecific antibody comprising a first antigen-binding region that binds to PD-1 and at least one additional antigen-binding region that binds to another antigen; or the antibody that binds to PD-1 is a bispecific antibody that comprises a first antigen-binding region that binds to PD-1 and a second antigen-binding region that binds to another antigen; The pharmaceutical composition according to claim 14.

17. the subject is a human subject, and / or the tumor or cancer is a solid tumor or cancer, and / or the tumor is a PD-L1 positive tumor; The pharmaceutical composition according to claim 1.

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

19. the tumor or cancer is lung cancer, in particular non-small cell lung cancer (NSCLC), e.g., squamous or non-squamous NSCLC; and / or the tumor or cancer is metastatic, e.g., metastatic NSCLC, and / or the lung cancer, particularly NSCLC, comprises cancer cells, and PD-L1 is expressed on ≥ 1% of the cancer or tumor cells, as assessed, for example, by immunohistochemistry (IHC); The pharmaceutical composition according to claim 17 or 18.

20. the subject has not received prior systemic treatment for metastatic disease, and / or the subject has not received prior treatment with a checkpoint inhibitor, e.g., a PD-1 or PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody; and / or the subject has not received prior treatment with a 4-1BB (CD137) targeting agent, e.g., an anti-4-1BB (CD137) antibody, an anti-tumor vaccine, or an autologous cellular immunotherapy; and / or the subject has not received prior treatment with a taxane chemotherapy agent, e.g., docetaxel, e.g., prior treatment for NSCLC with a taxane chemotherapy agent, e.g., docetaxel; The pharmaceutical composition according to claim 1.

21. the tumor or cancer has recurred and / or is resistant to treatment, such as systemic treatment with a checkpoint inhibitor, and / or the subject has received at least one prior line of systemic therapy, e.g., a systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody; and / or the cancer or tumor has relapsed and / or is refractory, or the subject has progressed after treatment with a PD-1 or PD-L1 inhibitor, such as an anti-PD-1 or anti-PD-L1 antibody, where the PD-1 or PD-L1 inhibitor is administered as monotherapy or as part of a combination therapy; and / or the last prior treatment was with a PD1 or PD-L1 inhibitor, such as an anti-PD-1 or anti-PD-L1 antibody, where the PD-1 or PD-L1 inhibitor was administered as monotherapy or as part of a combination therapy; and / or the cancer or tumor has recurred and / or is resistant, or The target is, (i) platinum doublet chemotherapy followed by treatment with an anti-PD-1 antibody or anti-PD-L1 antibody; or (ii) Treatment with anti-PD-1 antibody or anti-PD-L1 antibody after platinum doublet chemotherapy showing progression during or after The pharmaceutical composition according to claim 1.

22. 2. The medicament of claim 1, wherein one dose of the bispecific antibody and one dose of the PD-1 inhibitor are administered every 2 weeks (1Q2W), every 3 weeks (1Q3W), every 4 weeks (1Q4W), every 5 weeks (1Q5W), or every 6 weeks (1Q6W), preferably one dose of the bispecific antibody and one dose of the PD-1 inhibitor are administered every 6 weeks (1Q6W).

23. the amount of said bispecific antibody administered in each dose and / or in each treatment cycle is 100 mg; and / or the amount of the PD-1 inhibitor administered at each dose and / or in each treatment cycle is 200 mg or 400 mg; The pharmaceutical composition according to claim 1.

24. a 100 mg dose of the bispecific antibody and a 200 mg dose of the PD-1 inhibitor administered every three weeks (1Q3W); or a 100 mg dose of the bispecific antibody and a 400 mg dose of the PD-1 inhibitor are administered every six weeks (1Q6W), The pharmaceutical composition according to claim 1.

25. The pharmaceutical composition of claim 1, wherein the gap between the end of administration of the bispecific antibody and the start of administration of the PD-1 inhibitor, or the gap between the end of administration of the PD-1 inhibitor and the start of administration of the bispecific antibody, is up to about 14 days.

26. 1. A kit comprising: (i) a bispecific antibody comprising a first binding region that binds to CD137 and a second binding region that binds to PD-L1; and (ii) a PD-1 inhibitor; (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, GAS, and SEQ ID NO: 8, respectively; and (b) the second binding region that binds to PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, DDN, and SEQ ID NO: 18, respectively; case, the PD-1 inhibitor is not an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively; kit.

27. A composition comprising: (i) a bispecific antibody comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1; and (ii) a PD-1 inhibitor, (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, GAS, and SEQ ID NO: 8, respectively; and (b) the second binding region that binds to PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, DDN, and SEQ ID NO: 18, respectively; case, the PD-1 inhibitor is not an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively; composition.

28. A combination comprising: (i) a bispecific antibody comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1; and (ii) a PD-1 inhibitor, (a) a first binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 6, GAS, and SEQ ID NO: 8, respectively; and (b) the second binding region that binds to PD-L1 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, DDN, and SEQ ID NO: 18, respectively; case, the PD-1 inhibitor is not an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively; combination.

29. 29. The kit of claim 26, the composition of claim 27, or the combination of claim 28, wherein the bispecific antibody is as defined in any one of claims 1 and 4 to 13, and / or the PD-1 inhibitor is as defined in any one of claims 1 to 3 and 14 to 16.

30. 29. The kit of claim 26, the composition of claim 27, or the combination of claim 28, for use in a method for reducing or inhibiting tumor progression or treating cancer in a subject.

31. The kit of claim 26, the composition of claim 27, or the combination of claim 28, wherein the bispecific antibody is formulated to be administered before, simultaneously with, or after administration of a PD-1 inhibitor.

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