Multispecific binders for PD-L1 and CD137 to treat cancer

Abstract

The present invention relates to therapeutic methods using conjugates that bind to human PD-L1 and human CD137 for reducing or inhibiting tumor progression or for treating cancer. TIFF2026513865000063.tif111160

Description

[Technical Field]

[0001] The present invention relates to therapeutic methods using conjugates that bind to human PD-L1 and human CD137 for reducing or inhibiting tumor progression or treating cancer. [Background technology]

[0002] background CD137(4-1BB) is a member of the TNFR family, and CD8 +CD137 is a costimulatory molecule on T cells, 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) (Non-patent Literature 1)). Stimulation via its natural ligand, 4-1BBL, or agonist antibodies results in signaling using TRAF-2 and TRAF-1 as adapters. Initial signaling by CD137 involves a K-63 polyubiquitination reaction, ultimately resulting in activation of the nuclear factor (NF)-κB and mitogenic factor-activated protein (MAP) kinase pathways. This signaling leads to increased T cell costimulation, proliferation, cytokine production, maturation, and extended CD8+ T cell survival. Agonist antibodies against CD137 have been shown to promote T-cell-mediated antitumor control in various preclinical models (Murillo et al., Clin Cancer Res 2008;14(21):6895-906 (Non-Patent Literature 2)). Antibodies that stimulate CD137 can induce T-cell survival and proliferation, thereby enhancing the antitumor immune response. Antibodies that stimulate CD137 have been disclosed in the prior art and include urelumab, a human IgG4 antibody (AU 2004279877 (Patent Literature 1)), and utomirumab, a human IgG2 antibody (Fisher et al., 2012, Cancer Immunol. Immunother. 61: 1721-1733 (Non-Patent Literature 3)).

[0003] Programmed death ligand 1 (PD-L1, PDL1, CD274, B7H1) is a 33 kDa single-pass type I membrane protein. Three isoforms of PD-L1 have been described based on alternative splicing. PD-L1 belongs to the immunoglobulin (Ig) superfamily and contains one Ig-like C2 domain and one Ig-like V domain. Newly isolated T cells and B cells express only very small amounts of PD-L1 and CD14 + A portion of monocytes (approximately 16%) constitutively express PD-L1. However, interferon-γ (IFNγ) is known to upregulate PD-L1 on tumor cells. PD-L1 tolerates tumor-reactive T cells by (1) binding to its receptor, programmed cell death protein 1 (PD-1) (CD279), on activated T cells; and (2) PD-1 signaling via PD-L1 expressed on tumor cells, thereby promoting CD8 +Antitumor immunity is interfered with by (3) making tumor cells resistant to T cells and Fas ligand-mediated lysis; by (4) tolerating T cells through reverse signaling via CD80(B7.1) expressed on T cells; and by (5) promoting the development and maintenance of induced T regulatory cells. PD-L1 is expressed in many human cancers, including melanoma, ovarian cancer, lung cancer, and colon cancer (Latchman et al., 2004 Proc Natl Acad Sci USA 101, 10691-6 (Non-patent Literature 4)). PD-L1 blocking antibodies have shown clinical activity in several cancers known to overexpress PD-L1 (including melanoma and NSCLC). For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. Atezolizumab is currently in clinical trials as an immunotherapy for several indications, including various types of solid tumors (see, for example, Rittmeyer et al., 2017 Lancet 389:255-265 (Non-Patent Literature 5)), and is approved for the indications of non-small cell lung cancer and bladder cancer. Avelumab, a PD-L1 antibody (Kaufman et al Lancet Oncol. 2016;17(10):1374-1385 (Non-Patent Literature 6)), is 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 renal cancer. Durvalumab, a PD-L1 antibody, is approved for locally advanced or metastatic urothelial carcinoma and is in clinical development for several solid tumors and hematological malignancies (see, for example, Massard et al., 2016 J Clin Oncol. 34(26):3119-25 (Non-Patent Document 7)). Further anti-PD-L1 antibodies are described, for example, in WO 2004004771 (Patent Document 2).

[0004] Horton et al (J Immunother Cancer. 2015; 3(Suppl 2): ​​O10) (Non-patent document 8) disclose the combined use of an agonist 4-1BB antibody and a neutralizing PD-L1 antibody. WO 2019 / 025545 (Patent document 3) provides a conjugate that binds to both human PD-L1 and human CD137, such as a bispecific antibody. GEN1046 (DuoBody®-PD-L1×4-1BB) is a PD-L1×4-1BB bispecific antibody that targets both PD-L1 and 4-1BB.

[0005] However, despite these advances in this field, there is a great need for improved treatments for cancer. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] AU 2004279877 [Patent Document 2] WO 2004004771 [Patent Document 3] WO 2019 / 025545 [Non-patent literature]

[0007] [Non-Patent Document 1] Gros et al., J. Clin Invest 2014;124(5):2246-59 [Non-Patent Document 2] Murillo et al., Clin Cancer Res 2008;14(21):6895-906 [Non-Patent Document 3] Fisher et al., 2012, Cancer Immunol. Immunother. 61: 1721-1733 [Non-Patent Document 4] Latchman et al., 2004 Proc Natl Acad Sci USA 101, 10691-6 [Non-Patent Document 5] Rittmeyer et al., 2017 Lancet 389:255-265 [Non-Patent Document 6] Kaufman et al Lancet Oncol. 2016;17(10):1374-1385 [Non-Patent Document 7] Massard et al., 2016 J Clin Oncol. 34(26):3119-25 [Non-Patent Document 8] Horton et al(J Immunother Cancer. 2015; 3(Suppl 2): ​​O10) [Overview of the project]

[0008] overview The inventors have surprisingly found that a binder that binds to both human PD-L1 and human CD137 can be used to treat tumors or cancers in humans that are characterized by high frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

[0009] Accordingly, in the first aspect, the present disclosure provides a method for treating a tumor or cancer in a subject, the method comprising administering to the subject a binder comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

[0010] In a second aspect, the present disclosure provides a conjugate for use in a method for treating a tumor or cancer in a subject, the method comprising the step of administering to the subject a conjugate comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

[0011] In a third aspect, the present disclosure provides a pharmaceutical composition for use in a method for treating a tumor or cancer in a subject, comprising a binder comprising a first binding domain for binding to CD137 and a second binding domain for binding to PD-L1, and optionally a pharmaceutically acceptable carrier, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

[0012] In a fourth aspect, the Disclosure provides the use of a binder for the manufacture of a medicament for treating a tumor or cancer in a subject, wherein the tumor or cancer is a high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), and the binder comprises a first binding region that binds to CD137 and a second binding region that binds to PD-L1.

[0013] In a fifth aspect, the Disclosure provides a kit for use in a method for treating a tumor or cancer in a subject, comprising (a) a conjugate comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, and (b) a PD1 inhibitor, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). [Brief explanation of the drawing]

[0014] [Figure 1]This shows the time-course changes in target lesions (spider plot) for all 40 subjects with endometrial cancer who received GEN1046 in the expanded cohort 4 of trial GCT1046-01. [Figure 2] The best overall change in target lesions is shown (waterfall plot) for all 40 subjects with endometrial cancer who received GEN1046 in the expanded cohort 4 of trial GCT1046-01. [Figure 3] This waterfall plot shows the best overall change in target lesions in 33 subjects with MSS tumors who received GEN1046 in the expanded cohort 4 of trial GCT1046-01. [Figure 4] This waterfall plot shows the best overall change in target lesions in seven subjects with MSI-H tumors who received GEN1046 in the expanded cohort 4 of trial GCT1046-01. [Figure 5] A schematic diagram of the expected mechanism of action of the CD137×PD-L1 bispecific antibody is shown. (A) PD-L1 is expressed on antigen-presenting cells (APCs) and tumor cells. PD-L1 binding to T cells expressing the negative regulatory molecule PD-1 effectively inactivates the T cell activation signal, ultimately leading to T cell inhibition. (B) Upon addition of the CD137×PD-L1 bispecific antibody, the inhibitory PD-1:PD-L1 interaction is blocked via the PD-L1 specific arm, and simultaneously, the bispecific antibody provides agonist signaling to CD137 expressed on T cells through cell-cell interactions, resulting in strong T cell costimulation. [Figure 6]This shows an MC38 syngeneic tumor model established by subcutaneous inoculation of 1 × 10⁶ MC38 cells into C57BL / 6 mice. Mice were randomized when the tumor reached a mean volume of 64 mm³ and treated with either mbsIgG2a-PD-L1×4-1BB (5 mg / kg), anti-mouse PD-1 antibody (anti-mPD-1; 10 mg / kg) alone or in combination, or PBS (all 2QW×3). A. The data shown are the median tumor volume per treatment group (n=10), and data are carried over for animals that reached the termination criteria. Growth curves were interrupted when <50% of animals in the treatment group remained alive (PBS, mbsIgG2a-PD-L1×4-1BB, anti-mPD-1) or up to day 35 (combination of mbsIgG2a-PD-L1×4-1BB and anti-mPD-1). Arrows indicate the day of treatment. B. Progression-free survival, defined as the percentage of mice with a tumor volume smaller than 500 mm3, is shown as a Kaplan-Meier curve. Survival between treatment groups at day 45 was compared using Mantel-Cox analysis (Table 9). [Figure 7A]This report presents an analysis of the proliferation dose-response of GEN1046, the anti-PD-1 antibody nivolumab, or the anti-PD-1 antibody pembrolizumab in an antigen-specific T cell assay with an active PD1 / PD-L1 axis. CFSE-labeled T cells electroporated with claudin-6-specific TCR-IVT-RNA and PD-1-IVT-RNA were incubated for 5 days with immature dendritic cells electroporated with claudin-6-IVT-RNA in the presence of (A) GEN1046 (3-fold serial dilutions from 1 to 0.00015 μg / mL), (B) nivolumab (4-fold serial dilutions from 0.8 to 0.00005 μg / mL), or (C) pembrolizumab (4-fold serial dilutions from 0.8 to 0.00005 μg / mL). CD8+ T cell proliferation was measured by flow cytometry. The data shown are the expansion index as a function of antibody concentration. Error bars (SD) indicate intra-experimental variability ((A) is a repeat of n=3; (B) and (C) are double sequences of n=2, using cells from one representative donor). The curves were fitted using 4-parameter logarithmic fitting with GraphPad Prism software v9.0 to determine the EC50 value and Hill slope (shown in Tables 10-12). [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 8]This study demonstrates the deactivation of PD-1 / PD-L1-mediated T cell inhibition and additional co-stimulation of CD8+ T cell proliferation by GEN1046, either in the absence or in the presence of the anti-PD-1 antibody nivolumab or the anti-PD-1 antibody pembrolizumab. CFSE-labeled T cells electroporated with claudin-6-specific TCR-in vitro translation (IVT)-RNA and PD-1-IVT-RNA were incubated for 5 days with immature dendritic cells electroporated with claudin-6-IVT-RNA and in the presence of fixed concentrations of 1.6 μg / mL nivolumab, fixed concentrations of 0.8 μg / mL pembrolizumab, or 0.8 μg / mL unbound control antibody IgG1-ctrl, in the presence of 0.2 μg / mL, 0.0067 μg / mL, or 0.0022 μg / mL GEN1046 (n=2 technical replicates per condition, using n=3 individual donor-derived cells). Baseline proliferation in the absence of GEN1046 was determined using only culture medium, only 0.8 μg / mL IgG1-ctrl, only 1.6 μg / mL nivolumab, and only 0.8 μg / mL pembrolizumab. CD8+ T cell proliferation was measured by flow cytometry. The bar graphs represent the mean ± SD of the expansion growth 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. The dotted line represents baseline proliferation in the presence of the anti-PD-1 antibody pembrolizumab. [Figure 9-1]This study demonstrates the binding of IgG1-PD1 to various species of PD-1. CHO-S cells transiently transfected with various species of PD-1 were incubated with IgG1-PD1, pembrolizumab, or the unbound control antibodies IgG1-ctrl-FERR and IgG4-ctrl, and binding was analyzed using flow cytometry. Untransfected CHO-S cells incubated with IgG1-PD1 were included as a negative control. A-B. The data shown are the geometric mean fluorescence intensity (gMFI) ± SD of a double-well cell from one representative experiment out of four. C-D. The data shown are the gMFI ± SD of a double-well cell from one representative experiment out of two. E. The data shown are the geometric mean fluorescence intensity (gMFI) ± SD of a double-well cell from one representative experiment out of four. Abbreviations: gMFI = geometric mean fluorescence intensity; PD-1 = programmed cell death protein 1; PE = R-phycoerythrin. [Figure 9-2] See the explanation in Figure 9-1. [Figure 10A]This study demonstrates the competitive binding of IgG1-PD1 to human PD-1, PD-L1, and PD-L2. CHO-S cells transiently transfected with human PD-1 were incubated with 1 μg / mL biotinylated recombinant human PD-L1 (A) or PD-L2 (B) in the presence of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR was included as a negative control. Cells were stained with streptavidin-allophycocyanin, and the percentage of cells binding to biotinylated PD-L1 or PD-L2 was determined by measuring the percentage of streptavidin-allophycocyanin+ cells using flow cytometry. The percentages of streptavidin-allophycocyanin+ cells in antibody-free controls and untransfected samples are shown by dashed lines. The data shown are derived from a single replicate from one representative experiment out of three separate experiments. Abbreviations: Ab = antibody; CHO-S = Chinese hamster ovary, suspended; ctrl = control; FERR = L234F / L235E / G236R-K409R; PD-1 = programmed cell death protein 1; PD-L1 = programmed cell death ligand 1; PD-L2 = programmed cell death ligand 2. [Figure 10B] See the explanation in Figure 10A. [Figure 11] This study demonstrates 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 bioluminescence PD-1 / PD-L1 blockade reporter assay. The data shown are the mean luminescence ± SD of double wells in one representative experiment out 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 ligand 1; RLU = relative luminescence; SD = standard deviation. [Figure 12]This study demonstrates 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 CLDN6-specific TCRs and RNA encoding PD-1, and then labeled with CFSE. The T cells were then co-cultured with iDCs electroporated with CLDN6-coding RNA in the presence of IgG1-PD1, pembrolizumab, nivolumab, or IgG1-ctrl-FERR. CFSE dilution in T cells was analyzed by flow cytometry after 4 days and used to calculate the expansion growth index. Data from one representative donor (26268_B) out of four donors evaluated in three independent experiments are shown. Error bars represent standard deviation in double-well cells. Curves were fitted using GraphPad Prism with a 4-parameter logarithmic fit. Abbreviations: CFSE = Carboxyfluorescein succinimimidyl ester; FERR = L234F / L235E / G236R-K409R; PD1 = Programmed Cell Death Protein 1; SD = Standard Deviation. [Figure 13] This study shows IgG1-PD1-induced IFNγ secretion in an allogeneic MLR assay. Three unique donor pairs of allogeneic human mDCs and CD8+ T cells were co-cultured for 5 days in the presence of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR and IgG4 isotype controls were included as negative controls. IFNγ secretion was analyzed in the supernatant using an IFNγ-specific immunoassay. The data shown are mean ± mean standard error (SEM) concentrations for the 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 = mean standard error. [Figure 14A]This shows IgG1-PD1-induced cytokine secretion in an allogeneic MLR assay. Three unique donor pairs of allogeneic human mDCs and CD8+ T cells were co-cultured for 5 days in the presence of 1 μg / mL 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 factor change relative to cytokine levels measured in untreated co-cultures. (B) Cytokine production levels of the three unique allogeneic donor pairs are shown, with horizontal lines indicating the mean, upper limit, and lower limit. Abbreviations: FC = magnification 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 14B] See the explanation in Figure 14A. [Figure 15] This study demonstrates 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 (no inactivating mutation, with hexamerization-enhancing mutation), cells were incubated with human serum as a source of C1q. C1q binding was detected with FITC-conjugated rabbit anti-C1q antibody. The data shown are geometric mean fluorescence intensity (gMFI) ± standard deviation (SD) from double-well cells derived from one representative donor out of seven donors across three comparable experiments. Abbreviations: FITC = fluorescein isothiocyanate; gMFI = geometric mean fluorescence intensity; PE = R-phycoerythrocyanine. [Figure 16-1]This shows the binding of IgG1-PD1 to FcγR. The binding of IgG1-PD1 to immobilized human recombinant FcγR constructs was analyzed by SPR in an approved assay (n=1). IgG1-PD1 binding to FcγRIa (A), FcγRIIa-H131 (B), FcγRIIa-R131 (C), FcγRIIb (D), FcγRIIIa-F158 (E), and FcγRIIIa-V158 (F). The antibody IgG1-ctrl (without FER inactivation mutation) 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 unit. [Figure 16-2] See the explanation in Figure 16-1. [Figure 17-1] This shows the FcγR binding of IgG1-PD1 and several other anti-PD-1 antibodies. Binding of IgG1-PD1, nivolumab, pembrolizumab, dostallimab, and semiprimab 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 test antibodies. 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 unit. [Figure 17-2] See the explanation in Figure 17-1. [Figure 18]This study demonstrates the binding of IgG1-PD1 and several other anti-PD-1 antibodies to FcγRIa. The binding of IgG1-PD1, nivolumab, pembrolizumab, dostallimab, and semiprimab 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 19] This figure shows total human IgG in mouse plasma samples. Mice were intravenously injected with 1 or 10 mg / kg of IgG1-PD1 at t=0, and a series of plasma samples were collected 10 minutes, 4 hours, 1 day, 2 days, 8 days, 14 days, and 21 days after injection. Total huIgG in the plasma samples was measured by ECLIA for each mouse. Data are expressed as the mean huIgG concentration ± SD of three individual mice. The dashed line shows the plasma concentration of wild-type (wt) huIgG predicted by a two-compartment model based on IgG clearance in humans (Bleeker et al., 2001, Blood. 98(10):3136-42). The dotted lines show 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 20A]This study demonstrates the antitumor activity of IgG1-PD1 in human PD-1 knock-in mice. An MC38 colon cancer syngeneic tumor model was established by SC transplantation in hPD-1 KI mice. Mice were administered 0.5, 2, or 10 mg / kg of IgG1-PD1 or pembrolizumab, or 10 mg / kg of IgG1-ctrl-FERR in 2QW × 3 doses (9 mice per group). (A) Mean tumor volume ± SEM in each group up to the final point in time when the group completed treatment. (B) Tumor volume in various groups on the final day (day 11) when all groups completed treatment. The data shown are the tumor volume of individual mice in each treatment group and the mean tumor volume ± SEM per treatment group. Mann-Whitney analysis was used to compare the tumor volumes of the treatment groups with those of the IgG1-ctrl-FERR treatment group. *p<0.05, **p<0.01, and ***p<0.001. C. Progression-free survival, defined as the percentage of mice with a tumor volume smaller than 500 mm3, is shown as a Kaplan-Meier curve. One mouse from the 2 mg / kg IgG1-PD1 group that died from an undetermined cause on day 16, before its tumor volume exceeded 500 mm3, was excluded from the analysis. Abbreviations: 2QW×3 = twice a week for 3 weeks; ctrl = control; FERR = L234F / L235E / G236R / K409R mutation; IgG = immunoglobulin G; KI = knock-in; PD-1 = programmed cell death protein 1; SC = subcutaneous; SEM = standard error of the mean. [Figure 20B] See the explanation in Figure 20A. [Figure 20C] See the explanation in Figure 20A. [Figure 21]This study demonstrates IL-2 secretion induced by IgG1-PD1 combined with GEN1046 in an allogeneic MLR assay. 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-L1×ctrl (30 μg / mL), bsIgG1-ctrl×4-1BB (30 μg / mL), and IgG1-ctrl-FEAL (30 μg / mL) were included as control antibodies. IL-2 secretion was analyzed in the supernatant using Luminex. The data shown represent the mean IL-2 levels ± SEM of two specific 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 ligand 1; SEM = standard error of the mean. [Figure 22]This study demonstrates the enhancement of CD8+ T cell proliferation by IgG1-PD1 combined with GEN1046 in an antigen-specific T cell stimulation assay. Human CD8+ T cells were electroporated with RNA encoding CLDN6-specific TCRs and RNA encoding PD1, and labeled with CFSE. The T cells were then co-cultured with iDCs electroporated with CLDN6 in the presence of either 0.8 μg / mL of IgG1-PD1, pembrolizumab, or IgG1-ctrl-FERR, either alone or in combination with the indicated concentrations of GEN1046. CFSE dilutions in the T cells were analyzed by flow cytometry after 4 days and used to calculate the growth index. Data from one representative donor out of four donors, evaluated in two independent experiments, are shown. Error bars represent standard deviations (SD) in double-well cells. The dotted line shows the growth index of CD8+ T cells co-cultured with iDCs electroporated with mock cells (i.e., not expressing CLDN6). Abbreviations: CFSE = Carboxyfluorescein succinimimidyl 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 ligand 1; RNA = Ribonucleic acid; SD = Standard deviation; TCR = T cell receptor. [Figure 23]This study demonstrates enhanced cytokine secretion by IgG1-PD1 combined with GEN1046 after antigen-specific CD8+ T cell stimulation. Human CD8+ T cells expressing CLDN6-specific TCR and PD1 were co-cultured with iDCs expressing CLDN6 in the presence of 0.8 μg / mL of IgG1-PD1, pembrolizumab, or IgG1-ctrl-FERR, either alone or in combination with the indicated concentrations of GEN1046. Cytokine concentrations in the culture supernatant were measured at 4 days by multiplex electrochemiluminescence immunoassay. Data from one representative donor out of four donors, evaluated in two independent experiments, are shown. Error bars represent standard deviation (SD) in double 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 ligand 1; RNA = ribonucleic acid; SD = standard deviation; TCR = T cell receptor. [Figure 24] This shows an MC38 colon cancer model established by SC inoculation of 1 × 10⁶ MC38 cells into C57BL / 6 mice. Mice were randomized and treated with the antibodies or combinations shown (all 2QW × 3) when the tumor reached an average volume of 60 mm³. A. The data shown are the median tumor volume per treatment group (n=10), and data are carried over for animals that reached the termination criteria. Growth curves were interrupted when <50% of animals in the treatment group remained alive (mIgG2a-ctrl-AAKR, mbsIgG2a-PD-L1 × 4-1BB, anti-mouse PD-1 antibody [anti-mPD-1]) or up to day 69 (combination of mbsIgG2a-PD-L1 × 4-1BB and anti-mPD-1). Downward-pointing triangles indicate the day of treatment. B. Progression-free survival, defined as the percentage of mice with a tumor volume smaller than 500 mm³, is shown as a Kaplan-Meier curve. [Figure 25]This shows the (re)challenge of mice that had complete tumor regression at the time of treatment and a control group of tumor-naive mice. Mice were (re)loaded with 1 × 10⁶ MC38 tumor cells injected via SC 121 days after the start of antibody treatment. The data shown are mean tumor volume ± SEM. [Figure 26] This shows cytokine levels in the peripheral blood of MC38 tumor-bearing C57BL / 6 mice treated with either mbsIgG2a-PD-L1×4-1BB, an anti-mPD-1 antibody (either monotherapy or in combination), or the unbound control antibody IgG2a-ctrl-AAKR. Peripheral blood samples were collected at baseline (1 day before treatment [-1 day], dotted line) and 2 days after each treatment (2 and 5 days). Cytokine analysis was performed by ECLIA. [Figure 27] This report presents quantitative IHC and ISH data for cellular immune markers and tumor markers expressed in tumor tissue resected from an MC38 colon cancer model. C57BL / 6 mice were inoculated with 1 × 10⁶ MC38 cells. Mice were randomized when the tumor reached an average volume of 50–70 mm³ and treated with mbsIgG2a-PD-L1 × 4-1BB, anti-mPD-1, or a combination thereof. Tumors were resected on day 7 (n=5 per treatment group) or day 14 (n=5 per treatment group) after the start of treatment. Some of the resected tumor samples were too small for IHC analysis, resulting in the analysis of 4–5 tumors per treatment group. Resected tumor sections (4 μm) were stained by immunohistochemistry (IHC) with anti-CD3, anti-CD4, anti-CD8, or anti-PD-L1 antibodies, or by in-situ hybridization (ISH) for 4-1BB or PD-L2. Data from IHC are shown as the percentage of marker-positive cells relative to the total cells counted on the slide, and as the mean ± SEM per treatment group. Data from ISH are shown as the RNAscope H-score per slide, and as the mean ± SEM per treatment group. [Figure 28]This study shows the expression of GzmB and Ki67 in a subset of CD8 T cells derived from tumor tissue dissected from an MC38 colon cancer model. C57BL / 6 mice were inoculated with 1 × 10⁶ MC38 cells. Mice were randomized when the tumor reached an average volume of 50–70 mm³ and treated with mbsIgG2a-PD-L1×4-1BB, anti-mPD-1, or a combination thereof. Tumors were resected 7 days after the start of treatment (n=5 per treatment group), dissected into single-cell suspensions, and analyzed by flow cytometry. The data shown are the percentage of GzmB+ cells (A) or Ki67+ cells (B) within the CD8+ T cell population of individual mice, and the mean ± SEM per treatment group. Mann-Whitney statistical analysis was performed to compare the percentage of GzmB+ cells or Ki67+ cells within the CD8+ T cell population between treatment groups. *p<0.05 and **p<0.01. [Figure 29] This shows the characterization of the exhausted phenotype of CD3+ T cells after two rounds of CD3 / CD28 stimulation. (A) In vitro exhausted CD3+ T cells or naive T cells were stimulated with CD3 / CD28 beads. IFNγ secretion was analyzed by ELISA. The data shown are the mean + standard deviation (SD) of two wells from one representative donor pair. (B) Expression of TIM3, LAG3, PD-1, and 4-1BB on naive and in vitro exhausted CD3+ T cells was measured by flow cytometry. The data shown are the median fluorescence intensity (ΔMFI) corrected for background fluorescence. (C) Expression of Ki67 on naive and in vitro exhausted CD3+ T cells was measured by flow cytometry. [Figure 30]This study shows IFNγ secretion induced by GEN1046 in combination with pembrolizumab in mixed lymphocyte reactions (MLR) of mature dendritic cells (mDCs) and in vitro exhausted CD3+ T cells (Tex). Tex cells were co-cultured for 5 days with allogeneic LPS-mature DCs (DC:T cell ratio of 1:4) in the presence of GEN1046 (0.001–30 μg / mL) or pembrolizumab (1 μg / mL) alone or in combination. Co-cultures treated with no antibody (no antibody) or with bsIgG1-PD-L1×ctrl (30 μg / mL), bsIgG1-ctrl×4-1BB (30 μg / mL), IgG4 isotype control (1 μg / mL), or IgG1-ctrl-FEAL (30 μg / mL) were included as controls. IFNγ secretion was analyzed by ELISA. The data shown represents the mean + standard deviation (SD) of the double wells of one representative donor pair out of the four donor pairs tested. [Figure 31] This report presents the highest single agent (HSA) synergy scores for the combination of GEN1046 and pembrolizumab in mDCs and Tex in the MLR (Myelocellular Regeneration) of cells. Tex was co-cultured for 5 days with allogeneic LPS-mature DCs (DC:T cell ratio of 1:4) in the presence of GEN1046 (0.001–30 μg / mL) or pembrolizumab (1 μg / mL) alone or in combination. The data shown are the HSA synergy scores for one representative donor pair out of the four donor pairs tested. A score > 10 indicates a synergistic effect in this model. [Modes for carrying out the invention]

[0015] (Table 1) Sequences: In the following, we will refer in particular to the sequences and SEQ ID NOs shown in the sequence listing. We will also refer to, but will not limit, the antibodies of the present invention as described herein. These exemplary but non-limiting antibodies of the present invention are designated herein by reference to their antibody names. Bold and underlined are F;E;G;A;L;R and G, corresponding to positions 234;235;236;265;405;409 and 430, respectively, where the positions follow EU numbering. SEQ ID NOs: In 83 and 84, the bolded amino acids represent the -AAKR or -AALT mutations required for controlled Fab arm exchange. In the variable region, the CDR region (unless otherwise specified or inconsistent with the context) is underlined and annotated according to the definition of IMGT. TIFF2026513865000002.tif204159TIFF2026513865000003.tif223159TIFF2026513865000004.tif229159TIFF2026513865000005.tif222159TIFF2026513865000006.tif229159TIFF2026513865000007.tif229159TIFF2026513865000008.tif223159TIFF2026513865000009.tif223159TIFF2026513865000010.tif210159TIFF2026513865000011.tif220159TIFF2026513865000012.tif223159TIFF2026513865000013.tif227159TIFF2026513865000014.tif217159TIFF2026513865000015.tif229159TIFF2026513865000016.tif225159TIFF2026513865000017.tif226159TIFF2026513865000018.tif226159TIFF2026513865000019.tif220159TIFF2026513865000020.tif229159TIFF2026513865000021.tif222159TIFF2026513865000022.tif228159TIFF2026513865000023.tif223159TIFF2026513865000024.tif154159

[0016] Detailed description of the invention While this disclosure is described in more detail below, it should be understood that the specific methodologies, protocols, and reagents described herein are subject to change and are not limited thereto. Furthermore, it should be understood that the technical terms used herein are intended solely to describe specific aspects and are not intended to limit the scope of this disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0017] The elements of this disclosure are described in more detail below. These elements are listed along with specific embodiments, but it should be understood that they may be combined in any form and in any number to create additional embodiments. The examples and preferred embodiments described in various forms should not be construed as limiting this disclosure to only the embodiments expressly described. This description should be understood as supporting and encompassing embodiments that combine the expressly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any rearrangement and combination of all elements described in this application should be considered disclosed by the description of this application unless the context specifically indicates otherwise. For example, in a preferred embodiment of the binder used herein, the first heavy chain comprises, or is essentially derived from, the amino acid sequence [IgG1-Fc_FEAR] shown in SEQ ID NO: 23 or 29, and in another preferred embodiment of the binder used herein, the second heavy chain comprises, or is essentially derived from, the amino acid sequence [IgG1-Fc_FEAL] shown in SEQ ID NO: 24 or 30, and in a further preferred embodiment of the binder used herein, the first heavy chain comprises, or is essentially derived from, the amino acid sequence [IgG1-Fc_FEAR] shown in SEQ ID NO: 23 or 29, and the second heavy chain comprises, or is essentially derived from, the amino acid sequence [IgG1-Fc_FEAL] shown in SEQ ID NO: 24 or 30.

[0018] Preferably, terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0019] The practices described herein refer to the literature in the field unless otherwise specified (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) It employs conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in (see 1989).

[0020] All methods described herein may be performed in any suitable order, unless otherwise indicated herein or unless it is particularly clearly inconsistent with the context. All examples or exemplary language provided herein (e.g., “such as”) are intended solely to better illustrate the disclosure and do not impose any limitation on the scope of the disclosure as otherwise claimed. No language herein should be construed as indicating any unclaimed element essential to the practice of the disclosure.

[0021] The enumeration of value ranges in this specification is intended solely as a simple way to refer to each individual value within that range individually. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually enumerated.

[0022] Several documents are referenced throughout the text of this Spec. Each of the documents referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, whether above or below. Nothing herein should be construed as an acknowledgment that the present invention does not have prior rights to such disclosures on the grounds of prior art.

[0023] definition The following definitions are provided that apply to all aspects of this disclosure. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has the meaning recognized in the art.

[0024] Throughout this specification and the accompanying claims, unless the context requires otherwise interpretation, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a member, integer, or process, or a group of members, integers, or processes, but not the exclusion of any other member, integer, or process, or a group of members, integers, or processes. The term “consisting essentially of” means the exclusion of any other member, integer, or process that has an essential significance. The term “comprising” encompasses the term “consisting essentially of,” which in turn encompasses the term “consisting of.” Thus, in each instance in this application, the term “comprise” may be replaced by the terms “consisting essentially of” or “consisting of.” Similarly, in each instance in this application, the term “consisting essentially of” may be replaced by the term “consisting of.”

[0025] In the context describing this disclosure (particularly in the context of the claims), the terms “a,” “an,” and “the,” as well as similar references, should be construed to encompass both singular and plural forms unless otherwise specifically indicated herein or unless explicitly contradicted by the context.

[0026] Where used herein, “and / or” should be interpreted as a specific disclosure of each of the two expressed features or components, whether or not they are accompanied by the other. For example, “X and / or Y” should be interpreted as a specific disclosure of (i)X, (ii)Y, and (iii)X and Y, as each of which is presented separately herein.

[0027] In the context of this disclosure, the term “approximately” means a range 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 refers to deviations 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% from the given numerical value. As recognized by a person skilled in the art, a particular such deviation of a numerical value for a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have larger such deviations than artificial or engineered technical effects.

[0028] In the context of this disclosure, the term “binding agent” refers to any agent capable of binding to a desired antigen. In certain embodiments of this disclosure, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also include a synthetic portion, a modified portion, or a portion not naturally occurring, particularly a non-peptide portion. Such a portion may, for example, link a desired antigen-binding function or antigen-binding region, e.g., an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0029] As used herein, “immune checkpoint” refers to a modulator of the immune system, in particular, co-stimulatory and inhibitory signals that modulate the amplitude and quality of antigen recognition by T cell receptors. In certain embodiments, an immune checkpoint is an inhibitory signal. In certain embodiments, an inhibitory signal is an interaction between PD-1 and PD-L1 and / or PD-L2. In certain embodiments, an inhibitory signal is an interaction between CTLA-4 and CD80 or CD86, which replaces CD28 binding. In certain embodiments, an inhibitory signal is an interaction between LAG-3 and an MHC class II molecule. In certain embodiments, an inhibitory signal is an interaction between TIM-3 and one or more of its ligands, e.g., galectin 9, PtdSer, HMGB1, and CEACAM1. In certain embodiments, an inhibitory signal is an interaction between one or more KIRs and their ligands. In certain embodiments, an inhibitory signal is an 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.

[0030] The terms “checkpoint inhibitor” (CPI) and “immune checkpoint (ICP) inhibitor” are used synonymously herein. These terms refer to molecules such as conjugates that inhibit immune checkpoints, in particular inhibit the suppressive signaling of immune checkpoints, thereby reducing, inhibiting, interfering with, or negatively modulating one or more checkpoint proteins, either entirely or partially, or reducing, inhibiting, interfering with, or negatively modulating the expression of one or more checkpoint proteins, either entirely or partially. In one embodiment, an immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, an immune checkpoint inhibitor binds to one or more molecules that modulate checkpoint proteins. In one embodiment, an immune checkpoint inhibitor binds to a precursor of one or more checkpoint proteins, for example, at the DNA level or RNA level. Any agent that functions as a checkpoint inhibitor according to this disclosure can be used. As used herein, the term “partially” means at a level, for example, at a level of inhibition of the checkpoint protein, 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%.

[0031] In one embodiment, a checkpoint inhibitor can be any compound that inhibits the inhibitory signal of an immune checkpoint, e.g., any binder, where the inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2; the interaction between CTLA-4 and CD80 or CD86 replacing CD28 binding; the interaction between LAG-3 and MHC class II molecules; the interaction between TIM-3 and one or more of its ligands, e.g., galectin 9, PtdSer, HMGB1, and CEACAM1; the interaction between one or more KIRs and their ligands; the interaction between TIGIT and one or more of its ligands, PVR, PVRL2, and PVRL3; and CD94 / NK The interaction is selected from the group consisting of: interactions between G2A and HLA-E; interactions between VISTA and its binding partner; interactions between one or more Siglecs and their ligands; interactions between GARP and one or more of its ligands; interactions between CD47 and SIRPα; interactions between PVRIG and PVRL2; interactions between CSF1R and CSF1; interactions between BTLA and HVEM; interactions between parts of the adenosinergic pathway, e.g., A2AR and / or A2BR, and adenosine produced by CD39 and CD73; interactions 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 PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, KIR inhibitors, LAG-3 inhibitors, TIGIT inhibitors, VISTA inhibitors, and GARP inhibitors. In one embodiment, the checkpoint inhibitor may be a blocking antibody, such as a PD-1 blocking antibody, a CTLA4 blocking antibody, a PD-L1 blocking antibody, a PD-L2 blocking antibody, a TIM-3 blocking antibody, a KIR blocking antibody, a LAG-3 blocking antibody, a TIGIT blocking antibody, a VISTA blocking antibody, or a GARP blocking antibody. Examples of PD-1 blocking antibodies include pembrolizumab, nivolumab, cemiprimab, and spartalizumab.Examples of CTLA4 blocking antibodies include ipilimumab and tremelimumab. Examples of PD-L1 blocking antibodies include atezolizumab, durvalumab, and avelumab.

[0032] In one embodiment, an anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 43, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 44.

[0033] In one embodiment, an anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) SEQ ID NO: CDR-H1 containing amino acid sequence 45; (ii) SEQ ID NO: CDR-H2 containing amino acid sequence 46; and (iii) CDR-H3 containing amino acid sequence SEQ ID NO: 47 The light chain variable region includes, (i) SEQ ID NO: CDR-L1 containing 48 amino acid sequences; (ii) SEQ ID NO: CDR-L2 containing amino acid sequence 49; and (iii) SEQ ID NO: CDR-L3 containing amino acid sequence 50 Includes.

[0034] In one embodiment of the anti-PD-1 antibody 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.

[0035] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antigen receptors such as antibodies or B cell receptors (BCRs). Immunoglobulins are characterized by structural domains having a characteristic immunoglobulin (Ig) fold, i.e., immunoglobulin domains. This term encompasses membrane-bound immunoglobulins and soluble immunoglobulins. Membrane-bound immunoglobulins are also referred to as surface immunoglobulins or membrane immunoglobulins, and these are generally part of the BCR. Soluble immunoglobulins are generally referred to as antibodies.

[0036] The structure of immunoglobulins is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2 nd ed. Raven Press, N.Y. (1989)). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are composed of immunoglobulin domains or regions, such as V L or VL (variable light chain) domains / regions, C L or CL (constant light chain) domains / regions, V H or VH (variable heavy chain) domains / regions, and C H or CH (constant heavy chain) domains / regions of C H 1 (CH1), C H 2 (CH2), C H 3 (CH3), and C HIt is primarily composed of 4(CH4). The heavy chain constant region typically consists 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 mobile. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in the IgG molecule. Each light chain typically consists of a VL and a CL. The light chain constant region typically consists of a single domain, the CL. The VH and VL regions can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs) (or hypervariable regions whose sequences can be hypervariable and / or in the form of structurally defined loops). Each VH and VL typically consists of three CDRs and four FRs aligned from the amino terminus to the carboxyl 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 inconsistent with the 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); also see the internet address www.imgt.org). Unless otherwise specified or inconsistent with the context, references to amino acid positions within constant regions 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).

[0037] There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which correspond to different classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane immunoglobulins or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxyl terminus. Mammals have two types of light chains, namely lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within different isotypes of immunoglobulins, while the variable region is highly diverse and responsible for antigen recognition.

[0038] The terms “amino acid” and “amino acid residue” may be used interchangeably herein and should not be understood as restrictive. Amino acids are organic compounds containing an amine (-NH2) functional group and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the context of this disclosure, amino acids may be classified based on their structural and chemical characteristics. Accordingly, the classes of amino acids may be reflected in one or both of the following tables.

[0039] (Table 2) Main classifications based on the structure of the R group and general chemical characterization TIFF2026513865000025.tif49128

[0040] (Table 3) Another physical and functional classification of amino acid residues TIFF2026513865000026.tif83152

[0041] For the purposes of this disclosure, the term "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 variants, splice variants, post-translational modified variants, conformations, isoforms, allele variants, species variants, and species homologs, in particular those occurring in nature. The term "variant" also includes, in particular, fragments of amino acid sequences.

[0042] Amino acid insertion variants involve the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, but random insertions are also possible, accompanied by appropriate screening of the resulting product.

[0043] The amino acid addition variant comprises amino-terminated and / or carboxyl-terminated fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0044] Amino acid deletion variants are characterized by the removal of one or more amino acids from a sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be located at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of a protein are also called N-terminal truncated variants and / or C-terminal truncated variants.

[0045] An amino acid substitution variant is characterized by the removal of at least one residue in a sequence and the insertion of another residue in its place. The substitution of one amino acid for another may be classified as a conserved or non-conserved substitution. Preferably, the modification is located in a position in the amino acid sequence that is not conserved among homologous proteins or peptides, and / or the amino acid is replaced by another amino acid having similar properties. Preferably, amino acid changes in peptide and protein variants are conserved amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of the families of amino acids related in their side chains. In the context of this disclosure, “conserved substitution” means the substitution of one amino acid for another amino acid having similar structural and / or chemical characteristics, and such substitution of one amino acid residue for another amino acid residue of the same class is as defined in either of the two tables above: for example, leucine may be substituted with isoleucine because they are both aliphatic branched hydrophobic substances. Similarly, aspartic acid may be substituted with glutamic acid because they are both small negatively charged residues. Naturally occurring amino acids can also generally be divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and non-charged amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). 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.

[0046] In this specification, the terms “amino acid corresponding to position…” and similar expressions refer to amino acid position numbers in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Therefore, an amino acid or segment in a sequence that “corresponds” to an amino acid or segment in another sequence is one that, when aligned with other amino acids or segments using a standard sequence alignment program such as ALIGN, ClustalW, or an analogue, typically with default settings, has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning sequences or segments within a sequence, thereby determining the positions in a sequence corresponding to the amino acid positions described herein, are well known in the art.

[0047] In the context of this disclosure, the term “antibody” (Ab) preferably refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which has the ability to specifically bind to an antigen (in particular an epitope on an antigen) under typical physiological conditions for a half-life of a significant 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 12 hours, at least about 24 hours or more, at least about 48 hours or more, at least about 3, 4, 5, 6, 7 days or more, or any other relevant functionally defined period (e.g., enough time to induce, promote, enhance, and / or modulate a physiological response related to antibody binding to an antigen, and / or enough time for the antibody to recruit effector activity). In particular, the term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. 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 region and constant region are also referred to herein as the variable domain and constant domain, respectively. The VH and VL regions can be further subdivided into more conserved, hypervariable regions called complementarity-determining regions (CDRs), interspersed with regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs aligned from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are designated as HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), and the CDRs of VL are designated as LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3). The variable regions of the heavy and light chains contain binding domains that interact with the antigen.The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (aligned from the amino terminus to the carboxyl terminus in the following order: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins 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. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or they can be the immunoactive moieties of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0048] 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 regions that interact with antigens and include both VH and VL regions. Antibodies as used herein include not only monospecific antibodies but also multispecific antibodies that contain two or more, for example, three or more different antigen-binding regions.

[0049] As stated above, the term "antibody" as used herein includes, unless otherwise specified or clearly contradicts the context, an antigen-binding fragment, i.e., a fragment of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include: (i) a monovalent fragment consisting of Fab' or Fab fragments, VL, VH, CL, and CH1 domains, or a monovalent antibody as described in WO 2007 / 059782 (Genmab); (ii) a bivalent fragment containing an F(ab')2 fragment, two Fab fragments linked by disulfide crosslinks at the hinge region; (iii) an Fd fragment essentially consisting of a VH domain and a CH1 domain; (iv) an Fv fragment essentially consisting of a single arm of an antibody's VL and VH domains; (v) a domain antibody essentially consisting of a VH domain (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90) also known as the dAb fragment (Ward et al., Nature) 341 , 544-546 (1989)); (vi) Camelid molecule or Nanobody molecule (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24); and (vii) an isolated complementarity-determining region (CDR). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but may be linked by a synthetic linker that allows the VL and VH regions to be paired and made into a single protein chain forming a monovalent molecule using recombination (known as a single-chain antibody or single-chain Fv (scFv), for example, Bird et al., Science 242 , 423-426 (1988) and Huston et al., PNAS USA 85See 5879-5883 (1988). Such single-chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by the context. While such fragments are generally included within the meaning of antibody, they are a distinctive feature of this disclosure, exhibiting a variety of biological properties and utility responsibilities, both collectively and independently. These and other useful antibody fragments in the context of this disclosure, as well as the bispecific forms of such fragments, will be discussed further herein. The term antibody should also be understood, unless otherwise explicitly stated, to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that possess the ability to specifically bind to antigens provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombination techniques.

[0050] The antibodies produced may harbor any isotype. As used herein, the term “isotype” refers to an immunoglobulin class encoded by a heavy chain constant region gene (e.g., IgG (IgG1, IgG2, IgG3, IgG4, etc.), IgD, IgA (IgA1, IgA2, etc.), IgE, IgM, or IgY). When a particular isotype, e.g., IgG1, is referred to herein, this term is used to indicate that the antibody sequence is closer to that isotype, e.g., IgG1, than to other isotypes. Therefore, for example, an IgG1 antibody disclosed herein may be a sequence variant of a naturally occurring IgG1 antibody, including variations in the constant region.

[0051] IgG1 antibodies can exist in multiple polymorphic variants, referred to as allotypes (as outlined in Jefferis and Lefranc 2009, mAbs Vol 1 Issue 4 1-7), any of which is suitable for use in some embodiments of this specification. Common allotype variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any embodiment of this specification, the antibody may include a heavy chain Fc region containing a human IgG Fc region. In a further embodiment, the human IgG Fc region contains human IgG1.

[0052] In the context of this disclosure, the term “multispecific antibody” refers to an antibody having at least two distinct antigen-binding regions defined by different antibody sequences. In some embodiments, the distinct antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the distinct antigen-binding regions bind to different target antigens. In one embodiment, a multispecific antibody is a “bispecific antibody” or “bs”. A multispecific antibody, such as a bispecific antibody, may be in any form, including either the bispecific antibody form or the multispecific antibody form described later herein.

[0053] When used in the context of antibodies, the term "full length" indicates that the antibody contains all of the domains of a particular isotype that are naturally and commonly found for that isotype, rather than just fragments. For example, for an IgG1 antibody, this would include the VH, CH1, CH2, CH3, hinge, VL, and CL domains.

[0054] As used herein, the term “human antibody” is intended to include antibodies having variable regions and framework regions derived from human germline immunoglobulin sequences, as well as human immunoglobulin constant domains. Human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced in vitro by random mutagenesis or site-directed mutagenesis, or in vivo by somatic mutation). However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as mouse, have been transplanted onto human framework sequences.

[0055] As used herein, the term “chimeric antibody” refers to an antibody in which the variable region originates from a non-human species (e.g., a rodent) and the 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 for various types of modification of antibodies, and the processes for antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be produced by 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 scope of knowledge of those skilled in the art, and therefore, the production of chimeric antibodies may be carried out by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic application in humans are developed to reduce the expected antibody immunogenicity of non-human antibodies, such as rodent antibodies. These may typically contain non-human (e.g., mouse or rabbit) variable regions that are specific to the antigen of interest, as well as human constant antibody heavy and light chain domains. In the context of chimeric antibodies, the term "variable region" or "variable domain" refers to a region containing both the CDR and framework regions of the heavy and light chains of immunoglobulins, as will be discussed later.

[0056] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to have a high level of sequence homology to the human variable domain. This can be achieved by transplanting six non-human antibody complementarity-determining regions (CDRs) that together form an antigen-binding site onto a homologous human acceptor framework region (FR) (see WO 92 / 22653 and EP 0 629 240). To completely reconstitute the binding affinity and specificity of the parental antibody, substitution (reverse mutation) of framework residues derived from the parental antibody (i.e., the non-human antibody) into the human framework region may be required. Structural homology modeling can help identify amino acid residues in the framework region that are important for the antibody’s binding properties. Thus, a humanized antibody may contain a non-human CDR sequence, a primarily human framework region optionally containing one or more amino acid reverse mutations into the non-human amino acid sequence, and a fully human constant domain. Optionally, additional amino acid modifications, not necessarily reverse mutations, may be applied to obtain humanized antibodies with desirable characteristics such as affinity and biochemical properties.

[0057] As used herein, a protein "derived" from another protein, for example, a parent protein, means that one or more amino acid sequences of the protein are identical or similar to one or more amino acid sequences in the other protein or parent protein. For example, in an antibody, binding arm, antigen-binding region, or constant region derived from another or parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to those of the other 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, VH CDRs and VL CDRs, and / or one or more or all of the framework region, VH, VL, CL, hinge, or CH region. For example, a humanized antibody may be described herein as "derived" from a non-human parent antibody, meaning that at least the VL CDR sequence and VH CDR sequence are identical or similar to the VH CDR sequence and VL CDR sequence 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 contains a VH CDR and / or VL CDR, or VH and / or VL sequence, that is identical or similar to that 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 performed on the CDR, constant region, or other parts of the antibody, binding arm, antigen-binding region, etc., to introduce desired features.When used in the context of one or more sequences derived from a first protein or parent protein, the “similar” amino acid sequences preferably have sequence identity of at least about 50%, for example, 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%.

[0058] Non-human antibodies can be produced in a number of different species, such as mice, rabbits, chickens, guinea pigs, llamas, and goats.

[0059] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using antibody gene libraries, are also available and are well known to those skilled in the art.

[0060] The creation of hybridomas in such non-human species is a very well-established procedure. Immunization protocols and techniques for isolating immunized animal / non-human splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0061] As used herein, unless otherwise inconsistent with the context, the terms “Fab arm” or “arm” refer to a single heavy-light chain pair and are used interchangeably with “half-chain” herein.

[0062] The term "binding arm containing an antigen-binding region" refers to an antibody molecule or fragment containing an antigen-binding region. Therefore, a binding arm can, for example, include six VH CDR sequences and VL CDR sequences, VH sequences and VL sequences, a Fab fragment or Fab' fragment, or a Fab arm.

[0063] As used herein, unless inconsistent with the context, the term “Fc region” refers to an antibody region consisting of two Fc sequences in the heavy chain of an immunoglobulin, wherein the Fc sequences include at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, as used herein, the term “Fc region” refers to a region of an antibody, from the N-terminus to the C-terminus, that includes at least a hinge region, a CH2 domain, and a CH3 domain. The Fc region of an antibody can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0064] In the context of this disclosure, the term "induces less Fc-mediated effector function" as used in relation to antibodies including multispecific antibodies means that the antibody induces less Fc-mediated effector function, particularly such function selected from the list of IgG Fc receptor (Fc-gamma-R, FcγR) binding, C1q binding, ADCC, or CDC, compared to (i) the antibody and a human IgG1 antibody containing the same CDR sequence, particularly including the same first and second antigen-binding regions, and (ii) two heavy chains containing the human IgG1 hinge, CH2, and CH3 regions.

[0065] Fc-mediated effector function can be measured by binding to FcγR, binding to C1q, or induction of Fc-mediated crosslinking via FcγR.

[0066] As used herein, the term “hinge region” refers to the hinge region of an immunoglobulin heavy chain. Therefore, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216–230 according to the EU numbering as shown in Kabat (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition—US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991)). However, the hinge region may also be any of the other subtypes described herein.

[0067] As used herein, the terms “CH1 region” or “CH1 domain” refer to the CH1 region of an immunoglobulin heavy chain. For example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118–215 according to the EU numbering as shown in Kabat (ibid.). However, the CH1 region may also be any of the other subtypes described herein.

[0068] As used herein, the terms “CH2 region” or “CH2 domain” refer to the CH2 region of an immunoglobulin heavy chain. For example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231–340 according to the EU numbering as shown in Kabat (ibid.). However, the CH2 region may also be any of the other subtypes described herein.

[0069] As used herein, the terms “CH3 region” or “CH3 domain” refer to the CH3 region of an immunoglobulin heavy chain. Therefore, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341–447 according to the EU numbering as shown in Kabat (ibid.). However, the CH3 region may also be any of the other subtypes described herein.

[0070] In the context of this disclosure, the term "monovalent antibody" means that an antibody molecule can bind to a single antigen molecule and therefore cannot crosslink the antigen.

[0071] A "CD137 antibody" or "anti-CD137 antibody" is an antibody that specifically binds to the antigen CD137, as described above.

[0072] A "CD137×PD-L1 antibody" or "anti-CD137×PD-L1 antibody" is a bispecific antibody that contains two different antigen-binding regions, one of which specifically binds to the antigen CD137, and the other specifically binds to the antigen PD-L1.

[0073] As used herein, the term “biosimilar” (for example, of an approved reference product / biological drug) means a biological product that is similar to a reference product based on data from one or more clinical studies (including evaluations of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate the safety, purity and efficacy of the biological product under one or more suitable conditions of use under which the reference product is approved and intended for use, and for which approval is sought (e.g., no clinically significant difference between the biological product and the reference product in terms of safety, purity and efficacy of the products). In some embodiments, the biosimilar biological product and the reference product utilize the same one or more mechanisms of action for one or more conditions of use prescribed, recommended or suggested in the proposed label, but to the extent that the one or more mechanisms of action are publicly known for the reference product. In some embodiments, one or more conditions of use prescribed, recommended, or suggested in the proposed label for the biological product are previously approved for the control product. In some embodiments, the route of administration, dosage form, and / or potency of the biological product are the same as those of the control product. The biosimilar may be, for example, a currently known antibody having the same primary amino acid sequence as a commercially available antibody, but may be produced in a different cell type or by a different production, purification, or formulation method.

[0074] As used herein, the terms “to bind” or “to be able to bind” in the context of the binding of an antibody to a given antigen or epitope typically mean approximately 10% when determined using biolayer interferometry (BLI), or, for example, when determined using surface plasmon resonance (SPR) technique in a BIAcore 3000 instrument with the antigen as a ligand and the antibody as an 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 smaller, K D This is binding at an affinity corresponding to the antigen. Antibodies bind to a given antigen, and to nonspecific antigens other than the given antigen or closely related antigens (e.g., BSA, casein) with K D K is at least 10 times lower than, 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 It binds with an affinity corresponding to the antibody's K. D It depends on the K of the antibody, and therefore the K D When the affinity is very low (i.e., the antibody is highly specific), the degree to which the affinity for the antigen is lower than the affinity for a nonspecific antigen can be at least 10,000 times.

[0075] The term "k" as used herein d (sec -1 The term refers to the dissociation rate constant of a particular antibody-antigen interaction. The aforementioned value is also known as k off Also called a value.

[0076] In this specification, "K D The term (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0077] Two antibodies have "same specificity" if they bind to the same antigen and the same epitope. Whether an antibody being tested recognizes the same epitope as a particular antigen-binding antibody, that is, whether the antibody binds to the same epitope, can be tested by various methods well known to those skilled in the art.

[0078] Antibody competition can be detected by cross-blocking assays. For example, a competitive ELISA assay can be used as a cross-blocking assay. For instance, the target antigen may be coated onto the wells of a microtiter plate, and antigen-binding antibodies and candidate competitive test antibodies may be added. The amount of antigen-binding antibody bound to the antigen in the wells is indirectly correlated with the binding ability of the competing candidate competitive test antibody that competes with it for binding to the same epitope. Specifically, the higher the affinity of the candidate competitive test antibody for the same epitope, the less antigen-binding antibody will be bound to the antigen-coated wells. The amount of antigen-binding antibody bound to the wells can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0079] With respect to binding to an antigen, an antibody that competes with another antibody, for example, an antibody containing heavy chain variable regions and light chain variable regions as described herein, or an antibody that is specific to the antigen of another antibody, for example, an antibody containing heavy chain variable regions and light chain variable regions as described herein, may be an antibody containing variants of said heavy chain variable regions and / or light chain variable regions, for example, modifications and / or some degree of identity in the CDR as described herein.

[0080] As used herein, “isolated multispecific antibody” is intended to refer to a multispecific antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated bispecific antibody that specifically binds to CD137 and PD-L1 substantially does not contain a monospecific antibody that specifically binds to CD137 or PD-L1).

[0081] As used herein, the term "monoclonal antibody" refers to a preparation of an antibody molecule with a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0082] As used herein, the term "heterodimer interaction between the first CH3 region and the second CH3 region" refers to the interaction between the first CH3 region and the second CH3 region in a first CH3 / second CH3 heterodimer antibody.

[0083] As used herein, the term "homodimer interaction between the first CH3 region and the second CH3 region" refers to the interaction between the first CH3 region and another first CH3 region in a first CH3 / first CH3 homodimer antibody, and the interaction between the second CH3 region and another second CH3 region in a second CH3 / second CH3 homodimer antibody.

[0084] As used herein, the term "homodimerated antibody" refers to an antibody comprising two first Fab arms or halves, wherein the amino acid sequences of the Fab arms or halves are identical.

[0085] As used herein, the term "heterodimerated antibody" refers to an antibody comprising a first Fab arm or half and a second Fab arm or half, wherein the amino acid sequences of the first Fab arm or half and the second Fab arm or half are different. In particular, the CH3 region or antigen-binding region, or the CH3 region and antigen-binding region of the first Fab arm / half and the second Fab arm / half are different.

[0086] The term "reducing conditions" or "reducing environment" refers to conditions or environments in which substrates, such as cysteine ​​residues in the hinge region of an antibody, are more likely to be reduced than oxidized.

[0087] This disclosure also describes multispecific antibodies, such as bispecific antibodies, which include functional variants of the VL, VH, or one or more CDR regions of an example bispecific antibody. Functional variants of VL, VH, or CDR used in the context of bispecific antibodies still allow each antigen-binding region of the bispecific antibody to 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.

[0088] Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The percentage of 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 percentage of 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), which is incorporated in the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0089] In the context of this disclosure, unless otherwise indicated, the following notation is used to describe mutations: (i) an amino acid substitution at a given position is described, for example, K409R, which means a substitution of lysine with arginine at position 409 of the protein; and (ii) for a particular variant, a specific three-letter code or one-letter code is used, including the codes Xaa and X, which indicate any amino acid residue. Thus, a substitution of lysine with arginine at position 409 is designated K409R, and a substitution of any amino acid residue of lysine at position 409 is designated K409X. A deletion of lysine at position 409 is indicated by K409*.

[0090] Exemplary variants include those that differ from the parent sequence's VH and / or VL and / or CDR primarily through conservative substitutions; for example, 12 substitutions in the variant, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, are substitutions of conservative amino acid residues.

[0091] In the context of this disclosure, a conservative substitution may be defined as a substitution within a class of amino acids, as defined in Tables 2 and 3.

[0092] As used herein, the term “CD137” refers to CD137(4-1BB), also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the 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, CD137, T cell antigen 4-1BB homolog, and T cell antigen ILA. In one embodiment, CD137(4-1BB) is human CD137(4-1BB) having UniProt accession number Q07011. The sequence of human CD137 is also shown as SEQ ID NO: 37. Amino acids 1-23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; amino acids 24-186 of SEQ ID NO: 37 correspond to the extracellular domain of human CD137; and the remainder of the protein, derived from amino acids 187-213 and 214-255 of SEQ ID NO: 37, are the transmembrane domain and cytoplasmic domain, respectively.

[0093] The “programmed death-1 (PD-1)” receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 (also known as CD279 or SLEB2) is primarily 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 having at least one epitope in common with hPD-1, in particular proteins having the amino acid sequence as shown in sequence listing SEQ ID NO: 113 (NCBI reference sequence: NP_005009.2), or preferably proteins encoded by the nucleic acid sequence as shown in sequence listing 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 being PD-L2) that downregulate T cell activation and cytokine secretion upon binding to PD-1.

[0094] As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, e.g., PD-L1 from macaques (cynomolgus macaques), African elephants, wild boars, and mice (see, for example, Genbank accession numbers NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), as well as analogs having at least one epitope in common with hPD-L1. The sequences of human PD-L1 are also shown in SEQ ID NO: 40 (mature sequence) and SEQ ID NO: 39, where amino acids 1-18 are predicted to be the signal peptide. As used herein, the term “PD-L2” includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, as well as analogs having at least one epitope common to hPD-L2. The ligands of PD-1 (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 can switch off T cells expressing PD-1, which results in suppression of the anti-cancer immune response. Interactions between PD-1 and its ligands result in a reduction of tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancer cells. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked.

[0095] As used herein, the term “dysfunctional” refers to immune cells in a state of reduced immune responsiveness to antigen stimulation. Dysfunctionality includes non-responsiveness to antigen recognition, as well as impaired ability to convert antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0096] As used herein, the term “anergy” refers to a state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signaling delivered through the T cell receptor (TCR). T cell anergy can also occur upon stimulation with an antigen in the absence of co-stimulation, resulting in the cell becoming unresponsive to subsequent activation by the antigen, even in the presence of co-stimulation. The unresponsive state can often be reversed by the presence of IL-2. Anergistic T cells do not undergo clonal expansion and / or acquire effector function.

[0097] As used herein, the term “exhaustion” refers to T-cell exhaustion as a state of T-cell dysfunction resulting from persistent TCR signaling, such as that occurring during many chronic infections and cancers. It is distinguished from anergy in that it results from persistent signaling, rather than through incomplete or deficient signaling. Exhaustion is defined by effector dysfunction, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector T cells or memory T cells. Exhaustion disrupts optimal control of disease (e.g., infections and tumors). Exhaustion can result from both exogenous negative regulatory pathways (e.g., immunomodulatory cytokines) and intracellular negative regulatory pathways (such as the suppressive immune checkpoint pathway described herein).

[0098] "Enhancing T cell function" means inducing, eliciting, or stimulating T cells to have sustained or amplified biological function, or renewing 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, or 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.

[0099] As used herein, the terms “inhibitory nucleic acid” or “inhibitory nucleic acid molecule” refer to nucleic acid molecules, such as DNA or RNA, that reduce, inhibit, interfere with, or negatively modulate one or more PD-1 proteins, either entirely or partially. 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).

[0100] As used herein, the term “oligonucleotide” refers to a nucleic acid molecule that can reduce protein expression, particularly the expression of PD-1 proteins such as the PD-1 protein described herein. Oligonucleotides are typically short DNA or RNA molecules containing 2 to 50 nucleotides. Oligonucleotides can be single-stranded or double-stranded. PD-1 inhibitor oligonucleotides can be antisense oligonucleotides.

[0101] Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, particularly to the nucleic acid sequence (or fragment thereof) of the PD-1 protein. Antisense RNA is typically used to inhibit protein translation of mRNA, for example, mRNA encoding the PD-1 protein, by binding to said mRNA. Antisense DNA is typically used to target specific and complementary (coding or non-coding) RNA. Once binding occurs, 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 blocks 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.

[0102] The terms “siRNA,” “small interfering RNA,” or “small inhibitory RNA” are used interchangeably herein and refer to double-stranded RNA molecules of a typical length of 20–25 base pairs that have complementary nucleotide sequences and interfere with the expression of certain genes, such as the gene encoding the PD-1 protein. In one embodiment, siRNA interferes with mRNA and therefore blocks translation, for example, the translation of the PD-1 protein. Transfection with exogenous siRNA can be used for gene knockdown, but its effect may be transient, especially in rapidly dividing cells. Stable transfection can be achieved, for example, by RNA modification or by using expression vectors. Useful modifications and vectors for stable transfection of siRNA into cells are known in the art. siRNA sequences can also be modified to produce “small hairpin RNA” or “shRNA” by introducing a short loop between the two strands. shRNA can be processed into functional siRNA by Dicer. shRNA has a relatively low degradation and turnover rate. Therefore, the PD-1 inhibitor may also be shRNA.

[0103] As used herein, the term “aptamer” refers to a single-stranded nucleic acid molecule, such as DNA or RNA, typically 25 to 70 nucleotides in length, that can bind to a target molecule, such as a polypeptide. In one embodiment, an aptamer binds to an immune PD-1 protein, such as the PD-1 checkpoint protein described herein. For example, the aptamers according to this 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 production and therapeutic use of aptamers are well known in the art (see, for example, U.S. Patent No. 5,475,096).

[0104] The terms “small molecule inhibitor” or “small molecule” are used interchangeably herein and refer to small molecular weight organic compounds, typically up to 1000 daltons, that reduce, inhibit, interfere with, or negatively modulate one or more PD-1 proteins as described above, either whole or partially. Such small molecule inhibitors are usually synthesized by organic chemistry, but may also be isolated from natural sources such as plants, fungi, and microorganisms. The low molecular weight allows small molecule inhibitors to diffuse rapidly across cell membranes. For example, various A2AR antagonists known in the art are organic compounds with molecular weights below 500 daltons.

[0105] The term "cell-based therapy" refers to the transplantation of cells expressing an immune PD-1 inhibitor (e.g., T lymphocytes, dendritic cells, or stem cells) into a subject for the purpose of treating a disease or disorder (e.g., cancer).

[0106] As used herein, the term “oncolytic virus” refers to a virus that, either in vitro or in vivo, selectively replicates in cancerous or hyperproliferating cells, slowing their growth or inducing their death, while having little or no effect on normal cells. An oncolytic virus for PD-1 inhibitor delivery comprises an expression cassette that may encode a PD-1 inhibitor, which is an inhibitory nucleic acid molecule such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or soluble PD-1 protein or fusion. The oncolytic virus is preferably replicable, and the expression cassette is under the control of a viral promoter, e.g., a synthetic early / late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picornavirus, e.g., Seneca Valley virus; SVV-001), coxsackievirus, parvovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza virus), measles virus, reovirus, cinbisvirus, vacciniaviruses (including Copenhagen, Western Reserve, and Wyeth strains) as exemplified 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 generation of recombinant oncolytic viruses containing soluble forms of PD-1 inhibitors and methods for their use are disclosed in WO 2018 / 022831, which is incorporated herein by reference in its entirety. The oncolytic viruses can be used as attenuated viruses.

[0107] In this specification, a “treatment cycle” is defined as the period within the additional effect of separate doses of a conjugate according to the pharmacodynamics of the conjugate, or, in other words, the period after the administered conjugate has been essentially removed from the subject’s body. Multiple small doses within a small time frame, for example, just 2 to 24 hours, for example, within 2 to 12 hours, or on the same day, may be equivalent to a larger single dose.

[0108] In this context, the terms “treatment,” “treatment,” or “therapeutic intervention” relate to the management and care of an object for the purpose of combating a condition such as a disease or disorder. The terms are intended to include all range of treatments for a given condition in which an object is afflicted, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, to slow the progression of a disease, disorder, or condition, to alleviate or reduce symptoms and complications, and / or to prevent a disease, disorder, or condition, where prevention should be understood as the management and care of the individual for the purpose of combating a disease, condition, or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications. In one embodiment, “treatment” refers to the administration of an effective amount of a therapeutically active conjugate of the present disclosure, such as a therapeutically active antibody, for the purpose of alleviating, improving, stopping, or eradicating (curing) a symptom or disease condition.

[0109] The response to treatment with the binders disclosed herein, as well as resistance to treatment, inability to respond to treatment, and / or recurrence from treatment, may be determined according to the Response Evaluation Criteria in Solid Tumors; version 1.1 (RECIST Criteria v1.1). The RECIST Criteria are shown in the table below (LD: longest dimension).

[0110] (Table 4) Definition of response (RECIST Criteria v1.1) TIFF2026513865000027.tif129161

[0111] "Best overall response" is the best response recorded from the start of treatment until disease progression / recurrence (for PD, the minimum measured value recorded after 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 be under disease control. Subjects with NE are counted as non-responders. Best overall response is the best response recorded from the start of treatment until disease progression / recurrence (for PD, the minimum measured value recorded after the start of treatment is used as the criterion). Subjects with CR, PR, or SD are considered to be under disease control. Subjects with NE are counted as non-responders.

[0112] "Duration of response (DOR)" is applicable only to subjects for whom the confirmed best overall response is CR or PR, and is defined as the time from when the objective tumor response (CR or PR) is first recorded until the date of the first PD or death due to the causative cancer.

[0113] "Progression-free survival (PFS)" is defined as the number of days from day 1 of cycle 1 until the first progression is observed or death from any cause.

[0114] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 until death from any cause. If it is unknown whether the subject has died, OS is truncated at the last day (cut-off date or earlier) on which the subject was known to be alive.

[0115] In the context of this disclosure, the term "treatment regimen" refers to a systematic treatment plan designed to improve and maintain health.

[0116] The term "effective dose" or "therapeutic effective dose" refers to the amount effective in achieving the desired therapeutic outcome at the required dosage and time. The therapeutic effective dose of a binder, such as a multispecific or monoclonal antibody, may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the binder's ability to elicit the desired response in the individual. The therapeutic effective dose is also the amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the binder or its fragments. If the response in a patient is insufficient with the initial dose, a higher dose (or an effective higher dose achieved by a different, more localized route of administration) may be used. If an undesirable side effect occurs in a patient at a certain dose, a lower dose (or an effective lower dose achieved by a different, more localized route of administration) may be used.

[0117] As used herein, the term “cancer” includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. “Cancer cell” means an abnormal cell that grows by rapid, uncontrolled proliferation and continues to grow after the stimulus that initiated new growth has ceased.

[0118] The term “cancer” as used in this disclosure also includes cancer metastasis. “Metastasis” means the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and depends on the detachment of malignant cells from the primary tumor, the invasion of the extracellular matrix, the penetration of the endothelial basement membrane to enter the body cavity and blood vessels, and then the invasion of the target organ after being transported by the blood. Finally, the growth of a new tumor, i.e., a secondary or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, because tumor cells or components may remain and give rise to metastatic potential. In one aspect, the term “metastasis” as used in this disclosure refers to “distant metastasis,” which refers to metastasis that is far away from the primary tumor and the regional lymph node system.

[0119] As used herein, terms such as “reduce,” “inhibit,” “interfere,” and “negatively modulate” mean the ability to cause an overall reduction of, for example, a level of 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 include complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0120] Terms such as “increase” or “strengthen” relate, in one aspect, to an increase or strengthening 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%.

[0121] As used herein, "physiological pH" refers to a pH of 7.5 or approximately 7.5.

[0122] As used in this disclosure, "weight percent" means weight percent, which is a unit of concentration that measures the amount of a substance in grams (g), expressed as a percentage of the total weight of the total composition in grams (g).

[0123] The term "freezing" usually refers to the solidification of a liquid by the removal of heat.

[0124] The terms "freeze-drying" or "freeze-drying" refer to the freeze-drying of a substance by freezing the substance and then reducing the ambient pressure (for example, below 15 Pa, below 10 Pa, below 5 Pa, or below 1 Pa) to directly sublimate the freezing medium in the substance from the solid phase to the gas phase. Therefore, the terms "freeze-drying" and "freeze-drying" are used interchangeably herein.

[0125] In the context of this disclosure, the term “recombinant” means “created through genetic engineering.” In one embodiment, “recombinant object” in the context of this disclosure does not exist in nature.

[0126] As used herein, the term “naturally occurring” refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in living organisms (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is considered naturally occurring. The term “naturally occurring” means “naturally occurring” and includes not only known objects but also objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from a natural source in the future.

[0127] According to this disclosure, the term “peptide” includes oligopeptides and polypeptides and refers to substances containing about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about 50, about 100, or about 150 consecutive amino acids linked to each other via peptide bonds. The term “protein” refers to a large peptide, in particular a peptide having at least about 151 amino acids, but the terms “peptide” and “protein” are generally used as synonyms herein.

[0128] A “therapeutic protein” has a positive or beneficial effect on a subject’s condition or disease state when delivered to the subject in a therapeutically effective amount. In one embodiment, a therapeutic protein may have a curative or palliative nature and may be administered to improve, reduce, alleviate, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have a prophylactic nature and may be used to delay the onset of a disease or to reduce the severity of such a disease or pathological condition. The term “therapeutic protein” may include the entire protein or peptide and may also refer to a therapeutically active fragment thereof. It may also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, immunostimulants such as antigens and cytokines for vaccination.

[0129] The term “part” refers to a single stroke. With respect to an amino acid sequence or a particular structure such as a protein, the term “part” may specify a continuous or discontinuous stroke of that structure.

[0130] The terms “part” and “fragment” are used interchangeably herein and refer to a contiguous element. For example, a part of an amino acid sequence or a part of a structure such as a protein refers to a contiguous element of that structure. When used in the context of a composition, the term “part” means a portion of the composition. For example, a part of a composition may be any portion derived from 0.1% to 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0131] A “fragment” of an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence corresponding to an amino acid sequence shortened at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translation of a shortened open reading frame that lacks the 3' end of the open reading frame. A N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translation of a shortened open reading frame that lacks the 5' end of the open reading frame, insofar as the shortened open reading frame contains a start codon that serves to initiate translation. An amino acid sequence fragment 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. Preferably, an amino acid sequence fragment contains at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0132] According to this disclosure, a part or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the fragment is derived. A part or fragment of a peptide or protein preferably contains at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acid sequences of the peptide or protein. A part or fragment of a peptide or protein preferably contains up to 8, in particular at most at least 10, at least 12, at least 15, at least 20, at least 30, or at least 55 consecutive amino acid sequences of the peptide or protein.

[0133] As used herein, "variant" means an amino acid sequence that is different from the parental amino acid sequence due to at least one amino acid modification. The parental amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence, or a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parental amino acid sequence, for example, from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or 1 to about 5 amino acid modifications compared to the parent.

[0134] As used herein, "wild-type" or "WT" or "native" means 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.

[0135] 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 to 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 total length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, for consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably with the best sequence alignment, for example, using Align with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0136] "Sequence similarity" refers to the percentage of amino acids that are either identical or correspond to a conserved amino acid substitution. "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.

[0137] The terms “% identical” and “identity%” or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may, but do not necessarily, be randomly distributed across the entire length of the sequences being compared. Comparison of two sequences is typically performed by comparing the sequences with respect to a segment or “window of comparison” after optimal alignment to identify local regions of the corresponding sequences. Optimal alignment for comparison can be performed manually, or with the help of local homology algorithms by Smith and Waterman, 1981, Ads App. Math. 2, 482, Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the help 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 percentage of identity between two sequences is determined using the BLASTN or BLASTP algorithm, which is available on the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi).In some embodiments, the algorithm parameters used in the BLASTN algorithm on the NCBI website include (i) an expected threshold set to 10; (ii) a word size set to 28; (iii) the maximum match in the query range set to 0; (iv) a match / mismatch score set to 1 or -2; (v) a gap cost set linearly; and (vi) the use of a filter for the low complexity region. In some embodiments, the algorithm parameters used in the BLASTP algorithm on the NCBI website include (i) an expected threshold set to 10; (ii) a word size set to 3; (iii) the maximum match in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to Existence: 11 and Extension: 1; and (vi) a conditional compositional score matrix adjustment.

[0138] The identity percentage is obtained by determining the number of corresponding 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 the result by 100.

[0139] In some embodiments, the degree of similarity or identity is given for 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 total 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 for 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, and in some embodiments, for consecutive amino acid residues. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0140] Homologous amino acid sequences, according to this disclosure, exhibit identity of at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98%, or at least 99% of amino acid residues.

[0141] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. Manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions, or deletions is described in detail, for example, Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared with the help of known peptide synthesis techniques, such as by solid-phase synthesis and similar methods.

[0142] 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 or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities presented by the amino acid sequence from which the fragment or variant is derived. As used herein, the term “functional fragment” or “functional variant” refers in particular to a variant molecule or sequence that includes an amino acid sequence in which one or more amino acids are modified compared to the amino acid sequence of the parent molecule or sequence, and which still performs one or more of the functions of the parent molecule or sequence, for example, which can induce an immune response. In one embodiment, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of a functional fragment or functional variant may be reduced but still significantly present; for example, the immunogenicity of a 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 a functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0143] The amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the original 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 that particular sequence or fragment. An amino acid sequence derived from a particular amino acid sequence may be a variant of that particular sequence or fragment. For example, it will be understood by those skilled in the art that antigens suitable for use herein can be modified to have a sequence different from the naturally occurring sequence from which they are derived or from the native sequence, while retaining the desired activity of the native sequence.

[0144] "Isolated" means modified or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from its natural coexisting substances is "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-native environment, such as a host cell. In a preferred embodiment, the binders used in this disclosure are in a substantially purified form.

[0145] The term “genetic modification” or simply “modification” includes the transfection of cells with nucleic acids. The term “transfection” relates to the introduction of nucleic acids, particularly RNA, into cells. For the 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 cells may be present in a subject, e.g., a patient. Accordingly, according to this disclosure, cells for the transfection of nucleic acids described herein may be present in vitro or in vivo, e.g., cells may form organs, tissues, and / or parts of a patient’s body. According to this disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is expressed only transiently. RNA can be transfected into cells to transiently express the protein it encodes. Since nucleic acids introduced in the transfection process are not typically integrated into the nuclear genome, foreign nucleic acids are diluted or degraded through mitosis. Cells that enable episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desirable that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, then stable transfection must occur. Such stable transfection can be achieved by using virus-based or transposon-based systems for transfection. Generally, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells to transiently express the protein it encodes.

[0146] According to this disclosure, an analogue of a peptide or protein is a modified form of the peptide or protein from which it is derived and possesses at least one functional property of the peptide or protein. For example, a pharmacologically active analogue of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the analogue is derived. Such modifications include any chemical modifications and include single or multiple substitutions, deletions, and / or additions of any molecule related to the protein or peptide, e.g., carbohydrates, lipids, and / or proteins or peptides. In one embodiment, “analogues” of proteins or peptides include their 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 “analogue” also extends to all functional chemical equivalents of the proteins and peptides.

[0147] As used herein, “activation” or “stimulation” refers to a state in which immune effector cells, such as T cells, are sufficiently stimulated to induce detectable cell proliferation. Activation may also relate to the initiation of signaling pathways, induced cytokine production, and detectable effector function. The term “activated immune effector cells” refers, in particular, to immune effector cells undergoing cell division.

[0148] The term "priming" refers to the process by which immune effector cells, such as T cells, come into contact with their specific antigen for the first time, triggering differentiation into effector cells, such as effector T cells.

[0149] The terms “clonal expansion” or “expansion” refer to the process by which a particular entity is doubled. In the context of this disclosure, the terms are preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, expand, and specific immune effector cells that recognize the antigen are amplified. Preferably, clonal expansion results in the differentiation of immune effector cells.

[0150] As used in this disclosure, “antigen” encompasses any substance that elicits an immune response, and / or any substance to 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 an antigenic peptide and an immune response or immune mechanism is directed to one or more antigenic peptides, particularly when presented in relation to MHC molecules. In particular, “antigen” relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or a T lymphocyte (T cell). As used in this disclosure, the term “antigen” includes any molecule containing at least one epitope, such as a T cell epitope. Preferably, in the context of this disclosure, an antigen is an immune-inducing molecule that is preferably specific to an antigen (including a cell expressing the antigen) after processing. In one embodiment, an antigen is a disease-associated antigen, e.g., a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0151] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, i.e., a part or fragment of a molecule that is recognized by the immune system, particularly when presented in relation to MHC molecules, for example, by antibodies, T cells, or B cells. In one embodiment, "epitope" means a protein determinant that can specifically bind to an antibody. Epitopes typically consist of surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural properties and specific charge properties. Constructive epitopes and non-constructive epitopes are distinguished in that, in the presence of a denaturing solvent, binding to the former is lost, but binding to the latter is not. Epitopes may include amino acid residues directly involved in binding, and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked or covered by peptides that specifically bind to the antigen (in other words, the amino acid residues are within the footprint of the peptides that specifically bind to the antigen).

[0152] A protein epitope preferably comprises a continuous or discontinuous portion of the protein, preferably having an amino acid length of 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. For example, an epitope may preferably have an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In the context of this disclosure, an epitope is particularly preferably a T cell epitope.

[0153] As used herein, the terms “optional” or “optional” mean that any event, situation, or condition described thereafter may or may not occur, and that such description includes both cases in which such event, situation, or condition occurs and cases in which it does not occur.

[0154] As used herein, the terms “linked,” “fused,” and “fused” are interchangeable. These terms refer to the joining of two or more elements, components, or domains.

[0155] The term “disease” (also referred to herein as “disorder”) refers to an abnormal condition affecting the body of an individual. Disease is often interpreted as a medical condition accompanied by specific symptoms and signs. Disease can be caused by factors of an original external source, such as infectious diseases, or by internal dysfunction, such as autoimmune diseases. In humans, “disease” is often used more broadly to refer to any condition that causes pain, impairment, suffering, social problems, or death in the affected individual, or similar problems in those in contact with that individual. In this broader sense, it sometimes includes injuries, physical disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, although in other contexts and for other purposes these may be considered distinct categories. Disease usually affects an individual not only physically but also emotionally, as suffering from and living with many diseases can alter their outlook on life and their personality.

[0156] The term “therapeutic treatment” refers to any treatment that improves the health of an individual and / or extends (increases) the lifespan of the individual. Such treatment may eliminate a disease in an individual, stop or delay the onset of a disease in an individual, suppress or delay the onset of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in individuals that currently have or have previously had a disease.

[0157] The terms “preventive measures” or “preventive measures” refer to any measures intended to prevent disease from occurring in an individual. The terms “preventive measures” or “preventive measures” are used interchangeably herein. Similarly, the term “methods for preventing” in the context of disease progression, e.g., the progression of a tumor or cancer, refers to any method intended to prevent disease progression in an individual.

[0158] The terms “individual” and “subject” are used interchangeably herein. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate) or any other non-mammal, including birds (chickens), fish, or any other animal species, that is susceptible to or prone to a disease or disorder (e.g., cancer). Unless otherwise specified, the terms “individual” and “subject” do not imply a specific age and therefore include adults, elderly, children, and newborns. In aspects of this disclosure, “individual” or “subject” is “patient.”

[0159] The term "patient" means an individual or subject for treatment, in particular an individual or subject suffering from a disease.

[0160] The term "microsatellite instability (MSI)" refers to a form of genomic instability associated with DNA mismatch repair (MMR) deficiency in tumors. See Boland et al., Cancer Research 58, 5258-5257, 1998. Based on the degree of instability, MSI can be classified as high-frequency microsatellite instability (MSI-H), low-frequency microsatellite instability (MSI-L), and microsatellite stability (MSS). In one embodiment, MSI analysis can be performed using the following five National Cancer Institute (NCI) recommended microsatellite markers: BAT25 (GenBank accession number 9834508), BAT26 (GenBank accession number 9834505), D5S346 (GenBank accession number 181171), D2S123 (GenBank accession number 187953), and D17S250 (GenBank accession number 177030). In another embodiment, MSI analysis can be performed using a marker panel of five poly(A) mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27). High-frequency microsatellite instability (MSI-H) can be determined when two or more of the five NCI markers listed above are unstable, or when 30% or more of all markers in another marker panel are unstable (i.e., have insertion / deletion mutations); low-frequency microsatellite instability (MSI-L) can be determined when one of the five NCI markers listed above is unstable, or when less than 30% of all markers in another marker panel are unstable (i.e., have insertion / deletion mutations); and microsatellite stability (MSS) can be determined when none of the five NCI markers listed above or any other marker panel are unstable (i.e., do not have insertion / deletion mutations). Commercially available tests for determining MMR status include, but are not limited to, the VENTANA MMR RxDx Panel.A proficient mismatch repair (pMMR) state refers to the normal expression of MMR proteins (MLH1, PMS2, MSH2, and MSH6) in tumor specimens determined by IHC, while a deficiency in mismatch repair (dMMR) state refers to a state of low or absent expression of one or more MMR proteins (MLH1, PMS2, MSH2, and MSH6) in tumor specimens determined by IHC, such as loss of nuclear expression. The MSI-H state is generally consistent with the dMMR state, while the MSI-L and MSS states are generally consistent with the pMMR state. Techniques for determining the MSI or MMR state (MSI-H, MSS, pMMR, dMMR, etc.) in tumors are within the knowledge of those skilled in the art and are not limited to the embodiments described herein. Examples of such techniques, including but not limited to those described in Gilson et al., Cancers (Basel), 2021 Mar 24;13(7):1491, are well known in the art. Commercially available tests for MSI or MMR analysis include, but are not limited to, the Promega® MSI multiplex PCR assay, FoundationOne® CDx (F1CDx), Guardant360® CDx, Idylla® MSI test, and the VENTANA MMR RxDx Panel. In some embodiments, the VENTANA MMR RxDx Panel can be used to determine the MSI or MMR status. In certain embodiments, the MSI or MMR status is determined by the results of mismatch repair (MMR) / microsatellite instability (MSI) tests using immunohistochemistry (IHC), polymerase chain reaction (PCR), or next-generation sequencing (NGS), performed in tests approved by the U.S. Food and / or Drug Administration (FDA) / Conformité Europeenne (CE) marked.Many types of cancer or tumors, including colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, pancreatic cancer, urinary tract cancer, bladder cancer, thyroid cancer, breast cancer, prostate cancer, central nervous system (CNS) cancer, and skin cancers such as melanoma, have been found to be associated with microsatellite instability (Han et al, Front Genet. 2022 Dec 1;13:933475).

[0161] Aspects and aspects of this disclosure In the first aspect, the present disclosure provides a method for treating a tumor or cancer in a subject, the method comprising administering to the subject a conjugate comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

[0162] A binder that binds to CD137 and PD-L1. In one embodiment, CD137 is human CD137, in particular human CD137 containing the sequence shown in SEQ ID NO: 38. In one embodiment, PD-L1 is human PD-L1, in particular human PD-L1 containing the sequence shown in SEQ ID NO: 40. In one embodiment, CD137 is human CD137 and PD-L1 is human PD-L1. In one embodiment, CD137 is human CD137 containing the sequence shown in SEQ ID NO: 38 and PD-L1 is human PD-L1 containing the sequence shown in SEQ ID NO: 40.

[0163] In one embodiment of the binder according to the first aspect, (a) The first binding region for human CD137 comprises a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1 or 9, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 5 or 10; and (b) The second antigen-binding region for binding to human PD-L1 includes a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 11, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 15.

[0164] In one embodiment of the binder according to the first aspect, (a) The first binding region for human CD137 includes a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 2, 3, and 4, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, 7, and 8, respectively; and (b) The second antigen-binding region for binding to human PD-L1 includes a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 16, 17, and 18, respectively.

[0165] In one embodiment of the binder according to the first aspect, the first binding region that binds to human CD137 includes a heavy chain variable region (VH) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 1 or 9, and a light chain variable region (VL) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 5 or 10.

[0166] In a further embodiment of the binder according to the first aspect, the second binding region for binding to human PD-L1 includes a heavy chain variable region (VH) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 25-100% sequence identity with respect to SEQ ID NO: 11, and a light chain variable region (VL) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with respect to SEQ ID NO: 15.

[0167] In one embodiment of the binder according to the first aspect, (a) The first binding region for human CD137 comprises a heavy chain variable region (VH) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 1 or 9, and a light chain variable region (VL) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 5 or 10; and (b) The second binding region for human PD-L1 includes a heavy chain variable region (VH) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 25-100% sequence identity with SEQ ID NO: 11, and a light chain variable region (VL) containing an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 15.

[0168] In one embodiment of the binder according to the first aspect, the first binding region that binds to human CD137 includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 1 or 9, and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 5 or 10.

[0169] In a further embodiment of the binder according to the first aspect, the second binding region for binding to human PD-L1 includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 15.

[0170] In one embodiment of the binder according to the first aspect, (a) The first binding region for human CD137 includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 1 or 9, and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 5 or 10; and (b) The second binding region for human PD-L1 includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 15.

[0171] In one embodiment of the binder according to the first aspect, (a) The first binding region for human CD137 includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 1, and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 5; and (b) The second binding region for human PD-L1 includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 15.

[0172] The binder may be an antibody, for example, a multispecific antibody, or a bispecific antibody. Alternatively, the binder may be in the form of a full-length antibody or an antibody fragment.

[0173] It is even more preferable that the binder is a human antibody or a humanized antibody.

[0174] Each variable region may include three complementarity determination regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0175] The complementarity-determining regions (CDRs) and framework regions (FRs) can be aligned from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0176] In one aspect of the first phase, the binder is (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and (ii) Polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH) Includes.

[0177] In one aspect of the first phase, the binder is (i) A polypeptide comprising the first light chain variable region (VL) and further comprising the first light chain constant region (CL), and (ii) A polypeptide comprising the second light chain variable region (VL) and further comprising the second light chain constant region (CL). Includes.

[0178] In one embodiment of the first aspect, the binder is an antibody comprising a first binding arm and a second binding arm, where the first binding arm is (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and (ii) A polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). including; and the second connecting arm is (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and (iv) Polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL) Includes.

[0179] In one aspect of the first phase, 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; and (ii) A second heavy chain and 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. Includes.

[0180] Each of the first heavy chain steady region (CH) and the second heavy chain steady region (CH) may include one or more of the steady heavy chain 1 (CH1) region, the hinge region, the steady heavy chain 2 (CH2) region, and the steady heavy chain 3 (CH3) region, preferably at least the hinge region, the CH2 region, and the CH3 region.

[0181] Each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) may contain a CH3 region, where the two CH3 regions contain an asymmetric mutation. An asymmetric mutation means that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at non-identical positions. For example, one of the first CH3 region and the second CH3 region contains a mutation at the position corresponding to position 405 in the human IgG1 heavy chain according to EU numbering, and the other of the first CH3 region and the second CH3 region contains a mutation at the position corresponding to position 409 in the human IgG1 heavy chain according to EU numbering.

[0182] In the first heavy chain constant region (CH), at least one amino acid at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the EU-numbered human IgG1 heavy chain may be substituted, and in the second heavy chain constant region (CH), at least one amino acid at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the EU-numbered human IgG1 heavy chain may be substituted. In certain embodiments, the first and second heavy chains are not substituted at the same position (i.e., the first and second heavy chains contain asymmetric mutations).

[0183] In one embodiment of the binder according to the first aspect, (i) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

[0184] In one embodiment of the first aspect, the binder induces Fc-mediated effector function to a lesser extent compared to another antibody containing the same first and second antigen-binding regions, as well as two heavy chain constant regions (CH) including the human IgG1 hinge, CH2, and CH3 regions.

[0185] In one specific embodiment of the binder according to the first aspect, the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an antibody that is otherwise identical except for containing the unmodified first heavy chain constant region (CH) and the second heavy chain constant region (CH). In particular, each or both of the unmodified first heavy chain constant region (CH) and the second heavy chain constant region (CH) may, may be, or essentially be, the amino acid sequence shown in SEQ ID NO: 19 or 25.

[0186] Fc-mediated effector function can be determined by measuring the binding of the binder to the Fcγ receptor, binding to C1q, or induction of Fc-mediated crosslinking of the Fcγ receptor. In particular, Fc-mediated effector function can be determined by measuring the binding of the binder to C1q.

[0187] The first and second heavy chain constant regions of the binder may be modified to reduce the binding of C1q to the antibody compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, where C1q binding is preferably determined by ELISA.

[0188] In one embodiment of the binder according to the first aspect, in at least one of the first heavy chain constant region (CH) and the second heavy chain constant region (CH), one or more amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

[0189] In one embodiment of the binder according to the first aspect, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering may be F and E in the first and second heavy chains, respectively.

[0190] In particular, the positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering may be F, E, and A in the first and second heavy chain steady regions, respectively.

[0191] In one embodiment of the binder according to the first phase, the positions corresponding to L234 and L235 in the human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F and E, respectively, where (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.

[0192] In one embodiment of the binder according to the first phase, 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, where (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.

[0193] In one embodiment of the binder according to the first phase, the steady regions of the first heavy chain and / or the second heavy chain are (a) Sequence [IgG1-FC] shown in SEQ ID NO: 19 or SEQ ID NO: 25; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution. It contains an amino acid sequence selected from the group consisting of the following.

[0194] In one embodiment of the binder according to the first phase, the steady region of the first or second heavy chain, for example, the second heavy chain, (a) Sequence [IgG1-F405L] shown in SEQ ID NO: 20 or SEQ ID NO: 26; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most nine substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most eight, at most seven, at most six, at most five, at most four, at most three, at most two, or at most one substitution. It contains, essentially consists of, or comprises an amino acid sequence selected from the group comprising the following.

[0195] In one embodiment of the binder according to the first phase, the steady region of the first heavy chain or the second heavy chain, for example, the first heavy chain, (a) Sequence shown in SEQ ID NO: 21 or 27 [IgG1-F409R]; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2, or at most 1 substitution. It contains, essentially consists of, or comprises an amino acid sequence selected from the group comprising the following.

[0196] In one embodiment of the binder according to the first phase, the steady regions of the first heavy chain and / or the second heavy chain are (a) The sequence [IgG1-Fc_FEA] shown in SEQ ID NO: 22 or SEQ ID NO: 28; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 7 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 6 substitutions, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution. It contains, essentially consists of, or comprises an amino acid sequence selected from the group comprising the following.

[0197] In one embodiment of the binder according to the first phase, the steady region of the first heavy chain and / or the second heavy chain, for example, the second heavy chain, (a) The sequence [IgG1-Fc_FEAL] shown in SEQ ID NO: 24 or SEQ ID NO: 30; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 6 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. It contains, essentially consists of, or comprises an amino acid sequence selected from the group comprising the following.

[0198] In one embodiment of the binder according to the first phase, the first heavy chain and / or the second heavy chain, for example, the steady region of the first heavy chain, (a) The sequence [IgG1-Fc_FEAR] shown in SEQ ID NO: 23 or SEQ ID NO: 29; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 6 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 5 substitutions, at most 4, at most 3, at most 2, or at most 1 substitution. It contains, essentially consists of, or comprises an amino acid sequence selected from the group comprising the following.

[0199] In one embodiment of the first aspect, the binder includes a kappa (κ) light chain constant region.

[0200] In one embodiment of the first aspect, the binder includes a constant region of the lambda (λ) light chain.

[0201] In one embodiment of the binder according to the first phase, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0202] In one embodiment of the binder according to the first phase, the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0203] In one embodiment of the binder according to the first phase, the first light chain steady region is a kappa (κ) light chain steady region and the second light chain steady region is a lambda (λ) light chain steady region, or the first light chain steady region is a lambda (λ) light chain steady region and the second light chain steady region is a kappa (κ) light chain steady region.

[0204] In one embodiment of the binder according to the first aspect, the kappa (κ) light chain is (a) Sequence shown in SEQ ID NO: 35; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2, or at most 1 substitution. It contains an amino acid sequence selected from the group consisting of the following.

[0205] In one embodiment of the binder according to the first aspect, the lambda (λ) light chain is (a) Sequence shown in SEQ ID NO: 36; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2, or at most 1 substitution. It contains an amino acid sequence selected from the group consisting of the following.

[0206] The binder (in particular, the antibody) in the first aspect is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In particular, the binder may be a full-length IgG1 antibody. In a preferred embodiment of the first aspect, the binder (in particular, the antibody) is an IgG1m(f) allotype.

[0207] In a preferred embodiment of the binder according to the first aspect, the binder is (i) A first heavy chain and a first light chain comprising the antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises the sequence shown in SEQ ID NO: 31 and the first light chain comprises the sequence shown in SEQ ID NO: 32; (ii) A second heavy chain and light chain comprising the antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises the sequence shown in SEQ ID NO: 33 and the second light chain comprises the sequence shown in SEQ ID NO: 34. Includes.

[0208] The binder for use in the first phase may, in particular, be akasanlimab or its biosimilar.

[0209] In the currently preferred embodiment, the amount of binder administered in each dose and / or in each treatment cycle is: (a) Approximately 0.3–5 mg / kg body weight or approximately 25–400 mg in total; and / or (b) Approximately 2.1×10 -9 ~3.4×10 -8 mol / kg body weight or total of approximately 1.7 × 10⁻⁶ -7 ~2.7×10 -6 mol That is the case.

[0210] According to these embodiments, the dose defined in mg / kg may be converted to a fixed dose based on the median body weight of the subject to whom the binder is administered being 80 kg, and vice versa.

[0211] The amount of binder administered at each dose and / or in each treatment cycle is, in particular, about 0.3–4.0 mg / kg body weight or about 25–320 mg in total; and / or about 2.1 × 10 -9 ~2.7×10 -8 mol / kg body weight or total of approximately 1.7 × 10⁻⁶ -7 ~2.2×10 -6 It could be in moles.

[0212] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 0.38–4.0 mg / kg body weight or about 30–320 mg in total; and / or about 2.6 × 10 -9 ~2.7×10 -8 mol / kg body weight or total of approximately 2.4 × 10⁻⁶ -7 ~2.2×10 -6 It could be in moles.

[0213] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 0.5–3.3 mg / kg body weight or about 40–260 mg in total; and / or about 3.4 × 10 -9 ~2.2×10 -8 mol / kg body weight or total of approximately 2.7 × 10⁻⁶ -7 ~1.8×10 -6 It could be in moles.

[0214] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 0.6–2.5 mg / kg body weight or about 50–200 mg in total; and / or about 4.3 × 10 -9 ~1.7×10 -8 mol / kg body weight or total of approximately 3.4 × 10 -7 ~1.4×10 -6 It could be in moles.

[0215] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 0.8–1.8 mg / kg body weight or about 60–140 mg in total; and / or about 5.1 × 10 -9 ~1.2 × 10-8 mol / kg body weight or total of approximately 4.1 × 10⁻⁶ -7 ~9.5×10 -7 It could be in moles.

[0216] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 0.9–1.8 mg / kg body weight or about 70–140 mg in total; and / or about 6.0 × 10 -9 ~1.2 × 10 -8 mol / kg body weight or total of approximately 4.8 × 10 -7 ~9.5×10 -7 It could be in moles.

[0217] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 1 to 1.5 mg / kg body weight or about 80 to 120 mg in total; and / or about 6.8 × 10 -9 ~1.0×10 -8 mol / kg body weight or total of approximately 5.5 × 10 -7 ~8.2×10 -7 It could be in moles.

[0218] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 1.1–1.4 mg / kg body weight or about 90–110 mg in total; and / or about 7.7 × 10 -9 ~9.4×10 -9 mol / kg body weight or total of approximately 6.1 × 10 -7 ~7.5×10 -7 It could be in moles.

[0219] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 1.2–1.3 mg / kg body weight or about 95–105 mg in total; and / or about 6.8 × 10 -9 ~8.9×10 -9 mol / kg body weight or total of approximately 6.5 × 10 -7 ~7.2×10 -7 It could be in moles.

[0220] The amount of binder 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 × 10 -9 ~1.0×10 -8 mol / kg body weight or total of approximately 4.4 × 10 -7 ~8.2×10 -7 It could be in moles.

[0221] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, about 0.9–1.3 mg / kg body weight or about 70–100 mg in total; and / or about 6.0 × 10 -9 ~8.5×10 -9 mol / kg body weight or total of approximately 4.8 × 10 -7 ~6.8×10 -7 It could be in moles.

[0222] Approximately 0.9–1.1 mg / kg body weight or approximately 75–90 mg in total; and / or Approximately 6.4×10 -9 ~7.7×10 -9 mol / kg body weight or total of approximately 5.1 × 10⁻⁶ -7 ~6.1×10 -7 mol.

[0223] Furthermore, the amount of binder administered at each dose and / or in each treatment cycle is, in particular, 0.3–4.0 mg / kg body weight or 25–320 mg in total; and / or 2.1 × 10 -9 ~2.7×10 -8 mol / kg body weight or total of 1.7 × 10 -7 ~2.2×10 -6 It could be in moles.

[0224] The amount of binder administered at each dose and / or in each treatment cycle is, in particular, 0.38–4.0 mg / kg body weight or 30–320 mg in total; and / or 2.6 × 10 -9 ~2.7×10 -8 mol / kg body weight or total of 2.4 × 10 -7~2.2×10 -6 may be in the range of mol.

[0225] The amount of the binder administered at each dose and / or in each treatment cycle may particularly be 0.5 - 3.3 mg / kg body weight or 40 - 260 mg in total; and / or 3.4×10 -9 ~2.2×10 -8 mol / kg body weight or 2.7×10 -7 ~1.8×10 -6 mol may be in the range of.

[0226] The amount of the binder administered at each dose and / or in each treatment cycle may particularly be 0.6 - 2.5 mg / kg body weight or 50 - 200 mg in total; and / or 4.3×10 -9 ~1.7×10 -8 mol / kg body weight or 3.4×10 -7 ~1.4×10 -6 mol may be in the range of.

[0227] The amount of the binder administered at each dose and / or in each treatment cycle may particularly be 0.8 - 1.8 mg / kg body weight or 60 - 140 mg in total; and / or 5.1×10 -9 ~1.2×10 -8 mol / kg body weight or 4.1×10 -7 ~9.5×10 -7 mol may be in the range of.

[0228] The amount of the binder administered at each dose and / or in each treatment cycle may particularly be 0.9 - 1.8 mg / kg body weight or 70 - 140 mg in total; and / or 6.0×10 -9 ~1.2×10 -8 mol / kg body weight or 4.8×10 -7 ~9.5×10 -7 mol may be in the range of.

[0229] The amount of binder administered at each dose and / or in each treatment cycle is, in particular, 1 to 1.5 mg / kg body weight or 80 to 120 mg in total; and / or 6.8 × 10 -9 ~1.0×10 -8 mol / kg body weight or total of 5.5 × 10 -7 ~8.2×10 -7 It could be in moles.

[0230] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 1.1–1.4 mg / kg body weight or 90–110 mg in total; and / or 7.7 × 10 -9 ~9.4×10 -9 mol / kg body weight or total of 6.1 × 10 -7 ~7.5×10 -7 It could be in moles.

[0231] The amount of binder administered at each dose and / or in each treatment cycle is, in particular, 1.2–1.3 mg / kg body weight or 95–105 mg in total; and / or 6.8 × 10 -9 ~8.9×10 -9 mol / kg body weight or total of 6.5 × 10 -7 ~7.2×10 -7 It could be in moles.

[0232] The amount of binder administered at each dose and / or in each treatment cycle is, in particular, 0.8–1.5 mg / kg body weight or 65–120 mg in total; and / or 5.5 × 10 -9 ~1.0×10 -8 mol / kg body weight or total of 4.4 × 10 -7 ~8.2×10 -7 It could be in moles.

[0233] The amount of binder administered in each dose and / or in each treatment cycle is, in particular, 0.9–1.3 mg / kg body weight or 70–100 mg in total; and / or 6.0 × 10 -9 ~8.5×10 -9 mol / kg body weight or total of 4.8 × 10-7 ~6.8×10 -7 It could be in moles.

[0234] The amount of binder administered at each dose and / or in each treatment cycle is, in particular, 0.9–1.1 mg / kg body weight or 75–90 mg in total; and / or 6.4 × 10 -9 ~7.7×10 -9 mol / kg body weight or total of 5.1 × 10 -7 ~6.1×10 -7 It could be in moles.

[0235] The amount of binder administered at each dose and / or in each treatment cycle is: (a) Approximately 1.1 mg / kg body weight or approximately 80 mg in total; and / or (b) Approximately 6.8×10 -9 mol / kg body weight or total of approximately 5.5 × 10 -7 mol It is possible.

[0236] The amount of binder 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 total of 5.5 × 10 -7 mol It is possible.

[0237] The amount of binder administered at each dose and / or in each treatment cycle is: (a) Approximately 1.25 mg / kg body weight or approximately 100 mg in total; and / or (b) Approximately 8.5×10 -9 mol / kg body weight or total of approximately 6.8 × 10 -7 mol It is currently preferable to be that way.

[0238] The amount of binder 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 total of 6.8 × 10 -7 mol It is equally preferable that this be the case.

[0239] The binder may be administered in any manner and by any route known in the art. In a preferred embodiment, the binder is administered systemically, for example parenterally, and particularly intravenously.

[0240] The binder may be administered in the form of any suitable pharmaceutical composition as described herein. In a preferred embodiment, the binder is administered in the form of an infusion.

[0241] The binder for use according to the present invention may be administered by intravenous (IV) infusion, for example, by intravenous infusion for a minimum of 30 minutes, for example, by intravenous infusion for a minimum of 60 minutes, or for example, by intravenous infusion for a minimum of 30 to 120 minutes. Preferably, the binder for use according to the present invention is administered by intravenous (IV) infusion over 30 minutes.

[0242] The binder can be administered before, simultaneously with, or after the administration of the PD-1 inhibitor.

[0243] In one embodiment, the binder is administered before the administration of the PD-1 inhibitor. For example, the gap between the end of the binder administration and the start of the PD-1 inhibitor administration is at least about 10 minutes, for example, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, and up to about 14 days (up to about 2 weeks), for example. For example, it could be up to approximately 13 days, up to approximately 12 days, up to approximately 11 days, up to approximately 10 days, up to approximately 9 days, up to approximately 8 days, up to approximately 7 days (up to approximately 1 week), up to approximately 6 days, up to approximately 5 days, up to approximately 4 days, up to approximately 3 days, up to approximately 2 days, up to approximately 1 day (up to approximately 24 hours), up to approximately 18 hours, up to approximately 12 hours, up to approximately 6 hours, up to approximately 5 hours, up to approximately 4 hours, up to approximately 3 hours, up to approximately 2.5 hours, or up to approximately 2 hours.

[0244] In one embodiment, the binder is administered after the 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 binder is at least about 10 minutes, for example, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, and up to about 14 days (up to about 2 weeks), for example For example, it could be up to approximately 13 days, up to approximately 12 days, up to approximately 11 days, up to approximately 10 days, up to approximately 9 days, up to approximately 8 days, up to approximately 7 days (up to approximately 1 week), up to approximately 6 days, up to approximately 5 days, up to approximately 4 days, up to approximately 3 days, up to approximately 2 days, up to approximately 1 day (up to approximately 24 hours), up to approximately 18 hours, up to approximately 12 hours, up to approximately 6 hours, up to approximately 5 hours, up to approximately 4 hours, up to approximately 3 hours, up to approximately 2.5 hours, or up to approximately 2 hours.

[0245] In one embodiment, the binder is administered simultaneously with the PD-1 inhibitor. For example, the binder and the PD-1 inhibitor may be administered using a composition containing both drugs. Alternatively, the binder may be administered to one limb of the subject, and the PD-1 inhibitor to the other limb of the subject.

[0246] PD-1 (Programmed Cell Death Protein 1, PD1, also known as CD279 or unknown) inhibitors In one embodiment, a PD-1 inhibitor blocks PD-1-related inhibitory signals. In one embodiment, a PD-1 inhibitor is an antibody or fragment thereof that disrupts or inhibits PD-1-related inhibitory signaling. In one embodiment, a PD-1 inhibitor is a small molecule inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, a PD-1 inhibitor is a peptide-based inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, a PD-1 inhibitor is an inhibitory nucleic acid molecule that disrupts or inhibits inhibitory signaling.

[0247] Inhibition or blockade of PD-1 signaling results in the prevention or reversal of immunosuppression and the establishment or enhancement of T-cell immunity against cancer cells, as described herein. In one embodiment, inhibition of PD-1 signaling reduces or inhibits dysfunction of the immune system, as described herein. In one embodiment, inhibition of PD-1 signaling reduces dysfunction of dysfunctional immune cells, as described herein. In one embodiment, inhibition of PD-1 signaling reduces dysfunction of dysfunctional T cells, as described herein.

[0248] In one embodiment, PD-1 inhibitors block the interaction between PD-1 and PD-L1.

[0249] A PD-1 inhibitor may be an antibody, its antigen-binding fragment, or a construct thereof, comprising an antibody moiety having an antigen-binding fragment of the required specificity. The antibody or its antigen-binding fragment is as described herein. An antibody or its antigen-binding fragment that is a PD-1 inhibitor includes, in particular, an antibody or its antigen-binding fragment that binds to PD-1. An antibody or its antigen-binding fragment that is a PD-1 inhibitor also includes an antibody or its antigen-binding fragment that binds to PD-L1. The antibody or antigen-binding fragment may also be conjugated to further portions as described herein. In particular, the antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody, or a human antibody.

[0250] In a preferred embodiment, the antibody that is a PD-1 inhibitor is an isolated antibody.

[0251] In one embodiment, a PD-1 inhibitor is an antibody, fragment thereof, or construct that blocks the interaction between PD-1 and PD-L1.

[0252] PD-1 inhibitors can be inhibitory nucleic acid molecules, such as oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers), particularly antisense oligonucleotides. In one embodiment, a PD-1 checkpoint inhibitor that is siRNA interferes with mRNA and therefore blocks translation, for example, the translation of the PD-1 protein.

[0253] In one embodiment, a PD-1 inhibitor is an antibody, its antigen-binding moiety, or a construct thereof that disrupts or inhibits the interaction between the PD-1 receptor and one or more of its ligands, PD-L1 and / or PD-L2. Antibodies that bind to PD-1 and disrupt or inhibit the interaction between PD-1 and one or more of its ligands are known in the art. In a particular embodiment, the antibody, its antigen-binding moiety, or a construct thereof specifically binds to PD-1.

[0254] 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, the combination of a conjugate containing a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, and an antibody that binds to PD-1, is thought to increase the response rate and improve the duration of response in subjects receiving combination therapy, because the combination therapy results in complete blockade of the PD-1 pathway with simultaneous conditional activation of 4-1BB. The PD-1 blocking antibody blocks interaction with both PD-L1 and PD-L2. Furthermore, combination therapy with an antibody that binds to PD-1 is thought to increase the amount of PD-L1 available for the conjugate to bind.

[0255] 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, WO 2015 / 35606, and U.S. 2015 / 0079109), lambrolizumab (disclosed, e.g., hPD109A and its humanized derivatives h409A1, h409A16, and h409A17 in WO2008 / 156712), AB137132 (Abcam), EH12.2H7, and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), and nivolumab (OPDIVO, BMS-936558; Bristol Myers). Squibb (see US Patent No. 8,008,449; WO 2013 / 173223; WO 2006 / 121168), pembrolizumab (KEYTRUDA; MK-3475; Merck; see WO 2008 / 156712), pizilizumab (CT-011; CureTech; Hardy et al., 1994, Cancer Res., 54(22):5793-6 and WO 2009 / 101611), PDR001 (Novartis; see WO 2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see WO 2012 / 145493), TSR-042 (WO See 2014 / 179664), semiprimab (REGN-2810; Regeneron; H4H7798N; see US 2015 / 0203579 and WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70: 136), AMP-224 (GSK-2661380; Li et al.See Int J mol Sci 17(7):1151 and WO 2010 / 027827 and WO 2011 / 066342, PF-06801591 (Pfizer), tislerizumab (see BGB-A317; BeiGene; WO 2015 / 35606, U.S. Patent No. 9,834,606 and US 2015 / 0079109), BI 754091, SHR-1210 (see WO 2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 as described in WO 2006 / 121168, INCSHR1210 (Jiangsu Hengrui Medicine (also known as SHR-1210; see WO 2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see W02014 / 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 WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), IBI308 (Innovent; see WO See 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540), cetrelimab (JNJ-63723283; JNJ-3283; see Calvo et al., J. Clin. Oncol. 36, no. 5_suppl (2018) 58), genolimuzumab (CBT-501; see Patel et al., J. ImmunoTher. Cancer, 2017, 5(Suppl 2):P242), sasanlimab (PF-06801591; Youssef et al., Proc. Am. Assoc. Cancer Res. Ann.Meeting 2017; Cancer Res 2017; 77(13 Suppl): See abstract), Tripalimab (JS-001; See US 2016 / 0272708), Camrelizumab (SHR-1210; INCSHR-1210; US 2016 / 376367; See Huang et al., Clin. Cancer Res. 2018; 24(6): 1296-1304), Spartalizumab (PDR001; WO 2017 / 106656; See Naing et al., J. Clin. Oncol. 34, no. 15_suppl (2016) 3060-3060), BCD-100 (JSC BIOCAD, Russia; WO See 2018 / 103017), valstilimab (AGEN2034; see WO 2017 / 040790), cintilimab (IBI-308; see WO 2017 / 024465 and WO 2017 / 133540), ezabenlimab (BI-754091; US ​​2017 / 334995; Johnson et al., J. Clin. Oncol. 36, no.See 5_suppl (2018) 212-212), Zimbererimab (GLS-010; see WO 2017 / 025051), LZM-009 (see US 2017 / 210806), AK-103 (see WO 2017 / 071625, WO 2017 / 166804, and WO 2018 / 036472), Retifanlimab (MGA-012; see WO 2017 / 019846), Sym-021 (see WO 2017 / 055547), CS1003 (see CN107840887), Dostallimab (JEMPERLI®, GlaxoSmithKline LLC, Philadelphia, PA), anti-PD-1 antibodies, e.g., U.S. Patent No. 7,488,802, U.S. Patent No. 8,008,449, U.S. Patent No. 8,168,757, WO 03 / 042402, WO 2010 / 089411 (further disclosing anti-PD-L1 antibodies), WO 2010 / 036959, WO 2011 / 159877 (further disclosing antibodies against TIM-3), WO 2011 / 082400, WO 2011 / 161699, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2012 / 145493 (further disclosing antibodies against PD-L1), WO 2015 / 035606, WO As described in US Patent Nos. 2014 / 055648 (further disclosing anti-KIR antibodies), US 2018 / 0185482 (further disclosing anti-PD-L1 antibodies and anti-TIGIT antibodies), US Patent Nos. 8,008,449, 8,779,105, 6,808,710, 8,168,757, US 2016 / 0272708, and US Patent No. 8,354,509, small molecule antagonists to the PD-1 signaling pathway, e.g., Shaabani et al., 2018, Expert Op Ther Pat.Examples of PD-1 inhibitors include those disclosed in 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, siRNAs against PD-1, soluble PD-1 proteins such as those disclosed in WO 2019 / 000146 and WO 2018 / 103501, and WO 2018 / 222711, as well as oncolytic viruses containing soluble forms of PD-1, such as those described in WO 2018 / 022831. Exemplary PD-1 inhibitors also include, non-limitingly, PD-L1 inhibitors such as atezolizumab, avelumab, durvalumab, emvafolimab, cosivelimab, AUNP12, CA-170, and BMS-986189.

[0256] In certain embodiments, the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558) or its biosimilar, pembrolizumab (KEYTRUDA; MK-3475) or its biosimilar, pizilizumab (CT-011), PDR001, MEDI0680 (AMP-514) or its biosimilar, TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI 754091, or SHR-1210.

[0257] PD-1 inhibitors may include pembrolizumab or its biosimilars. Alternatively, antibodies may include nivolumab or its biosimilars.

[0258] In a particular embodiment, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, dostallimab, voplaterimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, cosivelimab, AUNP12, CA-170, BMS-986189, or their respective biosimilars.

[0259] In a particular embodiment, the PD-1 inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof, comprising a CDR of one of the above-mentioned anti-PD-1 antibodies or antigen-binding fragments, such as pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, and cosivelimab, or their respective biosimilars.

[0260] In some embodiments, the CDRs of anti-PD-1 antibodies or anti-PD-L1 antibodies 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 a particular embodiment, the PD-1 inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof, comprising the heavy chain variable region and light chain variable region of one of the above-mentioned anti-PD-1 antibodies or antigen-binding fragments, for example, the heavy chain variable region and light chain variable region of one anti-PD-1 antibody or antigen-binding fragment selected from the group consisting of pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, cosiberimab, or biosimilars thereof.

[0262] In a particular embodiment, the PD-1 inhibitor is an anti-PD-1 antibody, anti-PD-L1 antibody, or antigen-binding fragment thereof, selected from the group consisting of pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, cosivelimab, or biosimilars thereof.

[0263] In a particular embodiment, the PD-1 inhibitor is an anti-PD-1 antibody or its antigen-binding fragment, selected from the group consisting of pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, or biosimilars thereof.

[0264] The CDR sequences of pembrolizumab are identified herein by SEQ ID NO: 59-61 (VH CDR 1, 2, and 3, respectively) and SEQ ID NO: 62-64 (VL CDR 1, 2, and 3, respectively). The VH and VL sequences are identified by SEQ ID NO: 65 and 66, respectively, and the heavy chain and light chain sequences are identified by SEQ ID NO: 67 and 68, respectively. Accordingly, in one embodiment, a PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown at SEQ ID NO: 59, 60, and 61, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown at SEQ ID NO: 62, 63, and 64, respectively.

[0265] In a further embodiment, the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) comprising, consisting of, or essentially comprising the sequence shown in SEQ ID NO: 65, and a light chain variable region (VL) comprising, consisting of, or essentially comprising the sequence shown in SEQ ID NO: 66. The PD-1 inhibitor may particularly be an antibody comprising a heavy chain comprising, consisting of, or essentially comprising the amino acid sequence shown in SEQ ID NO: 67, and a light chain comprising, consisting of, or essentially comprising the amino acid sequence shown in SEQ ID NO: 68.

[0266] The CDR sequences of nivolumab are identified herein by SEQ ID NO: 69-71 (VH CDR 1, 2, and 3, respectively) and SEQ ID NO: 72-74 (VL CDR 1, 2, and 3, respectively). The VH and VL sequences are identified by SEQ ID NO: 75 and 76, respectively, and the heavy chain and light chain sequences are identified by SEQ ID NO: 77 and 78, respectively. Accordingly, in one embodiment, a PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 69, 70, and 71, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 72, 73, and 74, respectively.

[0267] In a further embodiment, the PD-1 inhibitor is an antibody comprising a heavy chain variable region (VH) comprising, consisting of, or essentially comprising the sequence shown in SEQ ID NO: 75, and a light chain variable region (VL) comprising, consisting of, or essentially comprising the sequence shown in SEQ ID NO: 76. The PD-1 inhibitor may particularly be an antibody comprising a heavy chain comprising, consisting of, or essentially comprising the amino acid sequence shown in SEQ ID NO: 77, and a light chain comprising, consisting of, or essentially comprising the amino acid sequence shown in SEQ ID NO: 78.

[0268] The anti-PD-1 antibodies of this disclosure are preferably monoclonal and may be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by Fab expression libraries, and any of the above PD-1 binding fragments. In some embodiments, the anti-PD-1 antibodies described herein bind specifically to PD-1 (e.g., human PD-1). The immunoglobulin molecules of this disclosure may be 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 molecules.

[0269] In certain embodiments of this disclosure, the anti-PD-1 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) as described herein, including Fab, Fab' and F(ab')2, Fd, single-chain Fv(scFv), single-chain antibody, disulfide-linked Fv(sdFv), and V L Domain or V H This disclosure includes, but is not limited to, fragments containing any of the domains. Antigen-binding fragments containing a single-chain antibody may contain the variable region alone or in combination with all or part of the following: the hinge region, CH1, CH2, CH3, and CL domains. Antigen-binding fragments containing the variable region and any combination of the hinge region, CH1, CH2, CH3, and CL domains are also included in this disclosure. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken.

[0270] The anti-PD-1 antibodies disclosed herein may be monospecific, bispecific, triplicate, or more than multispecific. Multispecific antibodies may be specific to different epitopes of PD-1, or they may be specific not only to PD-1 but also to both heterologous proteins. For example, see PCT publications WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60 69; U.S. Patent No. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547 1553.

[0271] The anti-PD-1 antibodies disclosed herein may be described or explicitly stated with respect to the specific CDRs they contain.The precise boundaries of a given CDR or FR amino acid sequence can be found in: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003; This can be easily determined using any of the many well-known schemes, including those described by 27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun 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 CDR of a given antibody or its region (e.g., its variable region) or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass a CDR (or specific CDR) as defined by any of the schemes described above. For example, a specific CDR (e.g., CDR-H3) may be a given V. H Area or V L When it is stated that a region contains the amino acid sequence of a corresponding CDR, such a CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the schemes described above. Schemes for identifying one or more specific CDRs may be explicitly stated, such as CDRs as defined by Kabat, Chothia, AbM, or IMGT methods.

[0272] In some embodiments, the numbering of amino acid residues in the CDR sequence of the anti-PD-1 antibody or its antigen-binding fragment provided herein follows an IMGT numbering scheme as described in Lefranc, MP et al., Dev. Comp. Immunol., 2003, 27, 55-77.

[0273] In some embodiments, the anti-PD-1 antibodies disclosed herein include CDRs of the antibody nivolumab. See WO 2006 / 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). This disclosure encompasses anti-PD-1 antibodies or derivatives thereof that include a heavy chain variable domain or a light chain variable domain, wherein the variable domain includes (a) a set of three CDRs derived from the monoclonal antibody nivolumab, and (b) a set of four framework regions different from the set of framework regions in the monoclonal antibody nivolumab, and the anti-PD-1 antibody or derivative thereof binds to PD-1. In certain embodiments, the anti-PD-1 antibody is nivolumab.

[0274] Anti-PD-1 antibodies disclosed herein may also be described or expressed in relation to their binding affinity to PD-1 (e.g., human PD-1). A preferred binding affinity is 5 × 10⁻⁶. -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or 10 -15 This includes those with a dissociation constant or Kd of less than M.

[0275] Anti-PD-1 antibodies also include derivatives and constructs that are modified, i.e., modified by covalent bonding to the antibody of any type of molecule so that the covalent bond 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, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins. Any of the numerous chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and metabolic synthesis of tunicamycin. In addition, derivatives or constructs may contain one or more non-classical amino acids.

[0276] In a preferred embodiment, a 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, a PD-1 inhibitor is an antibody, particularly an antagonistic or blocking antibody, that disrupts or inhibits the interaction between PD-1 and PD-L1.

[0277] PD-1 inhibitors may be administered in the form of PD-1 inhibitors, for example, inhibitory nucleic acid molecules or nucleic acids encoding antibodies or fragments thereof, such as DNA or RNA molecules. For example, antibodies can be delivered encoded in an expression vector, as described herein. Nucleic acid molecules can thus be delivered, for example, in the form of plasmids or mRNA molecules, or in complex with a delivery vehicle, such as liposomes, lipoplexes, or nucleic acid lipid particles. PD-1t inhibitors may also be administered via oncolytic viruses containing expression cassettes encoding PD-1 inhibitors. PD-1 may also be administered, for example, in the form of cell-based therapies, by administration of endogenous or homogeneous cells capable of expressing PD-1 inhibitors.

[0278] Preferably, the PD-1 inhibitor is administered in an appropriate amount. The amount of the PD-1 inhibitor administered in each dose and / or treatment cycle may be in the range where 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 binds to PD-1.

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

[0280] In a particular embodiment, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the amount of the PD-1 inhibitor administered is, for example, 10 to 1000 mg in total at each dose and / or in each treatment cycle, for example, 100 to 600 mg in total, for example, 150 to 600 mg in total, for example, 150 to 500 mg in total, for example, 175 to 500 mg in total, for example, 175 to 450 mg in total, for example, 200 to 450 mg in total, or for example, 200 to 400 mg in total.

[0281] In a particular embodiment, the PD-1 inhibitor is pembrolizumab or its biosimilar, and the amount of PD-1 inhibitor administered is, for example, about 100-600 mg in total at each dose and / or in each treatment cycle; and / or about 6.84 × 10⁻⁶ mg in total. -7 ~4.11×10 -7 It is a mole.

[0282] In a particular embodiment, the PD-1 inhibitor is pembrolizumab or its biosimilar, and the amount of PD-1 inhibitor administered is, for example, about 100-400 mg in total at each dose and / or in each treatment cycle; and / or about 6.84 × 10⁻⁶ mg in total. -7 ~2.73 × 10 -6 mol, for example, 100-400 mg in total; and / or 6.84 × 10 in total -7 ~2.73 × 10 -6 It is a mole.

[0283] In a particular embodiment, the PD-1 inhibitor is pembrolizumab or its biosimilar, and the amount of PD-1 inhibitor administered is, for example, about 200-400 mg in total at each dose and / or in each treatment cycle; and / or about 6.84 × 10⁻¹⁴ mg in total. -7 ~2.73 × 10 -6 mol, for example, 200-400 mg in total; and / or 6.84 × 10 in total -7 ~2.73 × 10 -6 It is a mole.

[0284] In a particular embodiment, the amount of PD-1 inhibitor administered is, for example, approximately 200 mg or approximately 1.37 × 10¹⁶ mg in total for each dose and / or each treatment cycle. -6 moles, for example, 200 mg or 1.37 × 10⁻⁶ in total. -6 It is a mole.

[0285] In a particular embodiment, the PD-1 inhibitor is pembrolizumab or its biosimilar, and the amount of PD-1 inhibitor administered is, for example, about 200 mg or about 1.37 × 10¹¹ in total at each dose and / or in each treatment cycle. -6 moles, for example, 200 mg or 1.37 × 10⁻⁶ in total. -6 It is a mole.

[0286] In a particular embodiment, the amount of PD-1 inhibitor administered is, for example, approximately 400 mg in total or approximately 2.73 × 10⁶ mg in total at each dose and / or in each treatment cycle. -6 For example, a total of 400 mg or a total of 2.73 × 10 -6 That is the case.

[0287] In a particular embodiment, the PD-1 inhibitor is pembrolizumab or its biosimilar, and the amount of PD-1 inhibitor administered is, for example, about 400 mg in total or about 2.73 × 10⁻¹⁴ mg in total at each dose and / or in each treatment cycle. -6 For example, a total of 400 mg or a total of 2.73 × 10 -6 That is the case.

[0288] PD-1 inhibitors may be administered in any manner and by any route known in the art. The method and route of administration will depend on the type of PD-1 inhibitor used. In a preferred embodiment, the PD-1 inhibitor is administered systemically, for example parenterally, and in particular intravenously.

[0289] PD-1 inhibitors may be administered in the form of any suitable pharmaceutical composition as described herein. In a preferred embodiment, PD-1 inhibitors are administered in the form of infusions, such as intravenous infusions.

[0290] An antibody that binds to PD-1 may include a heavy chain variable region (VH) containing the HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) containing the LCDR1, LCDR2, and LCDR3 sequences, where the HCDR1, HCDR2, and HCDR3 sequences contain or have the sequences as shown in SEQ ID NO: 104, SEQ ID NO: 101, and SEQ ID NO: 100, respectively, and the LCDR1, LCDR2, and LCDR3 sequences contain or have the sequences as shown 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.

[0291] The terms “heavy chain variable region” (also known as “VH”) and “light chain variable region” (also known as “VL”) are used herein in their most common sense and include any sequences that may contain complementarity-determining regions (CDRs) interspersed with other regions also known as framework regions (FRs). The framework regions, in particular, space the CDRs so that they can form antigen-binding sites, especially after the folding and pairing of the VH and VL. Preferably, each VH and VL consists of three CDRs and four FRs aligned from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. That is, the terms “heavy chain variable region” and “light chain variable region” should not be interpreted as being limited to sequences that can be found in native antibodies or in the VH and VL sequences as illustrated herein (SEQ ID NO: 109-112 in the sequence listing). These terms include any sequence that includes and can accurately position the CDR, such as sequences derived from the VL and VH regions of the native antibody, or sequences as shown in sequence listing SEQ ID NO: 109-112. In particular, it is recognized by those skilled in the art that the framework region sequence can be modified without losing the characteristics of the VH and VL regions, respectively (including variants relating to amino acid substitutions and variants relating to sequence length, i.e., both insertion variants and deletion variants). In a preferred embodiment, any modification is limited to the framework region. However, those skilled in the art are also well aware that the CDR, hypervariable region, and variable region can also be modified without losing their ability to bind to PD-1. For example, the CDR region may be either identical or highly homologous to the region expressed herein. "Highly homologous" means that 1 to 5, preferably 1 to 4, e.g., 1 to 3 or 1 or 2 substitutions may be made in the CDR. In addition, the hypervariable region and the variable region may be modified to exhibit substantial homology with the regions specifically disclosed herein.

[0292] In antibodies that bind to PD-1, CDRs as explicitly stated herein have been identified using two different CDR identification methods. The first numbering scheme used herein follows Kabat (Wu and Kabat, 1970; Kabat et al., 1991), and the second scheme is IMGT numbering (Lefranc, 1997; Lefranc et al., 2005). A third approach utilizes the common elements of both identification schemes.

[0293] An antibody that binds to PD-1 may include one or more CDRs, sets of CDRs, or combinations of sets of CDRs as described herein, including the CDRs together with their intervening framework regions (also referred herein as framing regions or FRs) or portions thereof. Preferably, the portion includes at least about 50% of either or both of the first and fourth framework regions, the 50% being 50% of the C-terminus of the first framework region and 50% of the N-terminus of the fourth framework region. Construction of an antibody made by recombinant DNA techniques may result in the introduction of N-terminal or C-terminal residues into the variable region, encoded by a linker introduced to facilitate cloning or other manipulative steps, including the introduction of a linker to link the variable region of this disclosure to an immunoglobulin heavy chain, other variable domains (e.g., in the construction of a diabody), or further protein sequences including protein labeling.

[0294] An antibody that binds to PD-1 may include a heavy chain variable region (VH) containing 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 with the amino acid sequence of the VH sequence as shown in any one of SEQ ID NO: 111. In one embodiment, the antibody includes a heavy chain variable region (VH), where VH contains the sequence as shown in any one of SEQ ID NO: 111. In another embodiment, the antibody includes a light chain variable region (VL) containing 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 with the amino acid sequence of the VL sequence as shown in any one of SEQ ID NO: 112. In one embodiment, the antibody comprises a light chain variable region (VL), where the VL contains the sequence as shown in one of SEQ ID NO: 112.

[0295] An antibody that binds to PD-1 may include a heavy chain variable region (VH) and a light chain variable region (VL), where VH contains or has the sequence shown in SEQ ID NO: 111, and VL contains or has the sequence shown in SEQ ID NO: 112, or variants thereof. Another example of an antibody that binds to PD-1 may include a VH containing or having the sequence shown in SEQ ID NO: 111, or a variant thereof, and a VL containing or having the sequence shown in SEQ ID NO: 112, or a variant thereof. A specific, but non-limiting, example of such an antibody is MAB-19-0618. The antibody MAB-19-0618 is derived from MAB-19-0202. Furthermore, variants of the heavy chain variable region (VH) and the light chain variable region (VL), as well as each combination of these variants VH and VL, are also included in this disclosure.

[0296] The antibody that binds to PD-1 may include a heavy chain and a light chain, the heavy chain including a heavy chain constant region containing or having the sequence shown in SEQ ID NO: 93 or 90 and a heavy chain variable region (VH) containing or having the sequence shown in SEQ ID NO: 111, and the light chain including a light chain constant region containing or having the sequence shown in SEQ ID NO: 97 and a light chain variable region (VL) containing or having the sequence shown in SEQ ID NO: 112.

[0297] The antibody that binds to PD-1 may include a heavy chain and a light chain, the heavy chain including a heavy chain constant region including or having the sequence shown in SEQ ID NO: 93 or 90, and a heavy chain variable region (VH) including the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 111, and the light chain including a light chain constant region including or having the sequence shown in SEQ ID NO: 97, and a light chain variable region including the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 112. For example, the CDR1, CDR2, and CDR3 sequences are as expressly provided herein.

[0298] The antibody that binds to PD-1 may be a monoclonal, chimeric antibody, a monoclonal, humanized antibody, or a fragment of such an antibody. The antibody may be, for example, a bispecific antibody, a whole antibody, or an antigen-binding fragment thereof.

[0299] In an antibody that binds to PD-1, one or more, preferably both, heavy chain constant regions may be modified such that the 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%. In one embodiment, C1q binding can be determined by ELISA.

[0300] In this specification, “wild-type,” “WT,” or “native” means the naturally occurring amino acid sequence, including allele variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0301] In an antibody that binds to PD-1, one or more, preferably both, heavy chain constant regions may be modified such that the binding of the antibody to one or more IgG Fc-gamma receptors is preferably reduced 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, 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.

[0302] In one embodiment, an antibody that binds to PD-1 is unable to induce Fc-gamma RI-mediated effector function, or the induced Fc-gamma RI-mediated effector function is preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to a wild-type antibody.

[0303] In one embodiment, an antibody that binds to PD-1 is unable to induce at least one of complement-dependent cell-mediated lysis, antibody-dependent cell-mediated lysis, apoptosis, homotyped adhesion, and / or phagocytosis, or at least one of complement-dependent cell-mediated lysis, antibody-dependent cell-mediated lysis, apoptosis, homotyped adhesion, and / or phagocytosis is induced to a reduced degree, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%.

[0304] Antibody-dependent cell-mediated cytotoxicity is also referred to herein as "ADCC." ADCC describes the cytotoxicity of effector cells, particularly lymphocytes, as described herein, which preferably requires that the target cells are marked with an antibody.

[0305] ADCC preferably occurs when an antibody binds to an antigen on tumor cells and the antibody's Fc domain associates with an Fc receptor (FcR) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be seen as a mechanism for directly inducing varying degrees of immediate tumor destruction, resulting in antigen presentation and induction of antitumor T cell responses. Preferably, in vivo induction of ADCC results in antitumor T cell responses and host-derived antibody responses.

[0306] Complement-dependent cell injury is also referred to herein as "CDC." CDC is another method of cell toxicity that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also both very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex involves the C of an antibody molecule, such as an IgG molecule. H This results in the uncloaking of multiple adjacent C1q binding sites on the 2 domain (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert previous low-affinity C1q-IgG interactions to high-affinity ones, which induces a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of effector cell chemotactic / activating agents C3a and C5a. Preferably, the complement cascade terminates with the formation of membrane attack complexes, which can lead to apoptosis by creating pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0307] In one embodiment, the antibody that binds to PD-1 has reduced or depleted effector function. In one embodiment, the antibody does not mediate ADCC, CDC, or both.

[0308] In one embodiment, one or more, preferably both, heavy chain constant regions of an antibody that binds to PD-1 are modified compared to a wild-type antibody so as not to affect the binding of the antibody to the neonatal Fc receptor (FcRn).

[0309] In one embodiment, the PD-1 to which the antibody can bind is human PD-1. In one embodiment, PD-1 has or contains the amino acid sequence shown 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 shown in SEQ ID NO: 113 or SEQ ID NO: 114, or is an immunogenic fragment thereof. In one embodiment, the antibody has the ability to bind to the native epitope of PD-1 present on the surface of living cells.

[0310] In one embodiment, an antibody that binds to PD-1 includes a heavy chain constant region, where the heavy chain constant region includes an aromatic amino acid or a nonpolar amino acid at the position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at the position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering.

[0311] In this specification, the terms “amino acid corresponding to position…” and similar expressions refer to amino acid position numbers in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Therefore, an amino acid or segment in a sequence that “corresponds” to an amino acid or segment in another sequence is one that, when aligned with other amino acids or segments using a standard sequence alignment program such as ALIGN, ClustalW, or an analogue, typically with default settings, has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning sequences or segments within a sequence, thereby determining the positions in a sequence corresponding to the amino acid positions described herein, are considered to be well known in the art.

[0312] For example, referring to the amino acid sequence by SEQ ID NO: 93 in the sequence listing of this 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 in SEQ ID NO: 93, where F is located at position 117 (corresponding to position 234 in the human IgG1 heavy chain according to EU numbering), E is located at position 118 (corresponding to position 235 in the human IgG1 heavy chain according to EU numbering), and R is located at position 119 (corresponding to position 236 in the human IgG1 heavy chain according to EU numbering). In the sequence shown below, the FER amino acid sequence is underlined and shown in bold. TIFF2026513865000028.tif65151

[0313] Unless otherwise specified herein or unless explicitly stated otherwise in the context, all references to amino acid positions in the constant region of the antibody heavy chain throughout this disclosure refer to the positions corresponding to the respective positions in the human IgG1 heavy chain according to EU numbering as shown 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)).

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

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

[0316] As used herein, the term “Fc-mediated effector function” refers in particular to such functions 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 this 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 such function selected from the list of IgG Fc receptor (Fc-gamma-R, FcγR) binding, C1q binding, ADCC, or CDC, to a level of (i) the same CDR sequence including the same first antigen-binding region and second antigen-binding region, and (ii) two heavy chains including the human IgG1 hinge, CH2, and CH3 regions, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, compared to the human IgG1 antibody. “Depleted Fc-mediated effector function” or similar phrases include complete or essentially complete inhibition, i.e., reduction to zero or essentially zero.

[0318] In the context of this disclosure, the term "induces less Fc-mediated effector function" as used in relation to antibodies including multispecific antibodies means that the antibody induces less Fc-mediated effector function, particularly such function selected from the list of IgG Fc receptor (Fc-gamma-R, FcγR) binding, C1q binding, ADCC, or CDC, compared to (i) the antibody and a human IgG1 antibody containing the same CDR sequence, particularly including the same first and second antigen-binding regions, and (ii) two heavy chains containing the human IgG1 hinge, CH2, and CH3 regions.

[0319] Fc-mediated effector function can be determined by measuring the binding of the binder to the Fcγ receptor, to C1q, or the induction of Fc-mediated crosslinking of the Fcγ receptor. In particular, Fc-mediated effector function can be determined by measuring the binding of the binder to C1q and / or IgG FC-gamma RI.

[0320] In one embodiment of the use of antibodies that bind to PD-1, the amino acid at the position corresponding to position 236 in the 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 restrictive. Amino acids are organic compounds containing an amine (-NH2) functional group and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the context of this disclosure, amino acids may be classified based on their structure and chemical characteristics.

[0322] In this disclosure, amino acid residues are represented by the following abbreviations. Unless otherwise explicitly indicated, the amino acid sequences of peptides and proteins are identified from the N-terminus to the C-terminus (left to right), with the N-terminus being identified as the first residue. Amino acids are designated 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 divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and non-charged amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together 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 EU-numbered human IgG1 heavy chain 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 EU-numbered human IgG1 heavy chain is arginine (G236R). Such an amino acid substitution is also referred to herein as G236R. The term "G236R" indicates that at position 236 in the EU-numbered human IgG1 heavy chain, the amino acid glycine (G) is substituted with arginine (R). Within the scope of this disclosure, similar terms are used for other amino acid positions and amino acids. Unless otherwise indicated, the amino acid positions referred to in these terms are the amino acid positions in the EU-numbered human IgG1 heavy chain.

[0325] In one embodiment of the use of antibodies that bind to PD-1, the amino acid at the position corresponding to position 234 in the 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 antibodies that bind to PD-1, the amino acid at the position corresponding to position 234 in the 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 of the use of antibodies that bind to PD-1, the amino acid at the position corresponding to position 234 in the human IgG1 heavy chain according to EU numbering is phenylalanine (L234F).

[0328] The following table shows exemplary possible amino acid combinations for positions 234 and 236 in the human IgG mono-heavy chain, according to EU numbering.

[0329] (Table 5) TIFF2026513865000029.tif221106

[0330] For example, at positions 234 and 236 in the human IgG1 heavy chain according to EU numbering, the following amino acids may be present in the constant region of the heavy chain of antibodies that bind to 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 aforementioned amino acids or amino acid substitutions at positions 234 and 236 may be present in only one heavy chain of the antibody that binds to PD-1, or in both heavy chains of the antibody that binds to PD-1. Each amino acid present in the first and second heavy chains of the antibody may be selected independently of each other.

[0332] For example, at least one heavy chain of an antibody that binds to PD-1 may contain the following sequence (SEQ ID NO: 93). TIFF2026513865000030.tif58144

[0333] In one embodiment relating to an antibody that binds to PD-1, the amino acids at positions 234 and 236 of the human IgG1 heavy chain according to EU numbering are as explicitly stated above, and further, the amino acid at position 235 of the human IgG1 heavy chain according to EU numbering 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 position 235 of the human IgG1 heavy chain according to EU numbering is glutamic acid (L235E).

[0334] In one embodiment of an antibody that binds to PD-1, the amino acids at positions 234, 235, and 236 in the heavy chain constant region, 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] The following table shows exemplary possible amino acid combinations for positions 234, 235, and 236 in the human IgG mono-heavy chain, according to EU numbering.

[0336] (Table 6) TIFF2026513865000031.tif221163

[0337] For example, the following amino acids may be present in the constant region of the heavy chain of antibodies that bind to PD-1, particularly at positions 234, 235, and 236 of the 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 / 235D / 236R, 234F / 235L / 2 36R, 234W / 235L / 236R, 234Y / 235L / 236R, 234A / 235L / 236R, 234L / 235L / 23 6R, 234F / 235A / 236R, 234W / 235A / 236R, 234Y / 235A / 236R, 234A / 235A / 236R , 234L / 235A / 236R, 234F / 235E / 236K, 234W / 235E / 236K, 234Y / 235E / 236K, 2 34A / 235E / 236K, 234L / 235E / 236K, 234F / 235D / 236K, 234W / 235D / 236K, 234 Y / 235D / 236K, 234A / 235D / 236K, 234L / 235D / 236K, 234F / 235L / 236K, 234W / 235L / 236K, 234Y / 235L / 236K, 234A / 235L / 236K, 234L / 235L / 236K, 234F / 2 35A / 236K, 234W / 235A / 236K, 234Y / 235A / 236K, 234A / 235A / 236K, 234L / 235 A / 236K, 234F / 235E / 236H, 234W / 235E / 236H, 234Y / 235E / 236H, 234A / 235E / 236H, 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 aforementioned amino acids or amino acid substitutions at positions 234, 235, and 236 may 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 may be selected independently of each other.

[0339] For example, at least one heavy chain of an antibody that binds to PD-1 may contain the following sequence (SEQ ID NO: 90 or 93): TIFF2026513865000032.tif58144

[0340] For example, any rearrangement and combination of all described amino acid substitutions at positions 234, 236, and 235 in this application, as shown in Tables 5 and 6, should be considered disclosed by the description of this application where applicable, unless the context specifically indicates otherwise. For example, in one embodiment of the antibody, the first heavy chain contains the amino acid FER at positions 234-236 in the human IgG1 heavy chain as EU numbered, or the first heavy chain contains, is essentially, or consists of the amino acid sequence shown in SEQ ID NO: 93, and the second heavy chain of the antibody contains, is essentially, or consists of other amino acids, such as amino acids AAG or LLG, at positions 234-236 in the human IgG1 heavy chain as EU numbered, or the second heavy chain of the antibody contains, is essentially, or consists of the amino acid sequence shown in SEQ ID NO: 92 or 98. In another embodiment of the antibody, the first and second heavy chains include the same amino acids at positions 234-236 in the EU-numbered human IgG1 heavy chain, i.e., the same aromatic or nonpolar amino acid, e.g., F, at position 234 in the EU-numbered human IgG1 heavy chain, and the same amino acid other than glycine, e.g., R, at position 236 in the EU-numbered human IgG1 heavy chain, e.g., specific combinations such as FER or FLR.

[0341] In one embodiment, the antibody that binds to PD-1 comprises at least one or two heavy chain constant regions, where 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, the antibody that binds to PD-1 comprises one or more heavy chain constant regions (CH) 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 with the amino acid sequence of the heavy chain constant region sequence as shown in SEQ ID NO: 93.

[0343] In one embodiment, the antibody that binds to PD-1 comprises one or more, for example, two heavy chain constant regions (CH), where the heavy chain constant region comprises the sequence shown in SEQ ID NO: 93.

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

[0345] As used herein, the term "isotype" refers to an immunoglobulin class encoded by a heavy chain constant region gene. When IgG1 isotype is referred to herein, the term is used to indicate that an antibody sequence is closer to that isotype, e.g., IgG1, than to other isotypes, and is not limited to a specific isotype sequence, e.g., a particular IgG1 sequence. Thus, for example, an IgG1 antibody disclosed herein may be a sequence variant of a naturally occurring IgG1 antibody, including variations in the constant region.

[0346] IgG1 antibodies can exist in multiple polymorphic variants, referred to as allotypes (as outlined in Jefferis and Lefranc 2009, mAbs Vol 1 Issue 4 1-7), any of which is suitable for use in some embodiments of this specification. Common allotype variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any embodiment of this specification, the antibody may include a heavy chain Fc region containing a human IgG Fc region. In a further embodiment, the human IgG Fc region contains 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 within different isotypes of immunoglobulins, while the variable region is highly diverse and responsible for antigen recognition.

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

[0349] In one embodiment, the antibody that binds to PD-1 comprises a light chain having a light chain constant region (LC) containing a sequence that 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 with the amino acid sequence of the LC sequence as shown in SEQ ID NO: 97. In one embodiment, the antibody comprises a light chain having a light chain constant region (LC) containing a sequence as shown in SEQ ID NO: 97.

[0350] In one embodiment, the antibody that binds to PD-1 comprises a heavy chain containing 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 with the amino acid sequence shown in SEQ ID NO: 152, and a light chain containing 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 with the amino acid sequence shown in SEQ ID NO: 153. In one embodiment, the antibody that binds to PD-1 comprises a heavy chain containing the amino sequence shown in SEQ ID NO: 152, and a light chain containing the amino acid sequence shown in SEQ ID NO: 153.

[0351] In one embodiment of the present invention, the antibody that binds to PD-1 is a full-length IgG1 antibody, for example, IgG1,κ. In one embodiment of the present invention, the binder is a full-length human IgG1 antibody, for example, IgG1,κ.

[0352] In one embodiment, an antibody that binds to PD-1 can be derivatized, ligated, or co-expressed with other binding specificities. In another embodiment, the antibody can be derivatized, ligated, or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., a Fab' fragment). For example, it can be functionally ligated to one or more other molecular entities, e.g., another antibody (e.g., to produce a bispecific or multispecific antibody) (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise).

[0353] Antibodies that bind to PD-1 may 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 PD-1 may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random mutagenesis or site-directed mutagenesis, or in vivo by somatic mutation).

[0354] This disclosure includes the use of bispecific and multispecific molecules that have at least one first binding specificity to PD-1 and a second binding specificity (or further binding specificity) to a second target epitope (or further target epitopes).

[0355] In one embodiment, the first antigen-binding region of a multispecific antibody that binds to PD-1 includes a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown herein.

[0356] In one embodiment relating to the use of a multispecific antibody that binds to PD-1, the antibody comprises a first and second binding arm derived from a full-length antibody, for example, a full-length IgG1,λ (lambda) antibody or IgG1,κ (kappa) antibody as 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 isotype or IgG1,λ isotype, preferably comprising a human IgG1 / κ constant region or a human IgG1 / λ constant region.

[0357] The first antigen-binding region of the multispecific or bispecific antibody used in the present invention, which binds to PD-1, may include heavy-chain and light-chain variable regions of the antibody that compete for binding of PD-1 to PD-L1 and / or PD-L2. In one embodiment relating to the use of the multispecific or bispecific antibody, the first antigen-binding region that binds to PD-1 includes a heavy-chain variable region (VH) and / or a light-chain variable region (VL) as shown herein.

[0358] As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of the immune response, in contrast to the recognition and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., T cells including B cells and cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

[0359] "Target cells" means any undesirable cells in a subject (e.g., human or animal) that can be targeted by an antibody. In a preferred embodiment, the target cells are tumor cells.

[0360] The target of treatment and the tumor or cancer The subjects to be addressed by this disclosure are preferably human subjects.

[0361] In a preferred embodiment, the tumor or cancer is characterized by high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

[0362] 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. The tumor or cancer may be an unresectable tumor or cancer. The tumor or cancer may be a recurrent tumor or cancer.

[0363] In one embodiment, the tumor or cancer is a leukemia such as acute myeloid leukemia (AML).

[0364] 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, stomach cancer, breast cancer, kidney cancer, urothelial carcinoma, 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 lymphoma, non-Hodgkin 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.

[0365] Preferably, the tumor or cancer is selected from the group consisting of colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, pancreatic cancer, urinary tract cancer, bladder cancer, thyroid cancer, breast cancer, prostate cancer, ovarian cancer, central nervous system (CNS) cancer, and skin cancers such as melanoma, and more preferably, it is selected from the group consisting of colon cancer, gastric cancer, and endometrial cancer.

[0366] In a specific aspect, the tumor or cancer is endometrial cancer. Endometrial cancer is one of the most common gynecological malignancies globally, with over 400,000 new cases and over 97,000 deaths in 2020 (Globocan, 2020). The highest incidence rates are found in Northern America as well as Northern, Central, and Eastern Europe (Globocan, 2020). The global disease burden of endometrial cancer is also increasing, particularly in North America and Europe (Zhang et al., 2019). Approximately two-thirds of women with endometrial cancer present with early, localized uterine disease, which is typically treated surgically with or without radiotherapy and has a good outcome. However, women with recurrent and / or metastatic disease are incurable, with a 5-year survival rate of less than 20% and limited treatment options (SEER database, 2021). For many years, SoC frontline therapy for patients with previously untreated, unresectable and / or metastatic endometrial cancer has consisted of doublets or triplets of chemotherapy, with response rates of approximately 40%–50% and median survival in the range of 15 months (McMeekin et al., 2007; Miller et al., 2020), and there has been no significant progress in identifying subgroups with potentially different treatment needs. Endometrial cancer has historically been classified as either type I or type II cancer. Type I cancer accounts for approximately 85% of endometrial cancers and is generally characterized by a low to moderate endometrioid histological feature. Type II cancer includes non-endometrioid cases and is most commonly characterized by a papillary serous histological or clear cell histological feature. Importantly, studies have evaluated the variability of response rates across different histological subtypes, finding similar efficacy of SoC chemotherapy in both serous and endometrioid tumors, with an ORR of approximately 45% in both cases (McMeekin et al., 2007).Novel insights into the heterogeneity of endometrial tumors have emerged from genomic and transcriptome profiling of a large cohort of TCGA-treated endometrial tumors, resulting in a molecular classification of four subtypes of endometrial cancer, each with distinct molecular characteristics (Masood and Singh, 2021; Talhouk et al., 2015): POLE-mutated, ultramutated subtype (less than 10% of all endometrial cancers); dMMR or MSI-H, hyper-mutated subtype (25%-30% of all endometrial cancers); pMMR or MSS, CN low subtype (30%-40% of all endometrial cancers); pMMR or MSS, CN high subtype (25%-30% of all endometrial cancers). The PD-1 inhibitors dostallimab and pembrolizumab are currently approved by the FDA and EMA for second-line use in adult patients with recurrent or advanced endometrial cancer who are classified as dMMR or MSI-H as determined by FDA approval testing.

[0367] In one embodiment, the tumor is a PD-L1-positive tumor. In a particular embodiment, it is preferable that PD-L1 is expressed in 1% or more of cancer cells or tumor cells. In one embodiment, the tumor is a PD-L1-negative tumor. PD-L1 expression may be determined using techniques known to those skilled in the art, for example, by immunohistochemistry (IHC).

[0368] In one embodiment, the subject is progressing during or after at least one prior treatment regimen line for the unresectable and / or metastatic tumor or cancer. According to a preferred embodiment, the treatment regimen is systemic chemotherapy, such as platinum-based chemotherapy. According to this embodiment, the tumor or cancer is preferably endometrial cancer.

[0369] In one embodiment, the subject has previously received treatment with a checkpoint inhibitor. The checkpoint inhibitor is, for example, a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. The PD-1 inhibitor or PD-L1 inhibitor is administered as monotherapy or as part of combination therapy. In a particular embodiment, the subject is progressing after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. According to this embodiment, the tumor or cancer is preferably endometrial cancer.

[0370] In a further embodiment, the subject has not been previously treated with a checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG3 antibody, or an anti-TIGIT antibody. According to this embodiment, the tumor or cancer is preferably endometrial cancer.

[0371] Treatment regimen The binder and PD-1 inhibitor can be administered by any suitable method, for example, intravenously, intra-arterially, subcutaneously, intradermally, intramuscularly, intranodally, or intratumorally.

[0372] In one embodiment of the first aspect, the binder is administered to the subject by systemic administration. Preferably, the binder is administered to the subject by intravenous injection or infusion. In one embodiment, the binder is administered in at least one treatment cycle.

[0373] In one embodiment, the PD-1 inhibitor is administered to the subject, particularly by systemic administration. 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.

[0374] In one embodiment, the binder and the PD-1 inhibitor are administered to the subject, particularly by systemic administration. Preferably, the binder and the PD-1 inhibitor are administered to the subject by intravenous injection or infusion. In one embodiment, the binder and the PD-1 inhibitor are administered in at least one treatment cycle.

[0375] In one embodiment, each treatment cycle is approximately 2 weeks (14 days), 3 weeks (21 days), 4 weeks (28 days), 5 weeks (35 days), or 6 weeks (42 days). In a preferred embodiment, each treatment cycle is 3 weeks (21 days). In another preferred embodiment, each treatment cycle is 6 weeks (42 days).

[0376] In certain embodiments, one dose of the binder is administered or infused every two weeks (1Q2W), every three weeks (1Q3W), or every four weeks (1Q4W), every five weeks (1Q5W), or every six weeks (1Q6W), preferably every three weeks (1Q3W) or every six weeks (1Q6W).

[0377] In certain embodiments, one dose of the binder and one dose of the PD-1 inhibitor are administered or infused every two weeks (1Q2W), every three weeks (1Q3W), or every four weeks (1Q4W), every five weeks (1Q5W), or every six weeks (1Q6W), preferably every three weeks (1Q3W) or every six weeks (1Q6W).

[0378] In some embodiments, one dose or each dose is administered or infused on day 1 of each treatment cycle. For example, one dose of the binder and one dose of the PD-1 inhibitor may be administered on day 1 of each treatment cycle.

[0379] In some embodiments, a 100 mg dose (or approximately 1.25 mg / kg body weight) of the binder is administered every three weeks (1Q3W).

[0380] In some embodiments, a 100 mg dose (or approximately 1.25 mg / kg body weight) of the binder is administered every three weeks (1Q3W) over one or more treatment cycles, followed by a 500 mg dose (or approximately 6.25 mg / kg body weight) of the binder every six weeks (1Q6W) over one or more treatment cycles.

[0381] In some embodiments, a 100 mg dose (or approximately 1.25 mg / kg body weight) of a binder, preferably akasanlimab or its biosimilar, is administered every three weeks (1Q3W) over two treatment cycles, followed preferably by a 500 mg dose (or approximately 6.25 mg / kg body weight) of the binder every six weeks (1Q6W) over one or more treatment cycles, until complete tumor regression or disease progression.

[0382] In some embodiments, a 100 mg dose (or approximately 1.25 mg / kg body weight) of the binder is administered every 6 weeks (1Q6W).

[0383] In some embodiments, a 100 mg dose (or approximately 1.25 mg / kg body weight) of the binder and a 200 mg dose of the PD-1 inhibitor are administered every three weeks (1Q3W).

[0384] In some embodiments, a 100 mg dose (or approximately 1.25 mg / kg body weight) of the binder and a 400 mg dose of the PD-1 inhibitor are administered every six weeks (1Q6W).

[0385] In a particular embodiment, a 100 mg dose (or approximately 1.25 mg / kg body weight) of a conjugate, which is akasanlimab or a biosimilar thereof, and a 200 mg dose of a PD-1 inhibitor, which is pembrolizumab or a biosimilar thereof, are administered every three weeks (1Q3W), for example, on day 1 of each three-week treatment cycle.

[0386] In a particular embodiment, a 100 mg dose (or approximately 1.25 mg / kg body weight) of a conjugate, which is akasanlimab or a biosimilar thereof, and a 400 mg dose of a PD-1 inhibitor, which is pembrolizumab or a biosimilar thereof, are administered every six weeks (1Q6W), for example, on day 1 of each six-week treatment cycle.

[0387] The PD-1 inhibitor may be administered first, followed by the conjugate. Alternatively, the conjugate may be administered first, followed by the PD-1 inhibitor.

[0388] Each dose may be administered or infused over a minimum of 30 minutes, for example, a minimum of 60 minutes, a minimum of 90 minutes, a minimum of 120 minutes, or a minimum of 240 minutes.

[0389] The binder may be administered over 30 minutes, for example, over a minimum of 40 minutes, a minimum of 50 minutes, or for example, a minimum of 60 minutes, particularly by intravenous (IV) infusion.

[0390] PD-1 inhibitors may be administered, in particular, as intravenous fluids, over a period of 30 minutes, for example, over a minimum of 40 minutes, a minimum of 50 minutes, or for example, a minimum of 60 minutes.

[0391] The binder and the PD-1 inhibitor may be administered simultaneously. In an alternative, preferred embodiment, the binder and the PD-1 inhibitor are administered separately.

[0392] The binder and the PD-1 inhibitor may be administered in any suitable form (e.g., naked). However, it is preferable that the binder and the PD-1 inhibitor be administered in the form of any suitable pharmaceutical composition as described herein. In one embodiment, at least the binder and the PD-1 inhibitor are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binder and one pharmaceutical composition for the PD-1 inhibitor), and preferably the binder and the PD-1 inhibitor are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binder and one pharmaceutical composition for the PD-1 inhibitor).

[0393] The composition or pharmaceutical composition, together with carriers, excipients, and / or diluents, and any other components suitable for a pharmaceutical composition including known adjuvants, Remington: The Science and Practice of Pharmacy, 19 th The formulation may be prepared according to conventional techniques, such as those disclosed in Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. A pharmaceutically acceptable carrier or diluent, and any known adjuvants and excipients, should be suitable for the binder and / or PD-1 inhibitor, as well as the chosen mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined based on the absence of significant negative effects on the desired biological properties of the chosen compound or pharmaceutical composition (e.g., less than substantial effect at antigen binding [e.g., relative inhibition of ≤10%, relative inhibition of ≤5%]).

[0394] The compositions, in particular the pharmaceutical compositions of binders and the pharmaceutical compositions of PD-1 inhibitors, may include diluents, expanders, salts, buffers, surfactants (e.g., nonionic surfactants, e.g., Tween-20 or Tween-80), stabilizers (e.g., sugar or protein-free amino acids), preservatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition.

[0395] Pharmacokinetically 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).

[0396] Pharmaceutical carriers, excipients, or diluents may be selected considering the intended route of administration and standard pharmaceutical practices.

[0397] Pharmacopoecially acceptable carriers include any and all suitable solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption retarders that are physiologically compatible with the active compound, particularly binders and PD-1 inhibitors.

[0398] Examples of suitable aqueous and non-aqueous carriers that may be used in (pharmaceutical) compositions include water, saline solution, phosphate-buffered saline solution, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil), carboxymethylcellulose colloidal solutions, tragacanth gum, and organic esters for injection (e.g., ethyl oleate), and / or various buffers. Other carriers are well known in the pharmaceutical art.

[0399] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in (pharmaceutical) compositions is intended unless any conventional media or agent is incompatible with the active compound.

[0400] As used herein, the term “excipient” refers to a substance that may be present in the (pharmaceutical) compositions of this 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 colorants.

[0401] The term “diluent” refers to an agent that dilutes and / or reduces viscosity. Furthermore, the term “diluent” includes any one or more fluids, liquids, or solid suspensions and / or mixtures. Examples of suitable diluents include ethanol, glycerol, and water.

[0402] The (pharmaceutical) composition may also contain pharmaceutically acceptable antioxidants, for example, (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0403] The (pharmaceutical) composition may also contain isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol, or sodium chloride.

[0404] The (pharmaceutical) composition may also contain one or more adjuvants suitable for a selected route of administration, such as preservatives, humectants, emulsifiers, dispersants, or buffers, which may enhance the shelf life or efficacy of the composition. The compositions used herein may be prepared with a carrier that protects the compound from rapid release, such as sustained-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with wax, or other materials well 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, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0405] "Pharmacologically acceptable salts" include, for example, acid addition salts that can be formed using pharmaceutically acceptable acids such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Furthermore, suitable pharmaceutically acceptable salts include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and ammonium (NH4) +); and may include salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations formed using counter anions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfons, and aryl sulfons).Examples of pharmaceutically acceptable salts include acetate, adipine, alginate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, tartrate, borate, bromide, butyrate, calcium edetate, canhorate, camphor sulfonate, cansilate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, and edetate. Toate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, arsanilate glycolyl, hemisulfate, heptaneate, hexanoate, hexylresorcinate, hydravamin, hydrobromide, hydrochloride, hydroiodide, 2-hydr Roxy-ethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mutinate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pa This includes, but is not limited to, motates (embonates), palmitates, pantothenates, pectates, persulfates, 3-phenylpropionates, phosphates / diphosphates, phthalates, picrates, pivalates, polygalacturonates, propions, salicylates, stearates, sulfates, suberates, succinates, tannates, tartrates, theoclates, tosylates, triethiozides, undecanoates, valersates, etc. (see, for example, SM 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 this is included in the disclosure.

[0406] In one embodiment, the binders and PD-1 inhibitors used herein may be formulated to ensure appropriate distribution in vivo. Pharmacopoeia-acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in compositions is intended unless any conventional media or agent is incompatible with the active compound. Other active or therapeutic compounds may also be incorporated into the compositions.

[0407] Pharmaceutical compositions for injection must typically be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Carriers may be aqueous or non-aqueous solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and organic esters for injection (e.g., ethyl oleate). Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Often, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols (e.g., glycerol, mannitol, sorbitol), or sodium chloride. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents (e.g., monostearate and gelatin) in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, along with one or a combination of components, if necessary, such as those listed above, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components, such as those listed above. For sterile powders for the preparation of sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying (lyophilization) of the active ingredient powder, with any additional desired components added, resulting from a pre-sterilized filtered solution.

[0408] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, along with one or a combination of the components listed above, if necessary, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying (lyophilization) of the active ingredient powder, with any additional desired components added, resulting from a pre-sterilized filtered solution.

[0409] In certain embodiments, the binder for use according to the present invention is formulated into a composition or formulation comprising histidine, sucrose, and polysorbate-80, and having a pH of about 5 to about 6, for example, 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, about 0.02% polysorbate-80, and having a pH of about 5.5, for example, in a composition or formulation comprising 20 mM histidine, 250 mM sucrose, 0.02% polysorbate-80, and having a pH of 5.5. In certain embodiments, the formulation may contain about 10 to about 30 mg of binder / mL, for example, 10 to 30 mg of binder / mL, in particular about 20 mg of binder / mL, for example, 20 mg of binder / mL.

[0410] The binder for use according to the present invention may be provided in a composition as defined above, and then diluted in 0.9% NaCl (saline solution) before administration.

[0411] In a second aspect, the Disclosure provides a conjugate for use in a method for treating a tumor or cancer in a subject, the method comprising the step of administering to the subject a conjugate comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). The aspects disclosed herein with respect to the first aspect (particularly with respect to conjugates, PD-1 inhibitors, treatment regimens, specific tumors / cancers, and subjects) also apply to conjugates for use in the second aspect.

[0412] In a third aspect, the present disclosure provides a pharmaceutical composition for use in a method for treating a tumor or cancer in a subject, comprising a binder comprising a first binding domain for binding to CD137 and a second binding domain for binding to PD-L1, and optionally a pharmaceutically acceptable carrier, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). The aspects disclosed herein with respect to the first aspect (particularly with respect to the binder, PD-1 inhibitor, treatment regimen, specific tumors / cancers, and subjects) also apply to the pharmaceutical composition for use in the third aspect.

[0413] In a fourth aspect, the Disclosure provides the use of a binder for the manufacture of a medicament for treating a tumor or cancer in a subject, wherein the tumor or cancer is a high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), and the binder comprises a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1. The aspects disclosed herein with respect to the first aspect (particularly with respect to the binder, PD-1 inhibitors, treatment regimens, specific tumors / cancers, and subjects) also apply to the use in the fourth aspect.

[0414] In a fifth aspect, the present disclosure provides a kit for use in a method for treating a tumor or cancer in a subject, comprising (a) a conjugate comprising a first binding domain for binding to CD137 and a second binding domain for binding to PD-L1, and (b) a PD1 inhibitor, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). The embodiments disclosed herein with respect to the first aspect (particularly with respect to the conjugate and the PD-1 inhibitor) also apply to the kit in the fifth aspect. In one embodiment, the kit comprises at least two containers, one of which contains the conjugate (either by itself or in the form of a (pharmaceutical) composition), and the second container contains the PD-1 inhibitor (either by itself or in the form of a (pharmaceutical) composition.

[0415] The references to documents and studies made herein are not intended as an endorsement that any of the foregoing are relevant prior art. All statements relating to the contents of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the contents of these documents.

[0416] The description (including the following embodiments) is presented so that those skilled in the art can construct and use various embodiments. Descriptions of specific apparatus, techniques, and applications are provided merely as examples. Various modifications to the embodiments 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 embodiments described and shown herein, but should be consistent with the scope of the claims.

[0417] Items of this disclosure Item 1 A method for treating a tumor or cancer in a subject, the method comprising administering to the subject a binder comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). Item 2 The method of item 1, wherein PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence shown in SEQ ID NO: 40; and / or CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO: 38. Item 3 (a) The method of item 1 or 2, wherein the first binding region of the binder comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 2, 3, and 4, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GAS, and SEQ ID NO: 8, respectively; and (b) the second binding region of the binder comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 12, 13, and 14, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 16, DDN, and SEQ ID NO: 18, respectively. Item 4 (a) The first binding region of the binder includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 5; and (b) The second binding region of the binder includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 11 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 15, One of the methods described above. Item 5 The method according to any one of the above items, wherein the binder is a multispecific antibody, for example, a bispecific antibody. Item 6 The method of any one of the above items, wherein the binder is in the form of a full-length antibody or an antibody fragment. Item 7 The binder is an antibody comprising a first binding arm and a second binding arm. The first connecting arm, (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and (ii) A polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). including; and the second connecting arm, (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and (iv) Polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL) including, One of the methods described above. Item 8 The aforementioned 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; and (ii) A second heavy chain and 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. One of the methods of the above items, including: Item 9 (i) The amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) The amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain. Method 7 or 8. Item 10 One of the methods described in items 7 to 9, 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 heavy chain and the second heavy chain, respectively. Item 11 One of the methods described in items 7-10, wherein the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A in the first heavy chain constant region (HC) and the second heavy chain constant region (HC), respectively. Item 12 The positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, and (i) The position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) The position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R, and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L. Choose one of the methods from items 7 through 11. Item 13 The EU numbering of positions L234, L235, and D265 in the human IgG1 heavy chain in both the first and second heavy chain constant regions corresponds to positions F, E, and A, respectively, and (i) The position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) The position of the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L. Choose one of the methods from items 7 to 12. Item 14 The steady region of the first heavy chain and / or the second heavy chain, for example, the second heavy chain, (a) Sequence shown in SEQ ID NO: 24 or 30 [IgG1-Fc_FEAL]; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 6 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. It contains, or is essentially, an amino acid sequence selected from the group consisting of, Choose one of the methods from items 7 to 13. Item 15 The first heavy chain and / or the second heavy chain, for example, the steady region of the first heavy chain, (a) Sequence shown in SEQ ID NO: 23 or 29 [IgG1-Fc_FEAR]; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and (c) A sequence having at most 6 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 5 substitutions, at most 4, at most 3, at most 2, or at most 1 substitution. It contains, or is essentially, an amino acid sequence selected from the group consisting of, Choose one of the methods from items 7 to 14. Item 16 The method according to any one of items 7 to 15, wherein the binder comprises a kappa (κ) light chain constant region. Item 17 The method according to any one of items 7 to 16, wherein the binder includes a constant region of the lambda (λ) light chain. Item 18 The method described in items 7-17, wherein the first light chain steady region is either a kappa (κ) light chain steady region or a lambda (λ) light chain steady region. Item 19 The method described above in any one of items 7-18, wherein the second light chain steady region is either a lambda (λ) light chain steady region or a kappa (κ) light chain steady region. Item 20 One of the methods described in items 7 to 19, wherein the first light chain steady region is a kappa (κ) light chain steady region and the second light chain steady region is a lambda (λ) light chain steady region, or the first light chain steady region is a lambda (λ) light chain steady region and the second light chain steady region is a kappa (κ) light chain steady region. Item 21 Kappa (κ) light chain, (a) Sequence shown in SEQ ID NO: 35; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 substitutions, or at most 1 substitution. One of items 16-20, comprising an amino acid sequence selected from the group consisting of the following. Item 22 The lambda (λ) light chain, (a) Sequence shown in SEQ ID NO: 36; (b) A subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 substitutions, or at most 1 substitution. One of the methods from items 17 to 21, comprising an amino acid sequence selected from the group consisting of the following. Item 23 The method of any one of the above items, wherein the binder is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Item 24 The method according to any one of the above items, wherein the binder is a full-length IgG1 antibody. Item 25 The method according to any one of the above items, wherein the binder is an antibody of the IgG1m(f) allotype. Item 26 The binder is a bispecific antibody that binds to CD137 and PD-L1, and the bispecific antibody is (i) A first heavy chain containing the amino acid sequence shown in SEQ ID NO: 31 and a first light chain containing the amino acid sequence shown in SEQ ID NO: 32, (ii) A second heavy chain containing the amino acid sequence shown in SEQ ID NO: 33 and a second light chain containing the amino acid sequence shown in SEQ ID NO: 34 A method having any one of the above items. Item 27 The method of any one of the above items, wherein the binder is akasanlimab or a biosimilar thereof. Item 28 The method of any one of the above items, wherein the binder is in a composition or formulation comprising histidine, sucrose, and polysorbate-80, and has a pH of 5 to 6. Item 29 The aforementioned binder is A composition or formulation containing approximately 20 mM histidine, approximately 250 mM sucrose, and approximately 0.02% polysorbate-80, and having a pH of approximately 5.5. One of the methods listed above. Item 30 Any one of the above methods, wherein the binder is present in a composition or formulation containing 10 to 30 mg of binder / mL, for example, 20 mg of binder / mL. Item 31 The method of any one of the items, wherein the binder is in a composition as defined in any one of items 28 to 30, and is diluted in 0.9% NaCl (saline solution) before administration. Item 32 Any one of the above items, further comprising the step of administering a PD-1 inhibitor to the subject. Item 33 PD-1 is human PD-1, and preferably PD-1 has or contains the amino acid sequence shown in SEQ ID NO: 113 or SEQ ID NO: 114, or The method of item 32, wherein the amino acid sequence of PD-1 is 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% identical to the amino acid sequence shown in SEQ ID NO: 113 or SEQ ID NO: 114, or is an immunogenic fragment thereof. Item 34 The method of item 32 or 33, wherein the PD-1 inhibitor is an antibody that binds to PD-1 or PD-L1, preferably an antibody that is a PD-1 / PD-L1 interaction antagonist, and / or an antibody that is a PD-1 or PD-L1 blocking antibody. Item 35 One of the methods described in items 32 to 34, wherein the PD-1 inhibitor is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, for example, an antibody of the IgG1 isotype. Item 36 The PD-1 inhibitor is a full-length antibody or an antibody fragment, for example, a full-length IgG1 antibody, according to any one of items 32-35. Item 37 The method according to any one of items 32-36, wherein the PD-1 inhibitor is a monospecific antibody. Item 38 One of the methods described in items 32 to 37, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 59, 60, and 61, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 62, LAS, and SEQ ID NO: 64, respectively. Item 39 One of the methods described in items 32 to 38, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising a VH region containing the amino acid sequence of SEQ ID NO: 65 and a VL region containing the amino acid sequence of SEQ ID NO: 66. Item 40 One of the methods described in items 32-39, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 67 and a light chain containing the amino acid sequence of SEQ ID NO: 68. Item 41 The method according to any one of items 32-40, wherein the PD-1 inhibitor is pembrolizumab or a biosimilar thereof. Item 42 The method according to any one of items 32 to 41, wherein the binder is akasanlimab or a biosimilar thereof, and the PD-1 inhibitor is pembrolizumab or a biosimilar thereof. Item 43 The method according to any one of items 32 to 37, wherein the PD-1 inhibitor is an antibody or antigen-binding fragment that binds to PD-1, and the antibody that binds to PD-1 comprises VH regions CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NO: 104, 101, and 100, respectively, and VL regions CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NO: 107, QAS, and SEQ ID NO: 105, respectively. Item 44 The method of item 43, wherein the antibody that binds to PD-1 includes a heavy chain variable region (VH) containing 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 with the amino acid sequence of the VH sequence as shown in SEQ ID NO: 111. Item 45 The method of item 44, wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH), and the VH comprises the sequence shown in SEQ ID NO: 111. Item 46 One of items 43 to 45, wherein the antibody that binds to PD-1 includes a light chain variable region (VL) 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 with the amino acid sequence of the VL sequence as shown in SEQ ID NO: 112. Item 47 The method of item 46, wherein the antibody that binds to PD-1 includes a light chain variable region (VL), and the VL includes the sequence shown in SEQ ID NO: 112. Item 48 The method according to any one of items 43 to 47, wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises or has the sequence shown in SEQ ID NO: 111, and the VL comprises or has the sequence shown in SEQ ID NO: 112. Item 49 The antibody that binds to PD-1 includes 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 L234 in the human IgG1 heavy chain according to EU numbering is phenylalanine, the amino acid corresponding to position L235 in the human IgG1 heavy chain according to EU numbering is glutamic acid, and the amino acid corresponding to position G236 in the human IgG1 heavy chain according to EU numbering is arginine (L234F / L235E / G236R), one of the methods described in items 43 to 48. Item 50 One of items 43 to 49, wherein the heavy chain constant region of the antibody that binds to PD-1 contains 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 with the amino acid sequence of the HC sequence as shown in SEQ ID NO: 93. Item 51 One of items 43-50, wherein the heavy chain constant region of the antibody that binds to PD-1 contains the sequence shown in SEQ ID NO: 93. Item 52 One of items 43-51, wherein the isotype of the heavy chain constant region of the antibody that binds to PD-1 is IgG1. Item 53 The method described in items 43-52, wherein the antibody that binds to PD-1 is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fragment of such an antibody. Item 54 Any one of the above methods, wherein the binder is akasanlimab or a biosimilar thereof, and the PD-1 inhibitor is an antibody that binds to PD-1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 152 and a light chain containing the amino acid sequence of SEQ ID NO: 153. Item 55 The PD-1 inhibitor is a multispecific antibody, for example, a bispecific antibody, according to any one of items 32 to 36. Item 56 The method according to any one of items 32-37, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, cosivelimab, AUNP12, CA-170, BMS-986189, or a biosimilar thereof. Item 57 The method described in item 32-37, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, cosivelimab, or any biosimilar thereof. Item 58 The method according to any one of items 32-37, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, or a biosimilar thereof. Item 59 Any one of the above methods, wherein the subject is a human subject. Item 60 The method of the item, wherein the binder is administered in at least one treatment cycle, each treatment cycle being 3 weeks (21 days) or 6 weeks (42 days). Item 61 One of the above methods, wherein one dose of the binder is administered every three weeks (1Q3W) or every six weeks (1Q6W). Item 62 Any one of the above methods, wherein one dose of the binder is administered on the first day of each treatment cycle. Item 63 One of the methods described above, wherein the amount of the binder administered in each dose and / or in each treatment cycle is 100 mg or 500 mg. Item 64 Any one of the above methods, wherein a 100 mg dose of the binder is administered every three weeks (1Q3W). Item 65 Any one of the above methods, wherein a 100 mg dose of the binder is administered every three weeks (1Q3W) over two treatment cycles, and then, preferably, a 500 mg dose of the binder is administered every six weeks (1Q6W) over one or more treatment cycles until complete tumor regression or disease progression. Item 66 The PD-1 inhibitor is administered in at least one treatment cycle, each treatment cycle being 3 weeks (21 days) or 6 weeks (42 days), according to one of items 32-64. Item 67 One of the methods described in items 32-64 and 66, in which one dose of the PD-1 inhibitor is administered every three weeks (1Q3W) or every six weeks (1Q6W). Item 68 One of the methods described in items 32-64, 66, and 67, wherein one dose of the PD-1 inhibitor is administered on day 1 of each treatment cycle. Item 69 One of the methods described in items 32-64, 66-68, wherein the amount of the PD-1 inhibitor administered at each dose and / or in each treatment cycle is 200 mg or 400 mg. Item 70 One of the methods described in items 32-64 and 66-69, wherein a 100 mg dose of the binder and a 200 mg dose of the PD-1 inhibitor are administered every three weeks (1Q3W). Item 71 One of the methods described in items 32-64, 66-70, in which a 100 mg dose of a conjugate, akasanlimab or its biosimilar, and a 200 mg dose of a PD-1 inhibitor, pembrolizumab or its biosimilar, is administered every three weeks (1Q3W), for example, on day 1 of each three-week treatment cycle. Item 72 One of the methods described in items 32-64 and 66-69, wherein a 100 mg dose of the binder and a 400 mg dose of the PD-1 inhibitor are administered every 6 weeks (1Q6W). Item 73 One of the following methods, items 32-64, 66-69, and 72, is used: a 100 mg dose of a conjugate, akasanlimab or its biosimilar, and a 400 mg dose of a PD-1 inhibitor, pembrolizumab or its biosimilar, administered every six weeks (1Q6W), for example, on day 1 of each six-week treatment cycle. Item 74 The method according to any one of items 32 to 73, wherein the PD-1 inhibitor is administered first, followed by the conjugate, preferably the administration of the conjugate begins at least 30 minutes after the end of the administration of the PD-1 inhibitor. Item 75 Any one of the above methods, wherein the tumor or cancer is a solid tumor or leukemia, preferably a solid tumor. Item 76 The tumor or cancer is selected from the group consisting of colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, pancreatic cancer, urinary tract cancer, bladder cancer, thyroid cancer, breast cancer, prostate cancer, ovarian cancer, central nervous system (CNS) cancer, and skin cancers such as melanoma, preferably selected from the group consisting of colorectal cancer, gastric cancer, and endometrial cancer, one of the above methods. Item 77 The method of item 76, wherein the tumor or cancer is endometrial cancer. Item 78 The method of item 76, wherein the tumor or cancer is colorectal cancer. Item 79 The method of item 76, wherein the tumor or cancer is gastric cancer. Item 80 Any one of the above methods of the tumor or cancer being unresectable, recurrent, and / or metastatic. Item 81 Any one of the above methods, wherein the subject is undergoing or following at least one prior treatment regimen for the unresectable and / or metastatic tumor or cancer, preferably during or after systemic chemotherapy such as platinum-based chemotherapy. Item 82 The method of any one of the above items, wherein the subject has not previously received treatment with a checkpoint inhibitor, such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG3 antibody, or anti-TIGIT antibody. Item 83 The subject has previously received prior treatment with a PD-1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, and the PD-1 inhibitor or PD-L1 inhibitor is administered either as monotherapy or as part of a combination therapy, according to one of items 1 to 81. Item 84 The method of item 83, wherein the subject is progressing after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. Item 85 A conjugate for use in a method for treating a tumor or cancer in a subject, the method comprising the step of administering to the subject a conjugate comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). Item 86 A binder for use in item 85, wherein the method is as defined in any one of items 1 to 84, and / or the binder is as defined in any one of items 1 to 84. Item 87 A pharmaceutical composition for use in a method for treating a tumor or cancer in a subject, comprising a binder having a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, and optionally a pharmaceutically acceptable carrier, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). Item 88 A pharmaceutical composition for use in item 87, wherein the method is as defined in any one of items 1 to 84, and / or the binder is as defined in any one of items 1 to 84. Item 89 Use of a binder for the manufacture of a pharmaceutical product for treating a tumor or cancer in a subject, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), and the binder comprises a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1. Item 90 The use of the binder of item 89, wherein the binder is as defined in any one of items 1 to 84. Item 91 A kit for use in a method for treating tumors or cancers in a subject, wherein the tumor or cancer is characterized by high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), (a) A binder comprising a first binding region that binds to CD137 and a second binding region that binds to PD-L1, and (b) PD-1 inhibitors A kit for the aforementioned use, including the above. Item 92 A kit for use of item 91, wherein the method is as defined in any one of items 1 to 84, and / or the binder is as defined in any one of items 1 to 84, and / or the PD-1 inhibitor is as defined in any one of items 1 to 84.

[0418] Further aspects of this disclosure are disclosed herein. [Examples]

[0419] Example 1 Clinical trial GCT1046-01 (ClinicalTrials.gov Identifier: NCT03917381) was designed as an open-label, multicenter, Phase I / IIa trial of GEN1046 (DuoBody®-PD-L1×4-1BB). The trial consisted of two parts: first-in-human (FIH) dose escalation (Phase I) and dose expansion (Phase IIa). In the expanded cohort 4 of the GCT1046-01 trial, women aged 18 years or older with endometrial cancer who had received up to four prior systemic treatment regimens for advanced / metastatic disease, had radiographic disease progression at or after the last prior treatment, had epithelial endometrial tissue including endometrioid, serous, squamous, clear cell carcinoma, or carcinosarcoma, and had not received prior treatment with PD-1 / L1 inhibitors were administered GEN1046 100 mg in 1Q3W. Subjects were treated until disease progression (PD), excessive toxicity, or withdrawal of consent. All treated subjects had measurable disease. Tumor response was assessed every 6 weeks (±7 days) for 50 weeks from the first administration date to PD, and then every 12 weeks (±7 days) thereafter, according to RECIST 1.1.

[0420] In the expanded cohort 4 of the GCT1046-01 trial, GEN1046 was administered to 40 subjects with endometrial cancer. Of these, 33 had microsatellite stability (MSS) disease, and 7 had high microsatellite instability (MSI-H) disease.

[0421] At the data cutoff date (January 12, 2023), preliminary data showed that 3 out of 40 subjects (7.5%) were still undergoing treatment, while 37 subjects (92.5%) had discontinued treatment. Of the 37 subjects who discontinued treatment, 28 (70%) had confirmed disease progression (PD) on radiography, 5 (12.5%) had clinically confirmed PD, 2 (5%) experienced adverse events (AEs), and 2 (5%) withdrew their consent.

[0422] Of the 40 subjects, 37 were eligible for evaluation of response. Best overall response (BOR), objective response rate (ORR), and disease control rate (DCR) are shown in Table 7.

[0423] The temporal changes in the target lesion are shown in Figure 1 (spider plot; all subjects), and the best overall changes in the target lesion are shown in Figure 2 (waterfall plot; all subjects), Figure 3 (waterfall plot; MSS subjects), and Figure 4 (waterfall plot; MSI-H subjects). Of the 7 subjects with MSI-H tumors, 3 (42.9%) achieved a partial response (PR). On the other hand, of the 33 subjects with MSS tumors, 1 (3%) achieved a PR. Preliminary data suggest that patients with MSI-H tumors have a higher response rate to GEN1046.

[0424] The safety of GEN1046 as monotherapy was evaluated in pooled analyses of 358 subjects, including those with endometrial cancer, in GCT1046-01 and GCT1046-02, with a data cutoff date of April 1, 2022. GEN1046 monotherapy was generally well-tolerated, and there were no indications that the safety profile of GEN1046 differed across tumor types.

[0425] (Table 7) Best overall response, objective response rate, and disease control rate - all subjects - FAS TIFF2026513865000033.tif89128cPR=Confirmed partial response;CR=Complete response;NE=Not quantifiable;PD=Disease progression;PR=Partial response;SD=Stable disease;uPR=Unconfirmed partial response

[0426] Example 2: Proliferation of colon cancer tumors in MC38 mice method MC38 mouse colon cancer cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. MC38 cells were collected from cell cultures undergoing logarithmic growth and quantified.

[0427] MC38 cells (1 × 10⁶ in 100 L PBS) 6 Individual tumor cells were subcutaneously injected into the right lower flank of female C57BL / 6 mice (obtained from Vital River Laboratories Research Models and Services; 6-8 weeks old at the start of the experiment).

[0428] Tumor growth was evaluated three times a week using calipers. Tumor volume (mm 3 ) from caliper measurements ([length] × [width] 2 The formula is calculated as () / 2, where length is the longest tumor dimension and width is the longest tumor dimension perpendicular to that length.

[0429] The procedure was performed when the tumor was 64 mm 3 The procedure was initiated when the median volume was reached. Mice were given equal mean tumor volumes (64 mm) before treatment. 3 Mice were randomized to a group having the following characteristics (n=10 / group). On the treatment day, mice were intraperitoneally injected with mbsIgG2a-PD-L1×4-1BB (5 mg / kg; 10 μL / g body weight injection volume; two doses weekly for 3 weeks [2QW×3]), anti-mouse PD-1 antibody (anti-mPD-1; 10 mg / kg; 10 μL / g body weight injection volume; 2QW×3; clone RMP1-14; Leinco Technologies, catalog no. P372), a combination of mbsIgG2a-PD-L1×4-1BB (5 mg / kg) and anti-mPD-1 (10 mg / kg) (two separate injections of 10 μL / g body weight injection volume [mbsIgG2a-PD-L1×4-1BB followed by anti-mPD-1 20 minutes later]; 2QW×3), or 10 μL / g body weight injection volume of PBS (Table 8).

[0430] Mice were monitored daily for clinical signs of the disease. Body weight was measured three times a week after randomization. Tumor volume was 1500 mm². 3The experiment was terminated for individual mice when the following conditions were exceeded, or when the animal reached a humane endpoint (e.g., when a mouse showed a 20% weight loss, when a tumor showed ulceration [>75%], when severe clinical signs were observed, and / or when tumor growth blocked the mouse's physical activity).

[0431] (Table 8) Treatment groups and medication regimens TIFF2026513865000034.tif89167 a 2QW×3: Two doses per week for three weeks.

[0432] result Rapid tumor growth was observed in MC38-carrying mice treated with PBS (Figure 6A). Delayed tumor growth was observed in mice treated with anti-mPD-1 (10 mg / kg) or mbsIgG2a-PD-L1×4-1BB (5 mg / kg), with a more pronounced delay in tumor growth induced by mbsIgG2a-PD-L1×4-1BB (Figure 6A). In mice treated with a combination of mbsIgG2a-PD-L1×4-1BB (5 mg / kg) and anti-mPD-1 (10 mg / kg; both 2QW×3), complete tumor regression was observed in 6 out of 10 mice at 21 days after the start of treatment, compared to the case where no complete tumor regression was observed with either agent alone in this model (Figure 6A). Kaplan-Meier analysis showed that treatment with the combination of mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 was superior to the PBS treatment group (p<0.001) and to either antibody alone (p≦0.001; Mantel-Cox; Figure 6B, Table 9) at 500 mm 3 It was shown that this combination induced a significant increase in progression-free survival, defined as the percentage of mice with a smaller tumor volume than the target group. Therefore, a therapeutic synergistic effect, defined as superior antitumor efficacy (p<0.05) compared to the activity shown by each agent as a monotherapy, was observed with this combination.

[0433] These results provide rationale for evaluating that the combination of GEN1046 and an anti-PD-1 antibody further amplifies the antitumor immune response in cancer patients, resulting in a longer-lasting, deeper clinical response and enhanced survival.

[0434] (Table 9) Mantel-Cox analysis of progression-free survival induced by mbsIgG2a-PD-L1×4-1BB and anti-mPD-1 (either alone or in combination) in the MC38 model of C57BL / 6 mice. TIFF2026513865000035.tif83164 1 Tumor volume <500 mm 3 This was used as the cutoff for progression-free survival. A Mantel-Cox analysis was performed on day 45.

[0435] Example 3: Antigen-specific CD8+ T cell proliferation assay for determining the proliferation dose-response of GEN1046 and anti-PD-1 antibody nivolumab or pembrolizumab in an antigen-specific T cell assay with an active PD1 / PD-L1 axis To measure the induction of T cell proliferation by GEN1046, nivolumab, or pembrolizumab, antigen-specific T cell proliferation assays with an active PD1 / PD-L1 axis were performed. TIFF2026513865000036.tif35158

[0436] HLA-A2+ peripheral blood mononuclear cells (PBMCs) were obtained from a healthy donor (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs using magnetically activated cell sorting (MACS) technology with anti-CD14 microbeads (Miltenyi; catalog number 130-050-201) according to the manufacturer's instructions. Peripheral blood lymphocytes (PBL, CD14-negative fraction) were frozen for future T cell isolation. 1 × 10⁶ cells were frozen for differentiation into immature dendritic cells (iDCs). 6Monocytes / ml were cultured for 5 days in RPMI GlutaMAX (Life technologies GmbH, catalog number 61870-044) containing 5% human AB serum (Sigma-Aldrich Chemie GmbH, catalog number H4522-100ML), sodium pyruvate (Life technologies GmbH, catalog number 11360-039), non-essential amino acids (Life technologies GmbH, catalog number 11140-035), 100 IU / mL penicillin-streptomycin (Life technologies GmbH, catalog number 15140-122), 1000 IU / mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi, catalog number 130-093-868), and 1000 IU / mL interleukin-4 (IL-4; Miltenyi, catalog number 130-093-924). Half of the culture medium was replaced with fresh medium once during these 5 days. iDCs were collected by collecting non-adherent cells, and adherent cells were detached by incubation at 37°C for 10 minutes with PBS containing 2 mM EDTA. After washing, the iDCs were frozen in RPMI GlutaMAX containing 10% v / v DMSO (AppliChem GmbH, catalog no. A3672,0050) + 50% v / v human AB serum for future antigen-specific T cell assays.

[0437] Frozen PBLs and iDCs from the same donor were thawed one day before the start of the antigen-specific CD8+ T cell proliferation assay. CD8+ T cells were isolated from PBLs using MACS technology with anti-CD8 MicroBeads (Miltenyi, catalog no. 130-045-201) according to the manufacturer's instructions. Approximately 10-15 × 10⁶ cells. 6To each CD8+ T cell, 10 μg of in vitro translation (IVT)-RNA encoding the α-chain of a claudin-6-specific mouse TCR (HLA-A2 restricted; described in WO 2015150327 A1) + 10 μg of IVT-RNA encoding the β-chain + 10 μg of IVT-RNA encoding PD-1 were electroporated using a BTX ECM® 830 electroporation system (BTX; 500 V, 1 × 3 ms pulse) in a 4-mm electroporation cuvette (VWR International GmbH, catalog no. 732-0023). Immediately after electroporation, the cells were transferred to fresh IMDM medium (Life Technologies GmbH, catalog no. 12440-061) supplemented with 5% human AB serum and allowed to rest at 37°C, 5% CO2 for at least 1 hour. T cells were labeled with 1.6 μM carboxyfluorescein succinimimidyl ester (CFSE; Invitrogen, catalog number C34564) in PBS according to the manufacturer's instructions and incubated overnight (O / N) in IMDM medium supplemented with 5% human AB serum.

[0438] Maximum 5 x 10 6 To each thawed iDC, either 1 μg (dose-response for GEN1046...

Claims

1. A method for treating a tumor or cancer in a subject, the method comprising administering to the subject a binder comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

2. The method according to claim 1, wherein PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence shown in SEQ ID NO: 40; and / or CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO:

38.

3. (a) The method according to claim 1 or 2, wherein the first binding region of the binder comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown at SEQ ID NO: 2, 3, and 4, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown at SEQ ID NO: 6, GAS, and SEQ ID NO: 8, respectively; and (b) the second binding region of the binder comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown at SEQ ID NO: 12, 13, and 14, respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown at SEQ ID NO: 16, DDN, and SEQ ID NO: 18, respectively.

4. (a) The first binding region of the binder includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 5; and (b) The second binding region of the binder includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 11 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 15, The method according to any one of the above claims.

5. The method according to any one of the claims, wherein the binder is a multispecific antibody, for example, a bispecific antibody.

6. The method according to any one of the claims, wherein the binder is in the form of a full-length antibody or an antibody fragment.

7. The binder is an antibody comprising a first binding arm and a second binding arm. The first connecting arm, (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and (ii) A polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). including; and the second connecting arm, (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and (iv) A polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). including, The method according to any one of the above claims.

8. The aforementioned 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; and (ii) A second heavy chain and a light chain comprising the antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region. The method according to any one of the claims, including

9. (i) The amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) The amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain. The method according to claim 7 or 8.

10. The method according to any one of claims 7 to 9, 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 heavy chain and the second heavy chain, respectively.

11. The method according to any one of claims 7 to 10, wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A in the first heavy chain constant region (HC) and the second heavy chain constant region (HC), respectively.

12. The positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, and (i) The position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) The position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R, and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L. The method according to any one of claims 7 to 11.

13. The EU numbering of positions L234, L235, and D265 in the human IgG1 heavy chain in both the first and second heavy chain constant regions corresponds to positions F, E, and A, respectively, and (i) The position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) The position of the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L. The method according to any one of claims 7 to 12.

14. The steady region of the first heavy chain and / or the second heavy chain, for example, the second heavy chain, (a) Sequence [IgG1-Fc_FEAL] shown in SEQ ID NO: 24 or 30; (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 6 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. It contains, or is essentially, an amino acid sequence selected from the group consisting of, The method according to any one of claims 7 to 13.

15. The first heavy chain and / or the second heavy chain, for example, the steady region of the first heavy chain, (a) Sequence [IgG1-Fc_FEAR] shown in SEQ ID NO: 23 or 29; (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and (c) A sequence having at most 6 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 5 substitutions, at most 4, at most 3, at most 2, or at most 1 substitution. It contains, or is essentially, an amino acid sequence selected from the group consisting of, The method according to any one of claims 7 to 14.

16. The method according to any one of claims 7 to 15, wherein the binder comprises a kappa (κ) light chain constant region.

17. The method according to any one of claims 7 to 16, wherein the binder comprises a lambda (λ) light chain constant region.

18. The method according to any one of claims 7 to 17, wherein the first light chain steady region is a kappa (κ) light chain steady region or a lambda (λ) light chain steady region.

19. The method according to any one of claims 7 to 18, wherein the second light chain steady region is a lambda (λ) light chain steady region or a kappa (κ) light chain steady region.

20. The method according to any one of claims 7 to 19, wherein the first light chain steady region is a kappa (κ) light chain steady region and the second light chain steady region is a lambda (λ) light chain steady region, or the first light chain steady region is a lambda (λ) light chain steady region and the second light chain steady region is a kappa (κ) light chain steady region.

21. The kappa (κ) light chain, (a) The sequence shown in SEQ ID NO: 35; (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 substitutions, or at most 1 substitution. The method according to any one of claims 16 to 20, comprising an amino acid sequence selected from the group consisting of the following.

22. The lambda (λ) light chain, (a) The sequence shown in SEQ ID NO: 36; (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having at most 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 substitutions, or at most 1 substitution. The method according to any one of claims 17 to 21, comprising an amino acid sequence selected from the group consisting of the following.

23. The method according to any one of the claims, wherein the binder is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

24. The method according to any one of the claims, wherein the binder is a full-length IgG1 antibody.

25. The method according to any one of the claims, wherein the binder is an antibody of the IgG1m(f) allotype.

26. The binder is a bispecific antibody that binds to CD137 and PD-L1, and the bispecific antibody is (i) A first heavy chain containing the amino acid sequence shown in SEQ ID NO: 31 and a first light chain containing the amino acid sequence shown in SEQ ID NO: 32, (ii) A second heavy chain containing the amino acid sequence shown in SEQ ID NO: 33 and a second light chain containing the amino acid sequence shown in SEQ ID NO: 34 A method according to any one of the claims, comprising:

27. The method according to any one of the claims, wherein the binder is akasanlimab or a biosimilar thereof.

28. The method according to any one of the claims, wherein the binder is in a composition or formulation comprising histidine, sucrose, and polysorbate-80, and has a pH of 5 to 6.

29. The aforementioned binder is A composition or formulation containing approximately 20 mM histidine, approximately 250 mM sucrose, approximately 0.02% polysorbate-80, and having a pH of approximately 5.

5. The method described in any one of the above claims.

30. The method according to any one of the claims, wherein the binder is in a composition or formulation containing 10 to 30 mg of binder / mL, for example, 20 mg of binder / mL.

31. The method according to any one of the claims, wherein the binder is in a composition as defined in any one of claims 28 to 30 and is diluted in 0.9% NaCl (saline solution) before administration.

32. The method according to any one of the claims, further comprising the step of administering a PD-1 inhibitor to the subject.

33. PD-1 is human PD-1, and preferably PD-1 has or contains the amino acid sequence shown in SEQ ID NO: 113 or SEQ ID NO: 114, or The method according to claim 32, wherein the amino acid sequence of PD-1 is 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% identical to the amino acid sequence shown in SEQ ID NO: 113 or SEQ ID NO: 114, or is an immunogenic fragment thereof.

34. The method according to claim 32 or 33, wherein the PD-1 inhibitor is an antibody that binds to PD-1 or PD-L1, preferably an antibody that is a PD-1 / PD-L1 interaction antagonist, and / or an antibody that is a PD-1 or PD-L1 blocking antibody.

35. The method according to any one of claims 32 to 34, wherein the PD-1 inhibitor is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, for example, an antibody of the IgG1 isotype.

36. The method according to any one of claims 32 to 35, wherein the PD-1 inhibitor is a full-length antibody or an antibody fragment, for example, a full-length IgG1 antibody.

37. The method according to any one of claims 32 to 36, wherein the PD-1 inhibitor is a monospecific antibody.

38. The method according to any one of claims 32 to 37, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 59, 60, and 61, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 62, LAS, and SEQ ID NO: 64, respectively.

39. The method according to any one of claims 32 to 38, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising a VH region containing the amino acid sequence of SEQ ID NO: 65 and a VL region containing the amino acid sequence of SEQ ID NO:

66.

40. The method according to any one of claims 32 to 39, wherein the PD-1 inhibitor is an antibody that binds to PD-1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 67 and a light chain containing the amino acid sequence of SEQ ID NO:

68.

41. The method according to any one of claims 32 to 40, wherein the PD-1 inhibitor is pembrolizumab or a biosimilar thereof.

42. The method according to any one of claims 32 to 41, wherein the binder is akasanlimab or a biosimilar thereof, and the PD-1 inhibitor is pembrolizumab or a biosimilar thereof.

43. The method according to any one of claims 32 to 37, wherein the PD-1 inhibitor is an antibody or antigen-binding fragment that binds to PD-1, and the antibody that binds to PD-1 comprises VH regions CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NO: 104, 101, and 100, respectively, and VL regions CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NO: 107, QAS, and SEQ ID NO: 105, respectively.

44. The method according to claim 43, wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH) 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 with the amino acid sequence of the VH sequence as shown in SEQ ID NO:

111.

45. The method according to claim 44, wherein the antibody that binds to PD-1 comprises a heavy chain variable region (VH), and the VH comprises the sequence shown in SEQ ID NO:

111.

46. The method according to any one of claims 43 to 45, wherein the antibody that binds to PD-1 includes a light chain variable region (VL) 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 with the amino acid sequence of the VL sequence as shown in SEQ ID NO:

112.

47. The method according to claim 46, wherein the antibody that binds to PD-1 comprises a light chain variable region (VL), the VL comprising the sequence shown in SEQ ID NO:

112.

48. The method according to any one of claims 43 to 47, 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 shown in SEQ ID NO: 111, and the VL comprises or has the sequence shown in SEQ ID NO:

112.

49. The method according to any one of claims 43 to 48, wherein the antibody that binds to PD-1 includes 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 L234 in the human IgG1 heavy chain according to EU numbering is phenylalanine, the amino acid corresponding to position L235 in the human IgG1 heavy chain according to EU numbering is glutamic acid, and the amino acid corresponding to position G236 in the human IgG1 heavy chain according to EU numbering is arginine (L234F / L235E / G236R).

50. The method according to any one of claims 43 to 49, wherein the heavy chain constant region of the antibody that binds to PD-1 contains 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 with the amino acid sequence of the HC sequence as shown in SEQ ID NO:

93.

51. The method according to any one of claims 43 to 50, wherein the heavy chain constant region of the antibody that binds to PD-1 includes the sequence shown in SEQ ID NO:

93.

52. The method according to any one of claims 43 to 51, wherein the isotype of the heavy chain constant region of the antibody that binds to PD-1 is IgG1.

53. The method according to any one of claims 43 to 52, wherein the antibody that binds to PD-1 is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fragment of such an antibody.

54. The method according to any one of the claims, wherein the binder is akasanlimab or a biosimilar thereof, and the PD-1 inhibitor is an antibody that binds to PD-1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 152 and a light chain containing the amino acid sequence of SEQ ID NO:

153.

55. The method according to any one of claims 32 to 36, wherein the PD-1 inhibitor is a multispecific antibody, for example, a bispecific antibody.

56. The method according to any one of claims 32 to 37, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, cosivelimab, AUNP12, CA-170, BMS-986189, or a biosimilar thereof.

57. The method according to any one of claims 32 to 37, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, atezolizumab, avelumab, durvalumab, emvafolimab, cosivelimab, or a biosimilar thereof.

58. The method according to any one of claims 32 to 37, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, semiprimab, dostallimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, retifanlimab, pizilizumab, AMP-514, or a biosimilar thereof.

59. The method according to any one of the claims, wherein the subject is a human subject.

60. The method according to any one of the claims, wherein the binder is administered in at least one treatment cycle, each treatment cycle being 3 weeks (21 days) or 6 weeks (42 days).

61. The method according to any one of the claims, wherein one dose of the binder is administered every three weeks (1Q3W) or every six weeks (1Q6W).

62. The method according to any one of the claims, wherein one dose of the binder is administered on the first day of each treatment cycle.

63. The method according to any one of the claims, wherein the amount of the binder administered in each dose and / or in each treatment cycle is 100 mg or 500 mg.

64. The method according to any one of the claims, wherein a 100 mg dose of the binder is administered every three weeks (1Q3W).

65. The method according to any one of the claims, wherein a 100 mg dose of the binder is administered every three weeks (1Q3W) over two treatment cycles, and then, preferably, a 500 mg dose of the binder is administered every six weeks (1Q6W) over one or more treatment cycles until complete tumor regression or disease progression.

66. The method according to any one of claims 32 to 64, wherein the PD-1 inhibitor is administered in at least one treatment cycle, each treatment cycle being 3 weeks (21 days) or 6 weeks (42 days).

67. The method according to any one of claims 32 to 64 and 66, wherein one dose of the PD-1 inhibitor is administered every three weeks (1Q3W) or every six weeks (1Q6W).

68. The method according to any one of claims 32 to 64, 66, and 67, wherein one dose of the PD-1 inhibitor is administered on the first day of each treatment cycle.

69. The method according to any one of claims 32 to 64, 66 to 68, wherein the amount of the PD-1 inhibitor administered in each dose and / or in each treatment cycle is 200 mg or 400 mg.

70. The method according to any one of claims 32 to 64, 66 to 69, wherein a 100 mg dose of the binder and a 200 mg dose of the PD-1 inhibitor are administered every three weeks (1Q3W).

71. The method according to any one of claims 32 to 64, 66 to 70, wherein a 100 mg dose of a conjugate, which is akasanlimab or a biosimilar thereof, and a 200 mg dose of a PD-1 inhibitor, which is pembrolizumab or a biosimilar thereof, are administered every three weeks (1Q3W), for example, on day 1 of each three-week treatment cycle.

72. The method according to any one of claims 32 to 64, 66 to 69, wherein a 100 mg dose of the binder and a 400 mg dose of the PD-1 inhibitor are administered every 6 weeks (1Q6W).

73. The method according to any one of claims 32-64, 66-69, and 72, wherein a 100 mg dose of a conjugate, which is akasanlimab or a biosimilar thereof, and a 400 mg dose of a PD-1 inhibitor, which is pembrolizumab or a biosimilar thereof, are administered every six weeks (1Q6W), for example, on day 1 of each six-week treatment cycle.

74. The method according to any one of claims 32 to 73, wherein the PD-1 inhibitor is administered first, followed by the administration of the binder, preferably the administration of the binder begins at least 30 minutes after the end of the administration of the PD-1 inhibitor.

75. The method according to any one of the claims, wherein the tumor or cancer is a solid tumor or leukemia, preferably a solid tumor.

76. The method according to any one of the claims, wherein the tumor or cancer is selected from the group consisting of colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, pancreatic cancer, urinary tract cancer, bladder cancer, thyroid cancer, breast cancer, prostate cancer, ovarian cancer, central nervous system (CNS) cancer, and skin cancers such as melanoma, and preferably selected from the group consisting of colorectal cancer, gastric cancer, and endometrial cancer.

77. The method according to claim 76, wherein the tumor or cancer is endometrial cancer.

78. The method according to claim 76, wherein the tumor or cancer is colorectal cancer.

79. The method according to claim 76, wherein the tumor or cancer is gastric cancer.

80. The method according to any one of the claims, wherein the tumor or cancer is unresectable, recurrent, and / or metastatic.

81. The method according to any one of the claims, wherein the subject is undergoing or following at least one prior treatment regimen for the unresectable and / or metastatic tumor or cancer, preferably during or after systemic chemotherapy such as platinum-based chemotherapy.

82. The method according to any one of the claims, wherein the subject has not been previously treated with a checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG3 antibody, or an anti-TIGIT antibody.

83. The method according to any one of claims 1 to 81, wherein the subject has previously received prior treatment with a PD-1 inhibitor or a PD-L1 inhibitor, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody, and the PD-1 inhibitor or PD-L1 inhibitor is administered as monotherapy or as part of a combination therapy.

84. The method according to claim 83, wherein the subject is progressing after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody.

85. A conjugate for use in a method for treating a tumor or cancer in a subject, the method comprising the step of administering to the subject a conjugate comprising a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

86. The binder for use according to claim 85, wherein the method is as defined in any one of claims 1 to 84, and / or the binder is as defined in any one of claims 1 to 84.

87. A pharmaceutical composition for use in a method for treating a tumor or cancer in a subject, comprising a binder having a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1, and optionally a pharmaceutically acceptable carrier, wherein the tumor or cancer is high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR).

88. The pharmaceutically acceptable composition for use according to claim 87, wherein the method is as defined in any one of claims 1 to 84, and / or the binder is as defined in any one of claims 1 to 84.

89. Use of a binder for the manufacture of a medicament for treating a tumor or cancer in a subject, wherein the tumor or cancer is a high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), and the binder comprises a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1.

90. The use of the binder according to claim 89, wherein the binder is as defined in any one of claims 1 to 84.

91. A kit for use in a method for treating tumors or cancers in a subject, wherein the tumor or cancer is characterized by high-frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), (a) A binder comprising a first binding region that binds to CD137 and a second binding region that binds to PD-L1, and (b) PD-1 inhibitors A kit for the aforementioned use, including the above.

92. The kit for use according to claim 91, wherein the method is as defined in any one of claims 1 to 84, and / or the binder is as defined in any one of claims 1 to 84, and / or the PD-1 inhibitor is as defined in any one of claims 1 to 84.

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