Combination dosing regimens of CD137- and PD-L1-binding agents

Abstract

The present invention relates to a method for reducing or inhibiting tumor progression in a subject or for treating cancer in a subject comprising administering to the subject a binding agent comprising a first antigen-binding region that binds human CD137 and a second antigen-binding region that binds human PD-L1.The present invention further provides a binding agent for use in reducing or inhibiting tumor progression or for use in treating cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for reducing or inhibiting the progression of a tumor in a subject or for treating cancer in a subject, comprising administering to the subject a binding agent comprising a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. [Background technology]

[0002] 2. Background of the Invention CD137 (4-1BB, TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor (TNFR) family. CD137 is involved in the expression of CD8 + and CD4 + It is a costimulatory molecule found on T cells, regulatory T cells (Treg), natural killer (NK) and NKT cells, B cells, and neutrophils. On T cells, CD137 is not constitutively expressed, but is induced upon T cell receptor (TCR) activation. Stimulation with the natural ligand 4-1BBL or agonistic antibodies results in signaling in which TNFR-associated factors (TRAF)-2 and TRAF-1 are used as adaptors. Initial signaling by CD137 involves K-63 polyubiquitination, which ultimately results in activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways. Signaling leads to T cell costimulation, proliferation, cytokine production, maturation, and CD8 expression. +This results in increased T cell longevity. Agonistic antibodies against CD137 have been shown to promote anti-tumor control by T cells in various preclinical models (Murillo et al. 2008 Clin.Cancer Res.14(21):6895-6906 (Non-Patent Document 1)). Antibodies that stimulate CD137 can induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art, including the human IgG4 antibody urelumab (WO2005035584 (Patent Document 1)) and the human IgG2 antibody utomirumab (Fisher et al. 2012 Cancer Immunol.Immunother.61:1721-1733 (Non-Patent Document 2)).

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

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

[0005] WO2019 / 025545 (Patent Document 2) shows a multispecific antibody that can bind to both PD-L1 and CD137. Such an antibody is designed to simultaneously bind to PD-L1-expressing antigen-presenting cells (APCs) or tumor cells and CD137-expressing T cells. By combining checkpoint blockade with 4-1BB-dependent T cell activation, the multispecific antibody enhances the proliferation and cytokine production of activated T cells, activates immune cells in tumor-draining lymph nodes, and induces in vivo tumor regression.

[0006] However, despite advances in the mode of action and proven in vivo efficacy of such multispecific antibodies, it remains desirable to provide means for further improving their in vivo efficacy. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2005035584 [Patent Document 2] WO2019 / 025545 [Non-patent literature]

[0008] [Non-Patent Document 1] Murillo et al.2008 Clin.Cancer Res.14(21):6895-6906 [Non-Patent Document 2] Fisher et al.2012 Cancer Immunol.Immunother.61:1721-1733 [Non-Patent Document 3] Latchman et al.,2004 Proc Natl Acad Sci USA 101,10691-6 Summary of the Invention

[0009] A clinical trial (ClinicalTrials.gov Identifier: NCT03917381) was designed, including dose escalation and expansion, to investigate, for example, the recommended phase II dose (RP2D) and the safety, tolerability, pharmacokinetics (PK), and antitumor activity of a multispecific antibody that binds PD-L1 and CD137. The inventors also developed a pharmacokinetic / pharmacodynamic (PK / PD) model to predict trimerization of PD-L1 and 4-1BB in tumors (crosslinking of the multispecific binding agent with PD-L1 and 4-1BB) and receptor occupancy (RO).

[0010] Using this PK / PD model, the inventors were able to show that when the multispecific antibody is administered, for example, every 3 weeks, a dose of 100 mg results in peak trimer formation in the tumor. Dosing the multispecific antibody at 500 mg on a less frequent basis, for example every 6 weeks, is predicted to result in a lower degree of trimer engagement compared to the 100 mg dose, leading to higher PD-L1 receptor occupancy and intermittent 4-1BB activation.

[0011] In clinical trials, the multispecific antibody has demonstrated a manageable safety profile and preliminary clinical activity in the population with advanced solid tumors. Pharmacodynamic clinical data showed greater and more consistent modulation of peripheral pharmacodynamic endpoints at dose levels ≦200 mg, and expansion clinical data showed that a dose of 100 mg administered every 3 weeks produced responses within the first 2 cycles. A dosing scheme combining frequent dosing of 100 mg of the multispecific antibody with a less frequent, higher maintenance dose, e.g., 500 mg, is expected to produce improved duration of response. Additionally, the high dose of the multispecific antibody is expected to have less trimer engagement in the liver compared to 100 mg Q3W, and therefore a better safety profile.

[0012] Thus, in a first aspect, the present invention provides a method for reducing or inhibiting the progression of a tumor in a subject or for treating cancer in a subject comprising administering to the subject a binding agent comprising a first antigen-binding region that binds to human CD137, such as human CD137 consisting of the amino acid sequence set forth in SEQ ID NO:24, and a second antigen-binding region that binds to human PD-L1, such as human PD-L1 consisting of the amino acid sequence set forth in SEQ ID NO:26, the binding agent is administered to the subject in a dosing schedule comprising administration of dose A in one or more treatment cycles and administration of dose B in one or more treatment cycles; The amount of binding substance in dose A is a) about 0.3 to 2.5 mg / kg body weight or about 25 to 200 mg in total; and / or b) Approximately 2.1 × 10-9 to 1.7 × 10-8 mol / kg body weight, or approximately 1.7 × 10-7 to 1.48 × 10-6 mol in total and The amount of binding substance in dose B is c) about 3.8-7.5 mg / kg body weight or about 300-600 mg in total; and / or d) about 2.6×10-8 to 5.1×10-8 mol / kg body weight, or about 2.0 to 4.1×10-6 mol in total That is, A method is provided.

[0013] The amount of said binding agent in dose A is preferably a) about 1.25 mg / kg body weight or about 100 mg in total; and / or b) Approximately 8.5×10 -9 mol / kg body weight, or about 6.8 × 10 total -7 Mol It is.

[0014] The amount of binding agent in dose B is preferably a) about 6.25 mg / kg body weight or about 500 mg in total; and / or b) Approximately 4.3×10 -8 mol / kg body weight, or about 3.4 × 10 total -6 Mol It is.

[0015] In a further aspect, the invention provides a binding agent for use in reducing or inhibiting the progression of a tumor or for use in the treatment of cancer, wherein the binding agent comprises a first antigen-binding region that binds human CD137, such as human CD137 consisting of the amino acid sequence set forth in SEQ ID NO:24, and a second antigen-binding region that binds human PD-L1, such as human PD-L1 consisting of the amino acid sequence set forth in SEQ ID NO:26, and wherein the binding agent is administered to a subject with a dosing schedule comprising administration of dose A for one or more treatment cycles and administration of dose B for one or more treatment cycles; The amount of binding substance in dose A is a) about 0.3 to 2.5 mg / kg body weight or about 25 to 200 mg in total; and / or b) Approximately 2.1 × 10-9 to 1.7 × 10-8 mol / kg body weight, or approximately 1.7 × 10-7 to 1.4 × 10-6 mol in total and The amount of binding substance in dose B is c) about 3.8-7.5 mg / kg body weight or about 300-600 mg in total; and / or d) about 2.6×10-8 to 5.1×10-8 mol / kg body weight, or about 2.0 to 4.1×10-6 mol in total That is, Regarding the binding substance.

[0016] These and other aspects and embodiments of the invention, including: [Brief description of the drawings]

[0017] [Figure 1A] Figure 1: Simultaneous binding of GEN1046 to PD-L1- and CD137-expressing K562 cells induces doublet formation with a bell-shaped dose-response curve. Equal numbers of CellTrace™ Far Red-labeled K562 cells transfected with CD137 (K562_h4-1BB) were co-incubated with CellTrace™ Violet-labeled K562 cells transfected with PD-L1 (K562_hPD-L1) for 15 minutes in the presence of 0.001-100 μg / mL of i) GEN1046 or ii) a combination of control antibodies PD-L1-547-FEALxb12-FEAR and b12-FEALxCD137-009-HC7LC2-FEAR. Samples were analyzed by flow cytometry and the percentage of CellTrace™ Far Red / CellTrace™ Violet double-positive doublets (A) was plotted as a function of GEN1046 concentration (B). Data shown are the mean ± standard deviation of n=3 technical replicates (symbols probably need to be smaller to show SD). [Figure 1B] See legend to Figure 1A. [Diagram 2]Schematic representation of the expected mode of action of CD137xPD-L1 bispecific antibody. (A) PD-L1 is expressed on antigen-presenting cells (APCs) as well as tumor cells. Binding of PD-L1 to T cells expressing the negative regulatory molecule PD-1 effectively neutralizes T cell activation signals, ultimately resulting in T cell inhibition. (B) When CD137xPD-L1 bispecific antibody is added, the inhibitory PD-1:PD-L1 interaction is blocked by the PD-L1 specific arm, while the bispecific antibody delivers agonistic signaling to CD137 expressed on T cells through cell-cell interactions, resulting in strong T cell co-stimulation. [Diagram 3] Relative light units (RLU) as a function of antibody concentration in a luciferase-based CD137 activation reporter assay performed in the presence of PD-L1-expressing tumor cell lines. Human ovarian cancer cell line ES-2 (A) and breast cancer cell line MDA-MB-231 (B), which endogenously express PD-L1, were co-cultured with NFkB-Luc2P / 4-1BB Jurkat reporter cells in the presence of 0.00128–100 μg / mL of i) GEN1046 or ii) b12-FEAL control antibody for 6 h. Induction of luciferase expression was examined by incubation with luciferase substrate and measurement of relative light units. Data shown are mean ± standard deviation of n=3 technical replicates. [Figure 4]Comparison of GEN1046 with control antibodies PD-L1-547-FEALxb12-FEAL or IgG1-b12-FEAL in polyclonal T cell proliferation assays. CFSE-labeled PBMCs were incubated with a suboptimal concentration of anti-CD3 antibody (0.03 μg / mL) and cultured for 4 days in the presence of 0.0032–10 μg / mL of i) GEN1046 ii) PD-L1-547-FEALxb12-FEAR or iii) b12-FEAL control antibodies. T cell proliferation was measured by flow cytometry in total T cells (A) and in CCR7+CD45RO+ central memory T cell subsets and CCR7-CD45RO+ effector memory T cell subsets in total T cells (B). Data from one representative donor are shown as the mean expansion index of two replicates calculated using FlowJo v10.4 software. Error bars (SD) indicate the variability of experiments (two replicates, one donor cell used). [Diagram 5] GEN1046 rescues PD-1 / PD-L1-mediated T cell inhibition and additional costimulation of CD8+ T cell proliferation in antigen-specific T cell assays with an active PD-1 / PD-L1 axis. CD8+ T cells were electroporated with 10 μg each of RNA encoding the alpha and beta chains of the CLDN6-specific TCR, either with (0.4–10 μg) or without (no PD-1) RNA encoding PD-1, labeled with CFSE, and cocultured with immature DCs electroporated with 0.3 μg (A) or 1 μg (B) of CLDN6-encoding RNA. Electroporated CD8+ T cells and iDCs were cocultured for 4 days in the presence of GEN1046 (0.00015–1 μg / mL) or b12-FEAL (1 μg / mL). T cell proliferation was assessed by analyzing CFSE dilution in CD8+ T cells using flow cytometry, and the T cell expansion index (e.g., how much the total T cell population expanded by proliferation) was automatically calculated by FlowJo (version 10.3). Data shown are the mean expansion index ± SD of triplicate wells from one donor out of four donors included in two experiments. [Figure 6] Effect of GEN1046 on the secretion of proinflammatory cytokines (IFNγ, TNFα, IL-13 and IL-8) in antigen-specific T cell assays with or without PD-1 electroporation of T cells. CD8+ T cells were electroporated with RNA encoding the alpha and beta chains of the CLDN6-specific TCR (10 μg each) either with (2 μg) or without (no PD-1) RNA encoding PD-1, labeled with CFSE, and cocultured with immature DCs electroporated with 1 μg of CLDN6-encoding RNA. Electroporated CD8+ T cells and iDCs were cocultured in the presence of GEN1046 (0.00015–1 μg / mL) or b12-FEAL (1 μg / mL). Supernatant cytokine levels were measured 48 hours after antibody addition by multiplex sandwich immunoassay using the MSD V-Plex Human Proinflammatory panel 1 (10-Plex) kit. Data shown are the mean concentrations ± SD of sextuplicate wells from one representative donor out of two included in the experiment. [Figure 7] Ex vivo expansion of tumor infiltrating lymphocytes (TILs) from human non-small cell lung cancer resected tissues by CD137-009-FEALxPD-L1-547-FEAR. Tumor pieces from resected tissues were cultured with 10 U / mL IL-2 and CD137-009-FEALxPD-L1-547-FEAR at the indicated concentrations. After 10 days of culture, cells were harvested and analyzed by flow cytometry. (A) TIL counts per 1,000 beads, (B) CD3+CD8+ T cell counts per 1,000 beads, (C) CD3+CD4+ T cell counts per 1,000 beads, (D) CD3-CD56+ NK cell counts per 1,000 beads. Data shown are the mean cell counts ± SD of 5 separate wells with 2 tumor pieces per well as starting material. *p<0.05 using ordinary one-way ANOVA with Dunnett's multiple comparison test. [Figure 8A]Figure 8: Pharmacodynamic assessments, including changes in circulating levels of interferon-gamma (IFN-γ) and interferon-γ-inducible protein 10 IP-10 (A-B), proliferating effector memory CD8 T cells, and total CD8 T cells (C-D), were performed with blood samples from patients with advanced solid tumors enrolled in the dose escalation phase of an open-label, multicenter safety trial of GEN1046 (NCT03917381; data cutoff: January 19, 2021). A-B. Circulating levels of IFN-γ and IP-10 were measured in serum samples at baseline and at multiple time points following dosing of GEN1046 in cycles 1 and 2 (day 1 [2 hours and 4-6 hours post-dose], days 2, 3, 8, and 15). IFN-γ and IP-10 levels in serum samples were measured by Meso Scale Discovery (MSD) multiplex immunoassays. Data shown are maximum fold changes from baseline measured in cycle 1. Statistical analysis was performed using the Wilcoxon-Mann-Whitney test. C-D. Peripheral blood immunophenotyping was performed on whole blood collected at baseline and at multiple time points (days 2, 3, 8, and 15) after administration of GEN1046 in cycles 1 and 2. The frequency of proliferating (Ki67+) total CD8 T cells and effector memory CD8 T cells (CD8+CD45RA-CCR7- T cells) was assessed by flow cytometry in whole blood samples. Data shown are maximum fold changes from baseline measured in cycle 1. Statistical analysis was performed using the Wilcoxon-Mann-Whitney test. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9] Illustrative outline of clinical trial design. [Figure 10]Dose escalation; Maximum percent change from baseline in tumor size, all patients. Data cutoff: September 29, 2020. Five patients did not have a post-baseline scan. aMinimum duration of response (5 weeks) per RECIST v1.1 was not achieved. bPR not confirmed on subsequent scan. NE, not evaluable; NSCLC, non-small cell lung cancer; PD, progressive disease; PD-(L)1, programmed cell death (ligand) 1; PR, partial response; SD, stable disease; SoD, sum of diameters; uPR, unconfirmed partial response. [Figure 11] Dose escalation; Maximum change from baseline in tumor size, patients with NSCLC. Data cutoff: September 29, 2020. aPR not confirmed by subsequent scan. bPD-L1 expression was assessed on archival tumor specimens. BOR, best overall response; CR, complete response; ICI, immune checkpoint inhibitor; NA, not available; PD, progressive disease; PD-(L)1, programmed cell death (ligand) 1; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; SoD, sum of diameters; TPS, tumor proportion score; uPR, unconfirmed partial response. [Figure 12] Maximal trimer formation and PD-L1 receptor occupancy for the 100 mg dose administered once every 3 weeks (Q3W) and the 500 mg dose administered once every 6 weeks (Q6W) predicted by the model. [Figure 13]Expansion cohort 1, data cutoff: January 29, 2021: A) Maximum change from baseline in tumor size: NE, not evaluable; PD, progressive disease; SD, stable disease; uPR, unconfirmed partial response, ◇=pre-PD-(L)1; PD-(L)1, programmed cell death (ligand) 1; RECIST, Response Evaluation Criteria in Solid Tumors. B) Change from baseline in sum of diameters of target lesions (SoD). Black lines represent subjects with progressive disease. Grey lines represent subjects with best overall response of stable disease (SD) or partial response (PR) if indicated. Black triangles, circles and squares indicate time-point effects for progressive disease, stable disease and partial response, respectively. Open circles represent "not evaluable"; i.e., patients are progressing but are being treated and are being followed by scans after progression based on RECIST; NL indicates "new lesions". [Figure 14] Expansion cohort 2, data cutoff: January 29, 2021: A) Maximum change from baseline in tumor size: PD, progressive disease; SD, stable disease; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; uPR, unconfirmed partial response; RECIST, Response Evaluation Criteria in Solid Tumors. B) Change from baseline in sum of target lesion diameters (SoD). Black lines represent subjects with progressive disease. Grey lines represent subjects with a best overall response of stable disease. Black triangles and circles indicate timepoint effects of progressive disease and stable disease, respectively. [Figure 15] Expansion cohort 3, data cutoff: January 29, 2021: A) Maximum change from baseline in tumor size: NE, not evaluable; PD, progressive disease; SD, stable disease; ◇=pre-PD-(L)1; PD-(L)1, programmed cell death (ligand) 1; RECIST, Response Evaluation Criteria in Solid Tumors. B) Change from baseline in sum of diameters of target lesions (SoD). Black lines represent subjects with progressive disease. Grey lines represent subjects with a best overall response of stable disease. Black triangles and circles indicate timepoint responses of progressive disease and stable disease, respectively. [Figure 16]Expansion cohort 4, data cutoff: January 29, 2021: A) Maximum change from baseline in tumor size: PD, progressive disease; SD, stable disease; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors. B) Change from baseline in sum of diameters of target lesions (SoD). Black lines represent subjects with progressive disease. Grey lines represent subjects with best overall response of stable disease or partial response (PR) if indicated. Black triangles, circles and squares indicate time-point effects for progressive disease, stable disease and partial response, respectively. Open circles represent "not evaluable"; i.e., patients are progressing but are being treated and are being followed by scans after progression per RECIST. [Figure 17] Expansion cohort 5, data cutoff: January 29, 2021: A) Maximum change from baseline in tumor size: NE, not evaluable; PD, progressive disease; SD, stable disease; uPR, unconfirmed partial response, ◇=pre-PD-(L)1; PD-(L)1, programmed cell death (ligand) 1; RECIST, Response Evaluation Criteria in Solid Tumors. B) Change from baseline in sum of diameters of target lesions (SoD). Black lines represent subjects with progressive disease. Grey lines represent subjects with best overall response of stable disease (SD) or partial response (PR) if indicated. Black triangles, circles and squares indicate time-point effects for progressive disease, stable disease and partial response, respectively. [Figure 18] Expansion cohort 6, data cutoff: January 29, 2021: A) Maximum change from baseline in tumor size; NE, not evaluable; PD, progressive disease; SD, stable disease; uPR, unconfirmed partial response, ◇=pre-PD-(L)1; PD-(L)1, programmed cell death (ligand) 1; RECIST, Response Evaluation Criteria in Solid Tumors. B) Change from baseline in sum of diameters of target lesions (SoD). Black lines represent subjects with progressive disease. Grey lines represent subjects with best overall response of stable disease (SD) or partial response (PR) if indicated. Black triangles, circles and squares indicate time-point effects for progressive disease, stable disease and partial response, respectively. Open circles represent "not evaluable"; i.e., patients are progressing but are being treated and are being followed by scans after progression based on RECIST. [Figure 19]Expansion cohort 7, data cutoff: January 29, 2021: A) Maximum change from baseline in tumor size: PD, progressive disease, PR, partial response; SD, stable disease; RECIST, Response Evaluation Criteria in Solid Tumors. B) Change from baseline in sum of diameters of target lesions (SoD). Black lines represent subjects with progressive disease. Grey lines represent subjects with best overall response of stable disease or partial response (PR) if indicated; black triangles, circles and squares indicate time-point effects for progressive disease, stable disease and partial response, respectively. Open circles represent "not evaluable"; i.e., patients are progressing but are being treated and are being followed by scans after progression per RECIST. [Figure 20] Waterfall plot showing progression-free survival in checkpoint inhibitor-pretreated subjects (grey line) and checkpoint inhibitor-naïve patients (black line). [Figure 21] Comparison of time from last prior anti-PD-(L)1 in subjects in the CPI-experienced expansion cohort (GEN1046 monotherapy) with clinical response (PR) compared to subjects with stable disease (SD) or progressive disease (PD). Response groups were compared using Wilcoxon test. PR vs PD: p=0.0017; PR vs SD: p=0.034. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention definition The term "immunoglobulin" refers to a class of structurally related glycoproteins that consist of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically comprises a heavy chain variable region (herein referred to as V Hor VH) and a heavy chain constant region (herein referred to as C H The heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically comprises a light chain variable region (herein referred to as V L or VL) and a light chain constant region (herein referred to as C L or CL). The light chain constant region is typically composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, also called complementarity determining regions (CDRs) (or hypervariable regions in which the sequence and / or the shape of structurally defined loops may be hypervariable), spaced by more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196 , 901-917 (1987). Unless otherwise indicated or contradicted by context, CDR sequences herein are identified using DomainGapAlign according to the IMGT rules (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also the internet http address www.imgt.org / Unless otherwise indicated or contradicted by context, references to amino acid positions within the constant regions herein are according to EU-numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0019] The term "amino acid corresponding to position ..." as used herein indicates the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be known by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that is aligned with the other amino acid or segment using a standard sequence alignment program, such as ALIGN, ClustalW or the like, typically with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain. It is considered well known in the art how to align sequences or segments within sequences, thereby determining positions within a sequence that correspond to amino acid positions according to the invention.

[0020] The term "binding agent" in the context of the present invention refers to any agent capable of binding to a desired antigen. In a particular embodiment of the present invention, the binding agent is an antibody, an antibody fragment or a construct thereof. The binding agent may also comprise synthetic modified or non-naturally occurring moieties, in particular non-peptide moieties. Such moieties may for example be linked to a desired antigen-binding function or antigen-binding region, for example an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0021] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, that has the ability to specifically bind to an antigen under typical physiological conditions, and has a half-life of a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other relevant functionally defined period of time (e.g., a sufficient time for a physiological response associated with the binding of the antibody to the antigen to be induced, promoted, enhanced, and / or modulated and / or a sufficient time for the antibody to recruit effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain a binding domain that interacts with an antigen. The term "antigen-binding region" as used herein refers to the region that interacts with an antigen and includes both the VH and VL regions. The term antibody, as used herein, includes not only monospecific antibodies, but also multispecific antibodies that contain multiple, e.g., two or more, e.g., three or more, different antigen-binding regions. The constant region of an antibody (Ab) may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation. As noted above, the term antibody herein includes, unless otherwise indicated or clearly contradicted by the context, fragments of antibodies that are antigen-binding fragments, i.e., that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO2007059782 (Genmab); (ii) F(ab'), which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region. 2(iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) an Fv fragment consisting essentially of the VL and VH domains of one antibody arm; (v) a domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90), also called dAb fragments (Ward et al., Nature 341 , 544-546(1989));(vi) Camelidae or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24) and (vii) isolated complementarity determining regions (CDRs). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be engineered using recombinant methods to pair the VL and VH domains into monovalent molecules (known as single-chain antibodies or single-chain Fvs (scFvs), see, e.g., Bird et al., Science 2003). 242 , 423-426 (1988) and Huston et al., PNAS USA 85, pp. 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term antibody unless otherwise stated or expressly stated in the text. Although such fragments are generally included in the meaning of antibodies, they are unique features of the present invention, collectively and each independently, exhibiting different biological properties and usefulness. These and other useful antibody fragments and bispecific forms of such fragments in the context of the present invention are further discussed herein. It should also be understood that the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, obtained by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques, unless otherwise stated. The antibodies produced can have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the sequence of the antibody is closer to said isotype, e.g., IgG1, than to other isotypes. Thus, for example, the IgG1 antibody of the present invention may be a sequence variant of a naturally occurring IgG1 antibody that contains diversity in the constant region.

[0022] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity with variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas that are obtained from transgenic or transchromosomal non-human animals, such as transgenic mice, that have genomes containing human heavy and light chain transgenes and that contain B cells fused with immortalized cells.

[0023] The term "bispecific antibody" or "bs" in the context of the present invention refers to an antibody having two different antigen-binding regions defined by different antibody sequences. In some embodiments, the different antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding regions bind to different target antigens. Bispecific antibodies can be of any format, such as any of the bispecific antibody formats described herein below.

[0024] As used herein, unless contradicted by context, the term "Fab arm" or "arm" comprises one heavy-light chain pair and is used interchangeably herein with "half molecule."

[0025] When a bispecific antibody is described as comprising a half molecule antibody "derived from" a first antibody and a half molecule antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody was generated by recombining the half molecules from each of the first and second antibodies by any known method to result in the bispecific antibody. In this context, "recombining" is not intended to be limited to any particular recombination method, and thus includes all methods for generating bispecific antibodies as described herein below, such as recombining by half molecule exchange, as well as recombining at the nucleic acid level and / or by co-expression of two half molecules in the same cell.

[0026] The term "monovalent antibody" means in the context of the present invention that an antibody molecule is capable of binding to one molecule of antigen and is therefore incapable of cross-linking antigens or cells.

[0027] The term "full length," when used in reference to an antibody, indicates that the antibody is not a fragment, but contains all domains of a particular isotype that are normally found in nature in that isotype, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains in an IgG1 antibody.

[0028] As used herein, unless contradicted by context, the term "Fc region" refers to the region of an antibody that consists of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least the hinge region, the CH2 domain and the CH3 domain.

[0029] As used herein, the term "heterodimeric interaction between a first CH3 region and a second CH3 region" refers to an interaction between a first CH3 region and a second CH3 region within a first CH3 / second CH3 heterodimeric protein.

[0030] As used herein, the term "homodimeric interaction between a first CH3 region and a second CH3 region" refers to an interaction between a first CH3 region and another first CH3 region within a first CH3 / first CH3 homodimeric protein and an interaction between a second CH3 region and another second CH3 region within a second CH3 / second CH3 homodimeric protein.

[0031] As used herein, the terms "bind" or "capable of binding to" in the context of antibody binding to a given antigen or epitope typically refer to a binding rate of about 10% or more when measured using Bio-Layer Interferometry (BLI) or, for example, using surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument, with the antigen as the ligand and the antibody as the analyte. -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even less than that, K D The antibody binds to the predetermined antigen with an affinity equivalent to the K of binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). D at least 10 times smaller, such as at least 100 times smaller, such as at least 1,000 times smaller, such as at least 10,000 times smaller, such as at least 100,000 times smaller, D The amount of affinity is determined by the antibody's K D Therefore, the K D If is very small (ie, the antibody is highly specific), this degree of affinity for the antigen may be at least 10,000 times less than the degree of affinity for a non-specific antigen.

[0032] The term "k" d ”(seconds -1) as used herein denotes the dissociation rate constant of a particular antibody-antigen interaction. The value k off Also called the value.

[0033] The term “K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0034] The antibody used according to the invention may be an isolated antibody. "Isolated antibody", as used herein, is intended to denote an antibody that is substantially free of other antibodies having different antigen specificities. In a preferred embodiment, an isolated bispecific antibody that specifically binds to PD-L1 and CD137 is substantially free of monospecific antibodies that specifically bind to PD-L1 or CD137. In another preferred embodiment, the antibody or pharmaceutical composition comprising the antibody is substantially free of naturally occurring antibodies that are unable to bind to PD-L1. In a further preferred embodiment, the antibody of the invention has a structural change in amino acid sequence compared to the structure of a naturally occurring anti-PD-L1 antibody, the structural change causing the antibody to exhibit altered functionality compared to the functionality exhibited by the naturally occurring anti-PD-L1 antibody, the functionality being selected from the group consisting of: (i) PD-L1 binding affinity, (ii) the ability to inhibit binding of PD-L1 to PD-1, (iii) the ability to induce Fc-mediated effector function, and (iv) the ability not to induce Fc-mediated effector function.

[0035] The term "PD-L1" as used herein refers to programmed cell death-ligand 1 protein. PD-L1 is found in humans and other species, and thus, the term "PD-L1" is not limited to human PD-L1 unless otherwise indicated by context. The sequences of human, macaque (Macaca fascicularis), African elephant, Sus scrofa and mouse PD-L1 can be found in Genbank accession numbers NP_054862.1, XP_005581836, XP_003413533, XP_005665023 and NP_068693, respectively. The sequence of human PD-L1 is also shown in SEQ ID NO:25, where amino acids 1-18 are predicted to be a signal peptide. The mature sequence of human PD-L1 is shown in SEQ ID NO:26.

[0036] The term "PD-1," as used herein, refers to the human programmed cell death-1 protein, also known as CD279.

[0037] The term "CD137" as used herein refers to human Cluster of Differentiation 137 protein. CD137 (4-1BB), also referred to as TNFRSF9, is the receptor for the ligand TNFSF9 / 4-1BBL. CD137 is believed to be involved in T cell activation. In one embodiment, CD137 is human CD137 and has UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO:23, where amino acids 1-23 are predicted to be a signal peptide. The mature sequence of human CD137 is shown in SEQ ID NO:24.

[0038] The percent identity between two sequences is a function of the number of identical positions that the sequences have in common (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) as incorporated in the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0039] In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes reflected in the table below.

[0040] (Table 1) Classification of amino acid residues for conservative substitutions TIFF2024523438000001.tif50133

[0041] In the context of the present invention, the following notation is used to indicate mutations, unless otherwise stated: i) substitution of an amino acid at a given position is described, for example, K409R, which means a substitution of lysine at position 409 of the protein with arginine; ii) specific variants use specific three-letter or one-letter codes, with the codes Xaa and X indicating any amino acid residue. Thus, a substitution of lysine at position 409 with arginine is indicated: K409R, and a substitution of lysine at position 409 with any amino acid residue is indicated: K409X. In case of deletion of lysine at position 409, K409R is indicated. * It is shown that:

[0042] In the context of the present invention, "inhibition of PD-L1 binding to PD-1" refers to any detectably significant reduction in the binding of PD-L1 to PD-1 in the presence of an antibody capable of binding to PD-L1. Typically, inhibition refers to at least about a 10% reduction in the binding between PD-L1 and PD-1 caused by the presence of an anti-PD-L1 antibody, such as at least about a 15%, such as at least about a 20%, such as at least a 40% reduction. Inhibition of PD-L1 binding to PD-1 may be measured by any suitable technique. In one embodiment, inhibition is measured as described in Example 6 of WO2019 / 025545.

[0043] The term "specificity", as used herein, unless contradicted by context, is intended to have the following meaning: Two antibodies have the "same specificity" if they bind to the same antigen and the same epitope.

[0044] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. An epitope usually consists of a group of molecules on a surface, such as amino acid side chains or sugar side chains, and usually has a specific three-dimensional structural characteristic as well as a specific charge characteristic. Structure-dependent and structure-independent epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not lost. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by a specific antigen-binding peptide (in other words, the amino acid residues are present within the footprint of the specific antigen-binding peptide).

[0045] The term "chimeric antibody" as used herein refers to an antibody whose variable region is derived from a non-human species (e.g., from a rodent) and whose constant region is derived from a different species, e.g., from a human. Chimeric monoclonal antibodies for therapeutic applications have been developed to reduce antibody immunogenicity. The term "variable region" or "variable domain" when used in the context of a chimeric antibody refers to the region of both the heavy and light chains of an immunoglobulin, including the CDRs and framework regions. 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. Chimeric antibodies can be genetically engineered or enzymatically engineered recombinant antibodies. It is within the knowledge of one skilled in the art to produce chimeric antibodies, and therefore the production of chimeric antibodies according to the invention may be performed by methods other than those described herein.

[0046] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology with the human variable domain. This can be done by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen binding site into a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parent antibody, it may be necessary to replace framework residues from the parent antibody (i.e., non-human antibody) with human framework regions (back mutations). Homology modeling of the structure can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise primarily human framework regions and fully human constant regions, including non-human CDR sequences, optionally one or more amino acid back mutations to the non-human amino acid sequences. Optionally, additional amino acid modifications may be applied, not necessarily back mutations, to obtain a humanized antibody with favorable properties, such as affinity and biochemical properties.

[0047] The term "human antibody" as used herein refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. A human antibody may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., mouse or rat, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are generally preferred, other techniques for producing monoclonal antibodies can also be used, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of human antibody genes. A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the mouse system. The production of hybridomas in mice is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be produced, for example, using transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat system. Thus, in one embodiment, human antibodies are obtained from transgenic animals, such as transgenic mice or rats, carrying human germline immunoglobulin sequences rather than the animal's immunoglobulin sequences.In such embodiments, the antibody originates from human germline immunoglobulin sequences introduced into the animal, but the final antibody sequence is the result of the human germline immunoglobulin sequences being further modified by somatic hypermutation and affinity maturation by the animal's endogenous antibody machinery, see e.g., Mendez et al. 1997 Nat Genet. 15(2):146-56. The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, here a cysteine ​​residue in the hinge region of an antibody, is more likely to become reduced than oxidized.

[0048] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject or administration of an active substance to a subject with the purpose of reversing, mitigating, ameliorating, arresting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease.

[0049] The term "first-line treatment" refers to the initial or primary treatment recommended for a disease or condition. It may also be referred to as first-line therapy, primary treatment, initial treatment, or induction therapy.

[0050] The term "second-line treatment" refers to treatment for a disease or condition after a subject's initial treatment (first-line treatment) has failed, after the subject has had a relapse or progressed with the disease, or after the subject has experienced unacceptable adverse or side effects.

[0051] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents, such as mice, rats, and guinea pigs. The terms "subject" and "patient" and "individual" are used interchangeably herein.

[0052] Response to treatment with a binding agent of the invention 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 Table 2 below.

[0053] Table 2. Definition of response (RECIST Criteria v1.1) TIFF2024523438000002.tif158151

[0054] "Best overall response" is the best response recorded from the start of treatment to disease progression / recurrence (the smallest measurement recorded since treatment began is used as the criterion for PD). Subjects with CR or PR are considered to be in objective response state. Subjects with CR, PR or SD are considered to be in disease control state. Subjects with NE are counted as non-responders. Best overall response is the best response recorded from the start of treatment to disease progression / recurrence (the smallest measurement recorded since treatment began is used as the criterion for PD).

[0055] "Duration of response (DOR)" applies only to subjects with a confirmed best overall response of CR or PR and is defined as the time from first documentation of an objective tumor response (CR or PR) to the date of first PD or death from the underlying cancer.

[0056] "Progression-free survival (PFS)" is defined as the number of days from Day 1 of Cycle 1 to first documented progression or death from any cause.

[0057] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 to death from any cause. If a subject is dead or unknown, OS is censored at the most recent date the subject was known to be alive (on or before the cutoff date).

[0058] The term "pharmacologically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or the mammal being treated therewith.

[0059] As noted above, in a first aspect, the present invention provides a method for reducing or inhibiting the progression of a tumor in a subject or for treating cancer in a subject, comprising administering to the subject a binding agent comprising a first antigen-binding region that binds to human CD137, such as human CD137 consisting of the amino acid sequence set forth in SEQ ID NO:24, and a second antigen-binding region that binds to human PD-L1, such as human PD-L1 consisting of the amino acid sequence set forth in SEQ ID NO:26, the binding agent is administered to the subject in a dosing schedule comprising administration of dose A in one or more treatment cycles and administration of dose B in one or more treatment cycles; The amount of binding substance in dose A is a) about 0.3 to 2.5 mg / kg body weight or about 25 to 200 mg in total; and / or b) Approximately 2.1×10 -9 ~1.7×10 -8 mol / kg body weight, or about 1.7 × 10 total -7 ~1.4×10 -6 Mol and The amount of binding substance in dose B is c) about 3.8-7.5 mg / kg body weight or about 300-600 mg in total; and / or d) Approximately 2,6×10 -8 ~5.1×10 -8 mol / kg body weight, or approximately 2.0-4.1 × 10 total -6 Mol That is, The present invention relates to the above method.

[0060] More specifically, the amount of binding agent in dose A is a) 0.3 to 2.5 mg / kg body weight or 25 to 200 mg in total; and / or b) 2.1 x 10 -9 ~1.7×10 -8 mol / kg body weight or 1.7 × 10 total -7 ~1.4×10 -6 Mol It could be; The amount of binding material in dose B is c) 3.8–7.5 mg / kg body weight or 300–600 mg in total; and / or d) 2.6×10 -8 ~5.1×10 -8 mol / kg body weight or 2.0-4.1×10 total -6 Mol It could be.

[0061] The amount of the binding agent in dose A is particularly about 0.4 to 2.3 mg / kg body weight, or about 30 to about 180 mg in total, and / or about 2.56×10 -9 ~Approx. 1.53×10 -8 mol / kg body weight, or about 2.04×10 total -7 ~Approx. 1.23×10 -6 It can be mol.

[0062] The amount of the binding agent in dose A is particularly about 0.5 to about 2.0 mg / kg body weight, or about 40 to about 160 mg in total, and / or about 3.41×10 -9 ~Approx. 1.36×10 -8 mol / kg body weight, or about 2.73 × 10 total -7 ~Approx. 1.09×10 -6 It can be mol.

[0063] The amount of the binding agent in dose A is particularly about 0.6 to about 1.9 mg / kg body weight, or about 50 to about 150 mg in total, and / or about 4.26×10 -9 ~Approx. 1.28×10 -8 mol / kg body weight, or about 3.41 × 10 total -7 ~About 1,02×10 -6 It can be mol.

[0064] The amount of the binding agent in dose A is particularly about 0.8 to about 1.8 mg / kg body weight, or about 60 to about 140 mg in total, and / or about 5.11×10 -9 ~Approx. 1.19×10 -8 mol / kg body weight, or about 4.09 × 10 total -7 ~Approx. 9.54×10 -7 It can be mol.

[0065] The amount of the binding agent in dose A is particularly about 0.9 to about 1.6 mg / kg body weight, or about 70 to about 130 mg in total, and / or about 5.96×10 -9 ~Approx. 1.11×10 -8 mol / kg body weight, or about 4.77 × 10 total -7 ~Approx. 8.86×10 -7 It can be mol.

[0066] The amount of the binding agent in dose A is particularly about 1 to about 1.5 mg / kg body weight, or about 80 to about 120 mg in total, and / or about 6.81×10 -9 ~Approx. 1.02×10 -8 mol / kg body weight, or about 5.45 × 10 total -7 ~Approx. 8.18×10 -7 It can be mol.

[0067] The amount of the binding agent in dose A is particularly about 1.1 to about 1.4 mg / kg body weight, or about 90 to about 110 mg in total, and / or about 7.67×10 -9 ~Approx. 9.37×10 -9 mol / kg body weight, or about 6.13 × 10 total -7 ~Approx. 7.49×10 -7 It can be mol.

[0068] The amount of the binding agent in dose A is particularly about 1.2 to about 1.3 mg / kg body weight, or about 95 to about 105 mg in total, and / or about 8.09×10 -9 ~Approx. 8.94×10 -9 mol / kg body weight, or about 6.47 × 10 total-7 ~Approx. 7.16×10 -7 It can be mol.

[0069] The amount of said binding substance in dose A is in particular between 0.4 and 2.3 mg / kg body weight, or between 30 and 180 mg in total, and / or between 2.56×10 -9 ~1.53×10 -8 mol / kg body weight or 2.04×10 total -7 ~1.23×10 -6 It can be mol.

[0070] The amount of the binding substance in dose A is in particular 0.5 to 2.0 mg / kg body weight, or a total amount of 40 to 160 mg, and / or 3.41×10 -9 ~1.36×10 -8 mol / kg body weight, or 2.73 × 10 total -7 ~1.09×10 -6 It can be mol.

[0071] The amount of said binding substance in dose A is in particular 0.6 to 1.9 mg / kg body weight, or a total amount of 50 to 150 mg, and / or 4.26×10 -9 ~1.28×10 -8 mol / kg body weight, or 3.41 × 10 total -7 ~1,02×10 -6 It can be mol.

[0072] The amount of said binding substance in dose A is in particular 0.8-1.8 mg / kg body weight, or a total amount of 60-140 mg, and / or 5.11×10 -9 ~1.19×10 -8 mol / kg body weight, or 4.09 × 10 total -7 ~9.54×10 -7 It can be mol.

[0073] The amount of said binding substance in dose A is in particular 0.9 to 1.6 mg / kg body weight, or a total amount of 70 to 130 mg, and / or 5.96×10 -9 ~1.11×10 -8mol / kg body weight, or 4.77 × 10 total -7 ~8.86×10 -7 It can be mol.

[0074] The amount of said binding substance in dose A is in particular 1-1.5 mg / kg body weight, or a total amount of 80-120 mg, and / or 6.81×10 -9 ~1.02×10 -8 mol / kg body weight, or 5.45 × 10 total -7 ~8.18×10 -7 It can be mol.

[0075] The amount of said binding substance in dose A is in particular 1.1 to 1.4 mg / kg body weight, or a total amount of 90 to 110 mg, and / or 7.67×10 -9 ~9.37×10 -9 mol / kg body weight, or 6.13 × 10 total -7 ~7.49×10 -7 It can be mol.

[0076] The amount of said binding substance in dose A is in particular 1.2-1.3 mg / kg body weight, or a total amount of 95-105 mg, and / or 8.09×10 -9 ~8.94×10 -9 mol / kg body weight, or 6.47 × 10 total -7 ~7.16×10 -7 It can be mol.

[0077] At present, the amount of binding material in dose A is a) about 1.25 mg / kg body weight or about 100 mg in total; and / or b) Approximately 8.5×10 -9 mol / kg body weight, or about 6.8 × 10 total -7 Mol It is preferable that:

[0078] The amount of binding material in dose A is a) 1.25 mg / kg body weight or 100 mg in total; and / or b) 8.5 x 10 -9 mol / kg body weight, or 6.8 × 10 total -7 Mol It is even more preferable that:

[0079] The amount of binding agent in dose B is particularly about 4.4 to about 7.4 mg / kg body weight, or a total amount of 350 to about 590 mg, and / or about 2.98×10 -8 ~Approx. 5.03×10 -8 mol / kg body weight, or about 2.39 × 10 total -6 ~Approx. 4.02×10 -6 It can be mol.

[0080] The amount of binding agent in dose B is particularly about 5.0 to about 7.25 mg / kg body weight, or about 400 to about 580 mg in total, and / or about 3.41×10 -8 ~Approx. 4.94×10 -8 mol / kg body weight, or about 2.73 × 10 total -6 ~Approx. 3.95×10 -6 It can be mol.

[0081] The amount of binding agent in dose B is particularly about 5.3 to about 7.1 mg / kg body weight, or a total amount of about 420 to about 570 mg, and / or about 3.58×10 -8 ~Approx. 4.86×10 -8 mol / kg body weight, or about 2.86 × 10 total -6 ~Approx. 3.88×10 -6 It can be mol.

[0082] The amount of binding agent in dose B is particularly about 5.4 to about 7.0 mg / kg body weight, or about 430 to about 560 mg in total, and / or about 3.66×10 -8 ~Approx. 4.77×10 -8 mol / kg body weight, or about 2.93 × 10 total -6 ~Approx. 3.82×10 -6 It can be mol.

[0083] The amount of binding agent in dose B is particularly about 5.5 to about 6.9 mg / kg body weight, or about 440 to about 550 mg in total, and / or about 3.75×10 -8 ~Approx. 4.69×10 -8 mol / kg body weight, or approximately 3.00 × 10 total -6 ~Approx. 3.75×10 -6 It can be mol.

[0084] The amount of binding agent in dose B is particularly about 5.6 to about 6.8 mg / kg body weight, or about 450 to about 540 mg in total, and / or about 3.83×10 -8 ~Approx. 4.60×10 -8 mol / kg body weight, or about 3.07 × 10 total -6 ~Approx. 3.68×10 -6 It can be mol.

[0085] The amount of binding agent in dose B is particularly about 5.8 to about 6.6 mg / kg body weight, or about 460 to about 530 mg in total, and / or about 3.92×10 -8 ~Approx. 4.51×10 -8 mol / kg body weight, or about 3.13 × 10 total -6 ~Approx. 3.61×10 -6 It can be mol.

[0086] The amount of binding agent in dose B is particularly about 5.9 to about 6.5 mg / kg body weight, or about 470 to about 520 mg in total, and / or about 4.00×10 -8 ~Approx. 4.43×10 -8 mol / kg body weight, or about 3.20 × 10 total -6 ~Approx. 3.54×10 -6 It can be mol.

[0087] The amount of binding agent in dose B is particularly about 6.0 to about 6.4 mg / kg body weight, or about 480 to about 515 mg in total, and / or about 4.09×10 -8 ~Approx. 4.39×10 -8 mol / kg body weight, or about 3.27 × 10 total -6 ~Approx. 3.51×10-6 It can be mol.

[0088] The amount of binding agent in dose B is particularly about 6.1 to about 6.4 mg / kg body weight, or about 490 to about 510 mg in total, and / or about 4.17×10 -8 ~Approx. 4.34×10 -8 mol / kg body weight, or about 3.34 × 10 total -6 ~Approx. 3.48×10 -6 It can be mol.

[0089] The amount of binding agent in dose B is particularly about 6.2 to about 6.3 mg / kg body weight, or about 495 to about 505 mg in total, and / or about 4.22×10 -8 ~Approx. 4.30×10 -8 mol / kg body weight, or about 3.37 × 10 total -6 ~Approx. 3.44×10 -6 It can be mol.

[0090] The amount of binding substance in dose B is in particular 4.4-7.4 mg / kg body weight, or a total amount of 350-590 mg, and / or 2.98×10 -8 ~5.03×10 -8 mol / kg body weight, or 2.39 × 10 total -6 ~4.02×10 -6 It can be mol.

[0091] The amount of binding substance in dose B is in particular 5.0-7.25 mg / kg body weight, or a total amount of 400-580 mg, and / or 3.41×10 -8 ~4.94×10 -8 mol / kg body weight, or 2.73 × 10 total -6 ~3.95×10 -6 It can be mol.

[0092] The amount of binding substance in dose B is in particular 5.3-7.1 mg / kg body weight, or a total amount of 420-570 mg, and / or 3.58×10 -8 ~4.86×10 -8mol / kg body weight, or 2.86 × 10 total -6 ~3.88×10 -6 It can be mol.

[0093] The amount of binding substance in dose B is in particular 5.4-7.0 mg / kg body weight, or a total amount of 430-560 mg, and / or 3.66×10 -8 ~4.77×10 -8 mol / kg body weight, or 2.93 × 10 total -6 ~3.82×10 -6 It can be mol.

[0094] The amount of binding substance in dose B is in particular 5.5-6.9 mg / kg body weight, or a total amount of 440-550 mg, and / or 3.75×10 -8 ~4.69×10 -8 mol / kg body weight or 3.00 x 10 total -6 ~3.75×10 -6 It can be mol.

[0095] The amount of binding substance in dose B is in particular 5.6-6.8 mg / kg body weight, or a total amount of 450-540 mg, and / or 3.83×10 -8 ~4.60×10 -8 mol / kg body weight, or 3.07 × 10 total -6 ~3.68×10 -6 It can be mol.

[0096] The amount of binding substance in dose B is in particular 5.8-6.6 mg / kg body weight, or a total amount of 460-530 mg, and / or 3.92 x 10 -8 ~4.51×10 -8 mol / kg body weight, or 3.13 × 10 total -6 ~3.61×10 -6 It can be mol.

[0097] The amount of binding substance in dose B is in particular 5.9-6.5 mg / kg body weight, or a total amount of 470-520 mg, and / or 4.00×10 -8~4.43×10 -8 mol / kg body weight, or 3.20 × 10 total -6 ~3.54×10 -6 It can be mol.

[0098] The amount of binding substance in dose B is in particular 6.0-6.4 mg / kg body weight, or a total amount of 480-515 mg, and / or 4.09 x 10 -8 ~4.39×10 -8 mol / kg body weight, or 3.27 × 10 total -6 ~3.51×10 -6 It can be mol.

[0099] The amount of binding substance in dose B is in particular 6.1-6.4 mg / kg body weight, or 490-510 mg total, and / or 4.17×10 -8 ~4.34×10 -8 mol / kg body weight, or 3.34 × 10 total -6 ~3.48×10 -6 It can be mol.

[0100] The amount of binding substance in dose B is in particular 6.2-6.3 mg / kg body weight, or 495-505 mg total, and / or 4.22×10 -8 ~4.30×10 -8 mol / kg body weight, or 3.37 × 10 total -6 ~3.44×10 -6 It can be mol.

[0101] At present, the amount of binding material in dose B is a) about 6.25 mg / kg body weight or about 500 mg in total; and / or b) Approximately 4.3×10 -8 mol / kg body weight, or about 3.4 × 10 total -6 Mol It is preferable that:

[0102] The amount of binding material in dose B is a) 6.25 mg / kg body weight or 500 mg in total; and / or b) 4.3 x 10 -8 mol / kg body weight, or 3.4 × 10 total -6 Mol It is even more preferable that:

[0103] More preferably, the dosing schedule comprises administration of dose A in one or more treatment cycles, followed by administration of dose B in one or more treatment cycles.

[0104] Dose A may be administered once in each treatment cycle, for example, on day 1 of each treatment cycle.

[0105] Dose B may also be administered once in each treatment cycle, for example, on day 1 of each treatment cycle.

[0106] Dose A may be administered in one or more treatment cycles, each treatment cycle having a duration of 3 weeks / 21 days, for example, 2, 3, 4, or 5 treatment cycles, each treatment cycle having a duration of 3 weeks / 21 days.Preferably, Dose A is administered in 2 treatment cycles, each treatment cycle having a duration of 3 weeks / 21 days.

[0107] Dose A is preferably administered once in each of the 3 week / 21 day treatment cycles (Q3W).

[0108] Dose A may specifically be administered on day 1 of each of said one or more 3 week / 21 day treatment cycles.

[0109] Dose B may be administered in one or more treatment cycles, each treatment cycle having a duration of 6 weeks / 42 days. In particular, dose B may be administered in 2-5 treatment cycles, each treatment cycle having a duration of 6 weeks / 42 days, e.g., in 2-10 treatment cycles, each treatment cycle having a duration of 6 weeks / 42 days, e.g., in 2-20 treatment cycles, each treatment cycle having a duration of 6 weeks / 42 days, or in 2-50 treatment cycles, e.g., each treatment cycle having a duration of 6 weeks / 42 days.

[0110] Dose B is preferably administered once in each of said one or more 6 week / 42 day treatment cycles (Q6W).

[0111] In the methods disclosed herein, Dose B may specifically be administered on day 1 of each of said one or more 6 week / 42 day treatment cycles.

[0112] In a further embodiment of the invention, the dosing schedule comprises administration of dose A for two treatment cycles, followed by administration of dose B for one or more treatment cycles.

[0113] In the context of the present invention, dose B may be considered a "maintenance therapy" and may therefore be continued until complete tumor regression or until disease progression. Thus, in the methods according to the invention, the dosing schedule comprises administration of dose A followed by administration of dose B until complete tumor regression or disease progression.

[0114] Further to dosing of a binding agent, the methods disclosed herein may include collecting a whole blood sample and assessing PD-L1 receptor occupancy by said binding agent.

[0115] The binding agent is preferably administered systemically, particularly by intravenous injection or infusion.

[0116] Each dose may be infused over a period of at least 30 minutes, for example, at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 240 minutes.

[0117] The binding agent used in the method of the present disclosure is a) the first antigen-binding region may comprise a heavy chain variable region (VH) comprising the sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) of SEQ ID NO:1, and a light chain variable region (VL) comprising the sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) of SEQ ID NO:5; and b) the second antigen-binding region may comprise a heavy chain variable region (VH) comprising the sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) of SEQ ID NO:8, and a light chain variable region (VL) comprising the sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) of SEQ ID NO:12; It may be a binding agent.

[0118] In particular, the binding agent is a) the first antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs:2, 3, and 4, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NOs:6, GAS, and SEQ ID NO:7, respectively; and b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:9, 10, 11, respectively, and a light chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:13, DDN, SEQ ID NO:14, respectively; It may be a binding agent.

[0119] Each variable region can include three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0120] The complementarity determining regions and framework regions are preferably arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0121] A binding agent for use in accordance with the present disclosure may comprise a first and a second antigen-binding region, a) the first antigen-binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:5; and b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:8, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:12.

[0122] In particular, the binding agents used in accordance with the present disclosure include a) the first antigen-binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:8 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:12; It may comprise a first and a second antigen-binding region.

[0123] The binding agent may in particular be an antibody. Examples of different types of binding agents according to the invention include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains for heterodimerization; (ii) recombinant IgG-like dual targeting molecules, where both molecules each contain a Fab fragment or a portion of said Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, where a full-length IgG antibody is fused with an additional Fab fragment or a portion of a Fab fragment; (iv) Fc fusion molecules, where a single chain Fv molecule or a stabilized diabody is fused with a heavy chain constant domain, an Fc region or a portion thereof; (v) Fab fusion molecules, where different Fab fragments are fused together and fused with a heavy chain constant domain, an Fc region or a portion thereof; and (vi) ScFv- and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) where different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused with each other or with another protein or carrier molecule fused with a heavy chain constant domain, an Fc region or a portion thereof.

[0124] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to, Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), so-called knob-into-hole molecules (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatically matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y molecules (Genentech, Wranik et al., J. Immunol. 20100263611; WO20100263611), WO20100263625, WO201002636364 ... al. J. Biol. Chem. 2012,287(52):43331-9,doi:10.1074 / jbc.M112.397869.Epub 2012 Nov 1), DIG-body and PIG-body molecules (Pharmabcine,WO2010134666, WO2014081202), strand-exchange engineered domain body (SEED body) molecules (EMD Serono, WO2007110205), Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254) and DuoBody® molecules (Genmab, WO2011131746).

[0125] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, dual targeting (DT)-Ig molecules (WO2009058383), two-in-one antibodies (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr; 5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), κλ Bodies (NovImmune, WO2012023053) and CovX-bodies (CovX / Pfizer; Doppalapudi, VR, et al. 2007.Bioorg.Med.Chem.Lett.17,501-506.

[0126] Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD)-Ig molecules (Abbott, US 7,612,181), dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like bispecific molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0127] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009(Abstract # 5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538) and Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine-China).

[0128] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15; 160(4): 1677-86.), dual action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotechnol, Schoonjans, J Immunol. 2000 Dec 15; 165(12): 7050-7.) and Fab-Fv molecules (UCB-Celltech, WO2009040562 A1).

[0129] Examples of ScFv-based, diabody-based antibodies and domain antibodies include, but are not limited to, bispecific T cell engager (BiTE) molecules (Micromet, WO2005061547), tandem diabody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66.), dual affinity retargeting technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), single chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO2010059315) and COMBODY molecules (Epigen These include dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual targeting heavy chain only domain antibodies (Ablynx, Hmila et al., FASEB J. 2010).

[0130] In a currently preferred embodiment, the binding agent is a multispecific antibody, such as a bispecific antibody. In particular, a binding agent used in accordance with the present invention may have up to two binding regions.

[0131] Many different formats and uses of bispecific antibodies are known in the art and have been reviewed in Kontermann; Drug Discov Today, 2015 Jul; 20(7): 838-47 and; Mabs, 2012 Mar-Apr; 4(2): 182-97.

[0132] Bispecific antibodies used according to the present invention are not limited to any particular bispecific format or method of production. Examples of bispecific antibody molecules that can be used in the present invention include (i) single antibodies with two arms containing different antigen-binding regions; (ii) single-chain antibodies with specificity for two different epitopes, e.g., by two scFvs linked in tandem by an additional peptide linker; (iii) dual variable domain antibodies (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem with a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin, 2003). (iv) chemically linked bispecific (Fab')2 fragments; (v) Tandabs, which are fusions of two single chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) flexibodies, which are combinations of scFvs and diabodies resulting in a multivalent molecule; (vii) so-called "dock-lock" molecules based on the "dimerization and docking domain" of protein kinase A, which when applied to Fabs can result in trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab arm; and (ix) diabodies.

[0133] In one embodiment, the binding agents used in the present invention are diabodies as well as cross-bodies. In one embodiment, the binding agents of the present invention are bispecific antibodies obtained by controlled Fab arm exchange (e.g., as described in WO2011131746 (Genmab)).

[0134] The binding agents used according to the invention are preferably human, humanized or chimeric antibodies. In the embodiment where the antibody is a bispecific antibody, both half molecules may be of human origin, humanized or chimeric, and the half molecules may be distinct in terms of sequence origin.

[0135] For example, in one embodiment, a binding agent, e.g., a bispecific antibody, comprises two half molecules, each half molecule comprising an antigen-binding region: (i) the half molecule is chimeric and contains an antigen-binding region capable of binding to human PD-L1; and / or (ii) If present, the half-molecule that contains an antigen-binding region capable of binding to human CD137 is chimeric.

[0136] For example, in another embodiment, a bispecific antibody comprises two half molecules, each half molecule comprising an antigen-binding region: (i) the half molecule containing the antigen-binding region capable of binding to human PD-L1 is humanized; and / or (ii) If present, the half molecule that contains an antigen-binding region capable of binding to human CD137 is humanized.

[0137] For example, a bispecific antibody comprises two half molecules, each of which may contain an antigen-binding region. (i) the half-molecule containing the antigen-binding region capable of binding to human PD-L1 is of human origin; and / or (ii) the half molecule containing the antigen-binding region capable of binding to human CD137 is of human origin;

[0138] Thus, for example, the antigen-binding region capable of binding to human PD-L1 may be humanized and the antigen-binding region capable of binding to human CD137 may be humanized.

[0139] Alternatively, the antigen-binding region capable of binding to human PD-L1 may be of human origin and the antigen-binding region capable of binding to human CD137 may be of human origin.

[0140] The binding agent may be a bispecific antibody comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD137, wherein the half molecule containing the antigen-binding region capable of binding to human PD-L1 is human-derived, humanized, or chimeric, and the half molecule containing the antigen-binding region capable of binding to human CD137 is humanized.

[0141] Preferably, the half molecule comprising the antigen-binding region capable of binding to human PD-L1 is of human origin, and the half molecule comprising the antigen-binding region capable of binding to human CD137 is humanized.

[0142] In the methods disclosed herein, the binding agent can be in the form of a full-length antibody or an antibody fragment.

[0143] In the method according to the present disclosure, the binding agent comprises: i) a polypeptide comprising, consisting of, or consisting essentially of said first heavy chain variable region (VH) and first heavy chain constant region (CH); and ii) a polypeptide comprising, consisting of, or consisting essentially of said second heavy chain variable region (VH) and said second heavy chain constant region (CH). may include.

[0144] In the method according to the present disclosure, the binding agent comprises: i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL); and ii) a polypeptide comprising said second light chain variable region (VL) and further comprising a second light chain constant region (CL). It may further include.

[0145] The binding agent is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising: i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH); and ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). Including, and the second binding arm is iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH); and iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). Including, It may be an antibody.

[0146] Each of the first and second heavy chain constant regions (CH) may include one or more of a heavy chain constant 1 (CH1) region, a hinge region, a heavy chain constant 2 (CH2) region, and a heavy chain constant 3 (CH3) region, preferably at least the hinge region, the CH2 region, and the CH3 region.

[0147] Each of the first and second heavy chain constant regions (CH) can comprise a CH3 region, wherein each or both of said CH3 regions comprise imbalanced mutations.

[0148] The bispecific antibody used according to the present invention may comprise a first Fc sequence comprising a first CH3 region and a second Fc sequence comprising a second CH3 region, wherein the sequence of the first CH3 region and the sequence of the second CH3 region are different, such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction of each of the first CH3 region and the second CH3 region. Further details regarding such interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0149] Stable bispecific PD-L1xCD137 antibodies can be obtained in high yield using certain methods based on one homodimeric starting PD-L1 antibody and one homodimeric starting CD137 antibody that contain only a few conservative imbalanced mutations within the CH3 regions. Imbalanced mutations mean that the sequence of the first CH3 region and the sequence of the second CH3 region contain amino acid substitutions at non-identical positions.

[0150] In the method according to the present disclosure, the binding agent may be a binding agent comprising first and second constant regions (CH), wherein in the first CH, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 according to EU numbering in a human IgG1 heavy chain is substituted, and in the second CH, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 according to EU numbering in a human IgG1 heavy chain is substituted, and wherein the first heavy chain and the second heavy chain are not substituted at the same positions.

[0151] In the methods according to the present disclosure, the binding agent may be (i) a binding agent in which the amino acid at a position corresponding to F405 according to EU numbering in a human IgG1 heavy chain is L in the first heavy chain constant region (CH) and the amino acid at a position corresponding to K409 according to EU numbering in a human IgG1 heavy chain is R in the second heavy chain constant region (CH), or (ii) a binding agent in which the amino acid at a position corresponding to K409 according to EU numbering in a human IgG1 heavy chain is R in the first heavy chain and the amino acid at a position corresponding to F405 according to EU numbering in a human IgG1 heavy chain is L in the second heavy chain.

[0152] In the methods according to the invention, the binding agent preferably induces Fc-mediated effector functions to a lesser extent than another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising the hinge, CH2, and CH3 regions of human IgG1.

[0153] The first and second heavy chain constant regions (CH) can be modified such that the antibody induces Fc-mediated effector function to a lesser extent than an otherwise identical antibody that contains unmodified first and second heavy chain constant regions (CH).

[0154] The unmodified first and second heavy chain constant regions (CH) may comprise the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:30.

[0155] The Fc-mediated effector functions can be measured by binding to Fcγ receptors, by binding to C1q, or by induction of Fc-mediated Fcγ receptor cross-linking.

[0156] Fc-mediated effector functions can be measured by measuring binding to C1q.

[0157] The first and second heavy chain constant regions may be modified such that C1q binding to the antibody is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, where C1q binding is preferably measured by ELISA.

[0158] In the method according to the invention, it is preferred that in at least one of the first and second heavy chain constant regions (CH), one or more amino acids at positions corresponding to L234, L235, D265, N297, and P331 according to EU numbering in a human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0159] Preferably, the amino acid residues at positions corresponding to L234 and L235 according to EU numbering in a human IgG1 heavy chain are F and E in said first and second heavy chains, respectively.

[0160] It is further preferred that the amino acid residues at positions corresponding to L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A, respectively, in said first and second heavy chain constant regions (HC).

[0161] Methods according to the present disclosure may use a binding agent in which the positions corresponding to L234 and L235 according to EU numbering in both the first and second heavy chain constant regions of a human IgG1 heavy chain are F and E, respectively, and (i) the position corresponding to F405 according to EU numbering in the first heavy chain constant region of a human IgG1 heavy chain is L and the position corresponding to K409 according to EU numbering in the second heavy chain of a human IgG1 heavy chain is R, or (ii) the position corresponding to K409 according to EU numbering in the first heavy chain constant region of a human IgG1 heavy chain is R and the position corresponding to F405 according to EU numbering in the second heavy chain of a human IgG1 heavy chain is L.

[0162] Provided herein is a binding agent for use in the method, in which the amino acid residues at positions corresponding to L234, L235, and D265 according to EU numbering in both the first and second heavy chain constant regions of a human IgG1 heavy chain are F, E, and A, respectively, and (i) the amino acid at a position corresponding to F405 according to EU numbering in the first heavy chain constant region of a human IgG1 heavy chain is L and the amino acid residue at a position corresponding to K409 according to EU numbering in the second heavy chain constant region of a human IgG1 heavy chain is R, or (ii) the position corresponding to K409 according to EU numbering in the first heavy chain of a human IgG1 heavy chain is R and the position corresponding to F405 according to EU numbering in the second heavy chain of a human IgG1 heavy chain is L.

[0163] Binders with the combination of the three amino acid substitutions L234F, L235E, and D265A plus the mutations K409R or F405L are referred to herein with the suffixes "FEAR" or "FEAL," respectively.

[0164] In the binding agent, the constant region of the first and / or second heavy chain comprises a) the sequence shown in SEQ ID NO:15 or SEQ ID NO:30 [IgG1-FC]; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 10 substitutions compared to the amino acid sequence defined in a) or b), for example up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 substitution. It may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0165] In the binding agent used according to the invention, the constant region of the first or second heavy chain, e.g. the first heavy chain, a) the sequence depicted in SEQ ID NO:16 or SEQ ID NO:31 [IgG1-F405L]; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 9 substitutions, e.g. up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 substitution, compared to the amino acid sequence defined in a) or b). It may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0166] The first or second heavy chain constant region, e.g., the second heavy chain constant region, a) the sequence depicted in SEQ ID NO:17 or SEQ ID NO:32 [IgG1-F409R]; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 10 substitutions compared to the amino acid sequence defined in a) or b), for example up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions, or up to 1 substitution. It may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0167] Additionally, the constant region of the first and / or second heavy chain comprises a) the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 33 [IgG1-Fc_FEA]; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 7 substitutions, e.g. up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions, or up to 1 substitution, compared to the amino acid sequence defined in a) or b). It may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0168] The constant region of the first and / or second heavy chain, e.g., the first heavy chain, a) the sequence depicted in SEQ ID NO: 19 or SEQ ID NO: 34 [IgG1-Fc_FEAL]; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 6 substitutions, e.g. up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions, or up to 1 substitution, compared to the amino acid sequence defined in a) or b). It may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0169] The first and / or second heavy chain constant region, e.g., the second heavy chain constant region, a) the sequence shown in SEQ ID NO:20 or SEQ ID NO:35 [IgG1-Fc_FEAR]; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 6 substitutions, e.g. up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions, or up to 1 substitution, compared to the amino acid sequence defined in a) or b). It may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0170] Although the constant region sequences set forth in SEQ ID NOs:15-20 describe a terminal lysine (K), this C-terminal lysine has been omitted from the sequences set forth in SEQ ID NOs:30-36, 38. The origin of this lysine is the naturally occurring sequence found in humans from which these Fc regions are derived. During production of recombinant antibodies in cell culture, this terminal lysine may be proteolytically cleaved off by endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking the C-terminal lysine. For purposes of antibody manufacturing, the DNA encoding this terminal lysine may be omitted from the sequence, allowing antibodies to be made that lack this lysine. Antibodies produced with nucleic acid sequences that either encode or do not encode a terminal lysine are substantially identical in sequence and function, since the degree of terminal lysine processing is typically high, for example, with antibodies produced in CHO-based production systems (Dick, LWet al. Biotechnol. Bioeng. 2008;100:1132-1143). It is therefore understood that antibodies according to the invention, for example as described herein, may be produced that do not encode or have a terminal lysine. Thus, for manufacturing purposes, antibodies may be produced that do not have a terminal lysine.

[0171] Either the human light chain constant region kappa (κ) or lambda (λ) can be used. Thus, in certain embodiments, a binding agent disclosed herein can comprise a kappa (κ) light chain constant region.

[0172] Alternatively, or additionally, a binding agent disclosed herein may comprise a lambda (λ) light chain constant region.

[0173] In a binding agent used according to the invention, the first light chain constant region may be a kappa (κ) light chain constant region.

[0174] In a binding agent used according to the invention, the second light chain constant region may be a lambda (λ) light chain constant region.

[0175] Alternatively, a binding agent for use according to the invention may comprise a first light chain constant region which is a lambda (λ) light chain constant region.

[0176] In a binding agent used according to the invention, said second light chain constant region may be a kappa (κ) light chain constant region.

[0177] In certain embodiments, the kappa (κ) light chain is a) the sequence shown in SEQ ID NO:21; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 10 substitutions compared to the amino acid sequence defined in a) or b), for example up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions, or up to 1 substitution. The amino acid sequence is selected from the group consisting of:

[0178] Lambda (λ) light chains are a) the sequence shown in SEQ ID NO:22; b) a subsequence of the sequence of a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having up to 10 substitutions compared to the amino acid sequence defined in a) or b), for example up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions, or up to 1 substitution. The amino acid sequence may be selected from the group consisting of:

[0179] Binding agents used in accordance with the methods of the present disclosure may be of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0180] The choice of isotype is typically guided by the desired Fc-mediated effector function, such as induction of ADCC or the requirement for an antibody lacking Fc-mediated effector function (an "inactive" antibody). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. The effector function of the antibody of the invention can be altered by isotype switching, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic applications.

[0181] In a currently preferred embodiment, the binding agent is a full-length IgG1 antibody.

[0182] The binding agent used in the methods according to the present disclosure may be an antibody that is of the IgG1m(f) allotype. Alternatively, the antibody may be of the IgG1m(za) allotype.

[0183] The binding agents used in accordance with the present disclosure include (i) a first binding arm comprising a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first VH comprises a first HCDR1 sequence, a first HCDR2 sequence, and a first HCDR3 sequence, wherein the first HCDR1 sequence is set forth in SEQ ID NO:2, the first HCDR2 sequence is set forth in SEQ ID NO:3, and the first HCDR3 sequence is set forth in and includes SEQ ID NO:4; and the first VL comprises a first LCDR1 sequence, a first LCDR2 sequence, and a first LCDR3 sequence, wherein the first LCDR1 sequence is set forth in SEQ ID NO:6, the first LCDR2 sequence is GAS, and the first LCDR3 sequence is set forth in SEQ ID NO:7; and (ii) a second binding arm comprising a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein said second VH comprises a second HCDR1 sequence, a second HCDR2 sequence, and a second HCDR3 sequence, said second HCDR1 sequence being set forth in SEQ ID NO:9, said second HCDR2 sequence being set forth in SEQ ID NO:10, and said second HCDR3 sequence being set forth in SEQ ID NO:11; and said second VL comprises a second LCDR1 sequence, a second LCDR2 sequence, and a second LCDR3 sequence, said second LCDR1 sequence being set forth in SEQ ID NO:13, said second LCDR2 sequence being DDN, and said second LCDR3 sequence being set forth in SEQ ID NO:14. may include; the first binding arm comprises a first heavy chain constant region (CH) and the second binding arm comprises a second CH, and positions L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A in the first CH and the second CH, respectively; and The amino acid at the position corresponding to F405 according to EU numbering in a human IgG1 heavy chain is L in the first CH, and the amino acid at the position corresponding to K409 according to EU numbering in a human IgG1 heavy chain is R in the second CH.

[0184] The binding agents used in accordance with the present disclosure include (i) a first binding arm comprising a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first VH comprises a first HCDR1 sequence, a first HCDR2 sequence, and a first HCDR3 sequence, wherein the first HCDR1 sequence is set forth in SEQ ID NO:2, the first HCDR2 sequence is set forth in SEQ ID NO:3, and the first HCDR3 sequence is set forth in SEQ ID NO:4, and the first VL comprises a first LCDR1 sequence, a first LCDR2 sequence, and a first LCDR3 sequence, wherein the first LCDR1 sequence is set forth in SEQ ID NO:6, the first LCDR2 sequence is GAS, and the first LCDR3 sequence is set forth in SEQ ID NO:7; and (ii) a second binding arm comprising a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second VH comprises a second HCDR1 sequence, a second HCDR2 sequence, and a second HCDR3 sequence, wherein the second HCDR1 sequence is set forth in SEQ ID NO:9, the second HCDR2 sequence is set forth in SEQ ID NO:10, and the second HCDR3 sequence is set forth in SEQ ID NO:11, and the second VL comprises a second LCDR1 sequence, a second LCDR2 sequence, and a second LCDR3 sequence, wherein the second LCDR1 sequence is set forth in SEQ ID NO:13, the second LCDR2 sequence is DDN, and the second LCDR3 sequence is set forth in SEQ ID NO:14. may include; the first binding arm comprises a first heavy chain constant region (CH) and the second binding arm comprises a second CH, and positions L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A in the first CH and the second CH, respectively; and The amino acid at the position corresponding to K409 according to EU numbering in a human IgG1 heavy chain is R in the first CH, and the amino acid at the position corresponding to F405 according to EU numbering in a human IgG1 heavy chain is L in the second CH.

[0185] The binding agents used in accordance with the present disclosure include (i) a first binding arm comprising a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first VH comprises a first HCDR1 sequence, a first HCDR2 sequence, and a first HCDR3 sequence, wherein the first HCDR1 sequence comprises the amino acid sequence set forth in SEQ ID NO:9, the first HCDR2 sequence comprises the amino acid sequence set forth in SEQ ID NO:10, and the first HCDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO:11, and the first VL comprises a first LCDR1 sequence, a first LCDR2 sequence, and a first LCDR3 sequence, wherein the first LCDR1 sequence comprises the amino acid sequence set forth in SEQ ID NO:13, the first LCDR2 sequence comprises the amino acid sequence GAS, and the first LCDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO:14; and (ii) a second binding arm comprising a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second VH comprises a second HCDR1 sequence, a second HCDR2 sequence, and a second HCDR3 sequence, wherein the second HCDR1 sequence comprises the amino acid sequence set forth in SEQ ID NO:18, the second HCDR2 sequence comprises the amino acid sequence set forth in SEQ ID NO:19, and the second HCDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO:20, and the second VL comprises a second LCDR1 sequence, a second LCDR2 sequence, and a second LCDR3 sequence, wherein the second LCDR1 sequence comprises the amino acid sequence set forth in SEQ ID NO:22, the second LCDR2 sequence comprises the amino acid sequence DDN, and the second LCDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO:23. may include; the first binding arm comprises a first heavy chain constant region (CH) and the second binding arm comprises a second CH, and positions L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A in the first CH and the second CH, respectively; and The amino acid at the position corresponding to F405 according to EU numbering in a human IgG1 heavy chain is L in the first CH, and the amino acid at the position corresponding to K409 according to EU numbering in a human IgG1 heavy chain is R in the second CH.

[0186] The binding agents used in accordance with the present disclosure include (i) a first binding arm comprising a first heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a first light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and (ii) a second binding arm comprising a second heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:8 and a second light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:12. may include; the first binding region comprises a first heavy chain constant region (CH) and the second binding arm comprises a second CH, and positions L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A in the first CH and the second CH, respectively; and The amino acid at the position corresponding to K409 according to EU numbering in a human IgG1 heavy chain is R in the first CH, and the amino acid at the position corresponding to F405 according to EU numbering in a human IgG1 heavy chain is L in the second CH.

[0187] The binding agents used in accordance with the present disclosure include (i) a first binding arm comprising a first heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a first light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and (ii) a second binding arm comprising a second heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:8 and a second light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:12. may include; the first binding arm comprises a first heavy chain constant region (CH), the second antigen-binding region comprises a second CH, and positions L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A in the first CH and the second CH, respectively; and The amino acid at the position corresponding to F405 according to EU numbering in a human IgG1 heavy chain is L in the first CH, and the amino acid at the position corresponding to K409 according to EU numbering in a human IgG1 heavy chain is R in the second CH.

[0188] The binding agents used in accordance with the present disclosure include i) a first heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:36, and a first light chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:37; and ii) a second heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:38, and a second light chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:39. may include.

[0189] Binding agents used in accordance with the present disclosure may be in the format of full-length antibodies or antibody fragments.

[0190] The binding agent used in accordance with the present disclosure may be acasunlimab or a biosimilar thereof.

[0191] The binding agent used in accordance with the present invention may be in a pharma- ceutically acceptable composition or formulation having a pH of 5-6, for example a composition or formulation comprising histidine, sucrose, and polysorbate-80.

[0192] In particular, the binding agent used in accordance with 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.

[0193] 2. The method of any one of the preceding claims, wherein the binding agent is in the form of a composition or formulation comprising 10-30 mg binding agent / mL, such as 20 mg binding agent / mL.

[0194] When used in the method according to the present disclosure, the binding agent can be diluted before administration to a subject.Dilution can be in saline; for example, 0.9% NaCl.The binding agent can be in the form of composition as defined above, and can be diluted in 0.9% NaCl (saline) before administration.

[0195] The subjects receiving the treatments disclosed herein may in particular be human subjects.

[0196] The tumor or cancer treated in accordance with the present disclosure may be a solid tumor.

[0197] In certain embodiments, the tumor is a PD-L1 positive tumor; that is, a tumor that expresses PD-L1.

[0198] The tumor or cancer may be selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, renal cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, ovarian cancer, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma.

[0199] The tumor or cancer may be selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urothelial carcinoma (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g., cancer of the oral cavity, pharynx, or larynx), and cervical cancer.

[0200] In a currently preferred embodiment, the tumor or cancer is lung cancer.

[0201] The lung cancer may in particular be non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

[0202] In certain embodiments, the NSCLC does not have an epidermal growth factor (EGFR) sensitizing mutation and / or an anaplastic lymphoma (ALK) gene translocation / ROS1 rearrangement.

[0203] Lung cancer is the most common malignant tumor and the most common cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for 85-90% of all lung cancer cases (Jemal et al., 2011). The 5-year survival rate for NSCLC is approximately 18% (SEER, 2018). The main histological subtypes of NSCLC include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large cell carcinoma, carcinoid tumors, and other less common subtypes, with adenocarcinoma being the most common.

[0204] Standard treatment for patients with advanced or metastatic NSCLC who have progressed on or are no longer candidates for targeted therapy typically includes platinum-based chemotherapy, which has been shown to produce overall response rates (ORR) of approximately 25-35%, time to progression (TTP) of 4-6 months, and median survival of 8-10 months.

[0205] Genetic mutations / alterations in tumors have been identified and have an impact on treatment choice. Identification of specific mutations or alterations in genes in tumors, such as anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR), ROS1 (c-ROS oncogene 1), BRAF, KRAS, and programmed cell death ligand-1 (PD-L1), can aid in the selection of potentially effective targeted therapies while avoiding the use of therapies unlikely to provide clinical benefit (NCCN, 2018c). Activating susceptible EGFR mutations predict response to EGFR tyrosine kinase inhibitors (TKIs) (e.g., gefitinib, erlotinib, afatinib, and osimertinib). Similarly, TKIs (e.g., alectinib, ceritinib, and crizotinib) are effective treatments for ALK and ROS1 mutations and are approved as first-line treatments for each mutation. Checkpoint inhibitor antibodies that block the interaction of PD1 and PD-L1 (e.g., pembrolizumab and nivolumab), alone or in combination with chemotherapy, have also been shown to be effective therapeutic agents for the treatment of patients with advanced or metastatic NSCLC whose tumors express PD-L1.

[0206] Despite multiple treatment options, patients with stage IV NSCLC ultimately have a poor prognosis, and lung cancer remains the number one cause of cancer death in both men and women. Treatment rates decline with each line of treatment as patients either succumb to their cancer or experience a decline in health that precludes further treatment.

[0207] The lung cancer may be NSCLC, but it does not have epidermal growth factor receptor (EGFR)-sensitive mutations and / or anaplastic lymphoma (ALK) gene translocations / ROS1 rearrangements. An EGFR-sensitive mutation refers to a mutation that confers sensitivity to EGFR tyrosine kinase inhibitors (TKIs), such as the approved tyrosine kinase inhibitors erlotinib, osimertinib, gefintinib, olmutinib, nazartinib, and avitinib.

[0208] The amino acid sequence of epidermal growth factor receptor (EGFR) is set forth herein as SEQ ID NO:27.

[0209] The subject who receives the treatment disclosed herein may be a subject who has undergone a pretreatment for shrinking or suppressing the progression of the tumor or a pretreatment for the cancer.The subject may have undergone, for example, one, two, three or four systemic pretreatment regimens for advanced / metastatic disease, and has experienced disease progression, for example radiographic disease progression, during or after the last systemic pretreatment.

[0210] In a particular embodiment, the treatment according to the present invention is provided to a subject who has undergone a prior treatment, e.g. a prior treatment as defined above, where the last prior treatment was a prior treatment with a PD1 inhibitor or a PD-L1 inhibitor, e.g. an anti-PD-1 antibody or an anti-PD-L1 antibody, where said PD-1 inhibitor or PD-L1 inhibitor is administered as a monotherapy or as part of a combination therapy. It will be understood that prior treatment in this context does not include treatment with a multispecific agent targeting PD-L1 and 4-1BB.

[0211] Preferably, treatment according to the invention is provided to a subject where the time since said subject's progression on last treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or such as 2 weeks or less.

[0212] By analogy, it may be preferable to provide treatment according to the invention to a subject where it has been 8 months or less since their last dose of a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or anti-PD-L1 antibody, as part of their last conditioning treatment, such as 8 months or less, for example 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, or for example 2 weeks or less.

[0213] Subjects have received prior treatment in the form of platinum-based chemotherapy.

[0214] Subjects undergoing the treatments disclosed herein may be those who are not eligible for platinum-based therapy and who have received prior treatment in the form of alternative chemotherapy, for example, treatment with a gemcitabine-containing regimen.

[0215] A subject receiving the treatments disclosed herein may have been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

[0216] The subject may have experienced disease progression during or after treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

[0217] A subject treated according to the present disclosure may have experienced disease progression during or after last prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

[0218] A subject treated as disclosed herein may have experienced disease progression, e.g., radiographically determined disease progression, during or after the last systemic conditioning treatment.

[0219] In other embodiments, the subject has not been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

[0220] The methods disclosed herein may be for first-line treatment of said tumor or cancer.

[0221] Alternatively, the method may be for second line treatment of the tumor or cancer.

[0222] In a further aspect, the invention provides a binding agent for use in reducing or inhibiting the progression of a tumor or for use in the treatment of cancer, said binding agent comprising a first antigen-binding region that binds human CD137, such as human CD137 consisting of the amino acid sequence set forth in SEQ ID NO:24, and a second antigen-binding region that binds human PD-L1, such as human PD-L1 consisting of the amino acid sequence set forth in SEQ ID NO:26, and wherein the binding agent is administered to a subject with a dosing schedule comprising administration of dose A for one or more treatment cycles and administration of dose B for one or more treatment cycles; The amount of binding substance in dose A is c) about 0.3 to 2.5 mg / kg body weight or about 25 to 200 mg in total; and / or d) Approximately 2.1×10 -9 ~1.7×10 -8 mol / kg body weight, or about 1.7 × 10 total -7~1.4×10 -6 Mol and The amount of binding substance in dose B is e) about 3.8 to 7.5 mg / kg body weight or about 300 to 600 mg in total; and / or f) Approximately 2,6×10 -8 ~5.1×10 -8 mol / kg body weight, or approximately 2.0-4.1 × 10 total -6 Mol That is, The binding agent is provided.

[0223] It will be appreciated that the above features relating to the methods of the present disclosure also apply in the context of binding agents for use in reducing or inhibiting tumor progression or for use in the treatment of cancer. In particular, the dosing schedule may be as further defined above.

[0224] The characteristics of the binding agents disclosed above may also apply in the context of a binding agent for use in reducing or inhibiting tumor progression or for use in the treatment of cancer; for example, the amino acid sequences of the CDRs and variable regions and the amino acid sequence of the constant region may be as defined above.

[0225] array (Table 3) In bold and underlined are F; E; A; L and R corresponding to positions 234 and 235; 265; 405 and 409, respectively, according to EU-numbering. In the variable region, the CDR regions annotated according to the IMGT definition are underlined. TIFF2024523438000003.tif238149TIFF2024523438000004.tif229149TIFF2024523438000005.tif223149TIFF20245234380 00006.tif215149TIFF2024523438000007.tif222149TIFF2024523438000008.tif222149TIFF2024523438000009.tif108149

[0226] The present invention is further illustrated by the following examples which should not be construed as limiting the scope of the invention. EXAMPLES

[0227] Example 1: Generation of CD137 antibody Antibodies CD137-005 and CD137-009 were generated as described in Example 1 of WO2016 / 110584. Briefly, rabbits were immunized with a protein mixture containing human CD137-Fc fusion protein. Single B cells were isolated from blood and screened for CD137-specific antibody production by ELISA and flow cytometry. RNA was extracted from the screened positive B cells and sequenced. Heavy and light chain variable regions were gene synthesized and cloned into human IgG1κ or human IgG1λ expression vectors containing human IgG1 heavy chain (corresponding to SEQ ID NO:20) with the following amino acid mutations according to EU numbering: L234F, L235E, D265A and F405L (FEAL) or F405L (FEAL). The variable region sequences of the chimeric CD137 antibody (CD137-009) are shown in SEQ ID NO:28 and SEQ ID NO:29 of the sequence listing herein.

[0228] Example 2: Humanization of rabbit (chimeric) CD137 antibody The rabbit anti-CD137-009 humanized antibody sequence was generated at Antitope (Cambridge, UK). The humanized antibody sequence was generated using germline humanization (CDR grafting) technology. Humanized V-region genes were designed based on human germline sequences with the closest homology to the rabbit antibody VH and Vκ amino acid sequences. A series of seven VH and three Vκ (VL) germline humanized V-region genes were designed. A structural model of the non-human parent antibody V-region was generated using the Swiss PDB and analyzed to identify amino acids within the V-region framework that may be important for the binding properties of the antibody. Such amino acids were recorded for incorporation into one or more variant CDR grafted antibodies. The germline sequences used as the basis for the humanized designs are shown in Table 4.

[0229] Table 4. Closest matching human germline V and J segment sequences TIFF2024523438000010.tif44150

[0230] Variant sequences with the lowest abundance of potential T cell epitopes were then selected using Antitope's proprietary in silico technology iTope™ and TCED™ (T cell epitope database) (Perry, LCA, Jones, TD and Baker, MP New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9(6):385-396; 20 Bryson, CJ, Jones, TD and Baker, MP Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24(1):1-8). Finally, the nucleotide sequences of the designed variants are codon-optimized.

[0231] The variable region sequences of the humanized CD137 antibody (CD137-009-HC7LC2) are shown in SEQ ID NO:1 and SEQ ID NO:5 of the sequence listing herein.

[0232] Example 3: Generation of PD-L1 antibodies Immunizations and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). cDNA encoding amino acids 19-238 of human PD-L1 was cloned into a proprietary expression plasmid from Aldevron. Antibody PD-L1-547 was generated by immunization of animals OmniRat (transgenic rats expressing a diversified repertoire of antibodies with fully human idiotypes; Ligand Pharmaceuticals Inc., San Diego, USA) using intradermal application of human PD-L1 cDNA-coated gold particles with a handheld device for particle bombardment ("gene gun"). Serum samples were collected after a series of immunizations and tested by flow cytometry in HEK cells transiently transfected with the aforementioned expression plasmid to express human PD-L1. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. RNA from PD-L1-specific antibody-producing hybridomas was extracted and sequenced. The heavy and light chain variable regions (SEQ ID NOs: 8 and 12) were gene synthesized and cloned into a human IgG1λ expression vector containing a human IgG1 heavy chain (corresponding to SEQ ID NO: 19) with the following amino acid mutations according to EU numbering: L234F, L235E, D265A, and K409R (FEAR).

[0233] Example 4: Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange Bispecific IgG1 antibodies were generated by Fab arm exchange under controlled reducing conditions. The method is based on the use of complementary CH3 domains to promote the formation of heterodimers under specific assay conditions as described in WO2011 / 131746. F405L and K409R (EU numbering) mutations were introduced into the corresponding antibodies to generate antibody pairs with complementary CH3 domains.

[0234] To generate bispecific antibodies, two complementary parent antibodies at a final concentration of 0.5 mg / mL of each antibody were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL PBS for 5 h at 31° C. The reduction reaction was stopped by removing the reducing agent 2-MEA using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol.

[0235] Bispecific antibodies were made by combining the following antibodies from Examples 1 and 4: - the CD137-009-FEAL antibody combined with the PD-L1-547-FEAR antibody; - the PD-L1-547-FEAL antibody combined with the CD137-009-FEAR antibody; - GEN1046 (PD-L1-547-FEAL antibody combined with CD137-009-HC7LC2-FEAR antibody), - b12-FEAL antibody combined with PD-L1-547-FEAR antibody, CD137-009-FEAR or CD137-009-HC7LC2-FEAR antibody, using as the first arm the antibody b12, a gp120 specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812-23); - PD-L1-547-FEAL or CD137-009-FEAL with the b12-FEAR antibody.

[0236] The following sequences were used for the heavy and light chains, respectively: PD-L1-547-FEAL: VH-PD-L1-547(SEQ ID NO:8), IgG1-FEAL-Fc(SEQ ID NO:20) VL- PD-L1-547(SEQ ID NO:12), λ-C(SEQ ID NO:22) PD-L1-547-FEAR: VH-PD-L1-547(SEQ ID NO:8), IgG1-FEAR-Fc(SEQ ID NO:19) VL-PD-L1-547(SEQ ID NO:12), λ-C(SEQ ID NO:22) CD137-009-FEAL: VH_CD137-009(SEQ ID NO: 28), IgG1-FEAL-Fc(SEQ ID NO:20) VL_CD137-009(SEQ ID NO: 29), κ-C(SEQ ID NO:21) CD137-009-HC7LC2-FEAR: VH_CD137-009-H7(SEQ ID NO:1), IgG1-FEAR-Fc(SEQ ID NO:19) VL_CD137-009-L2(SEQ ID NO:5), κ-C(SEQ ID NO:21) CD137-009-FEAR: VH_CD137-009(SEQ ID NO: 28), IgG1-FEAR-Fc(SEQ ID NO:19) VL_CD137-009(SEQ ID NO: 29), κ-C(SEQ ID NO:21)

[0237] Example 5: Simultaneous binding of GEN1046 to PD-L1-expressing and CD137-expressing cells To measure the dose response of GEN1046 simultaneous binding to cells expressing human PD-L1 and human CD137, transfected K562 cells were differentially labeled with fluorescent dyes and the formation of doublets was analyzed by flow cytometry.

[0238] Human PD-L1-transfected K562 cells (K562_hPD-L1; 6 × 10 6 6 × 10 cells) were fluorescently labeled in 2 mL of 2.5 μM staining solution for 10 min at 37 °C using the CellTrace™ Violet Cell Proliferation Kit (catalog no. C34557, Thermo Fisher Scientific GmbH, Dreieich, Germany). In parallel, human CD137-transfected K562 cells (K562_h4-1BB; 6 × 10 6 K562_hPD-L1 and K562_h4-1BB cells) were fluorescently labeled with the CellTrace™ Far Red Cell Proliferation Kit (cat. no. C34564, Thermo Fisher Scientific GmbH, Dreieich, Germany) in 2 mL of 0.5 μM staining solution for 10 min at 37°C. Staining was stopped by adding 4 mL of fetal bovine serum (FBS; cat. no. S0115, Biochrom GmbH, Berlin, Germany). After one wash in RPMI1640 supplemented with 10% FBS (cat. no. 11875093, Thermo Fisher Scientific GmbH, Dreieich, Germany), stained K562_hPD-L1 and K562_h4-1BB cells were combined in a 1:1 ratio and diluted to 1.25 × 10 in RPMI1640, 10% FBS. 6 The combined K562_hPD-L1 and K562_h4-1BB cells were transferred (1 × 10 cells / mL) into a polystyrene 5 mL round-bottom tube (cat. no. 10579511, Fisher Scientific, Schwerte, Germany). 6Cells were incubated with serial dilutions of antibody (ranging from 0.001 to 100 μg / mL in 10-fold dilution steps) in RPMI 1640, 10% FBS for 15 min at 37°C. Samples were immediately analyzed on a FACS Canto™ II flow cytometer (Becton Dickinson GmbH, Heidelberg, Germany) without prior mixing to preserve the doublets formed. K562_hPD-L1 / K562_h4-1BB doublets were identified as CellTrace™ Violet / CellTrace™ Far Red double positive populations by FlowJo 10.4 software. The percentage of double positive cells was plotted as a function of antibody concentration using GraphPad Prism version 8.01 (GraphPad Software, Inc).

[0239] Figure 1A shows that the addition of GEN1046 induced the formation of CellTrace™ Violet / CellTrace™ Far Red doublets. The K562_hPD-L1 / K562_h4-1BB co-culture incubated with the moderate concentration of GEN1046 at 0.1 μg / mL showed the most prominent doublet formation, while only moderate doublet formation was observed at the low GEN1046 concentration of 0.001 μg / mL, and minimal to no doublet formation was detectable at the high GEN1046 concentration of 100 μg / mL. This observation is consistent with the bell-shaped dose-response curve shown in Figure 1B, covering the range of antibody concentrations tested from 0.001 μg / mL to 100 μg / mL. In contrast to GEN1046, the combination of monovalent PD-L1 and CD137 control antibodies PD-L1-547-FEALxb12-FEAR and b12-FEALxCD137-009-HC7LC2-FEAR did not result in doublet formation at all antibody concentrations tested.

[0240] Example 6: Effect of GEN1046 in CD137 reporter assay A schematic diagram of the predicted mode of action of PD-L1xCD137 bispecific antibodies is shown in Figure 2.

[0241] To measure the dose response by which GEN1046 mediated PD-L1 binding-dependent CD137 agonist activity, a luciferase-based CD137 activation reporter assay was performed using adherent, growing human tumor cell lines as a source of PD-L1.

[0242] Human ES-2 (ovarian clear cell carcinoma; ATCC 62146) endogenously expresses PD-L1. (登録商標) CRL-1978™) and MDA-MB-231 (breast adenocarcinoma; ATCC (登録商標) HTB-26™ cells were cultured at 3 × 10 in DMEM (catalog no. 10566016, Thermo Fisher Scientific GmbH, Dreieich, Germany) in white flat-bottom 96-well plates (catalog no. 136101, Thermo Fisher Scientific GmbH, Dreieich, Germany). 4Cells were seeded at a density of 10000 cells / well and incubated overnight at 37° C. Cryo-conserved Thaw-and-use GloResponse™ NFkB-Luc2P / 4-1BB Jurkat reporter cells (catalog no. CS196003, Promega GmbH, Walldorf, Germany) were thawed the next day and the contents of a single vial were transferred to a 15 mL tube containing 9.5 mL of pre-warmed RPMI-1640 supplemented with 1% FBS. The culture medium of adherent ES-2 and MDA-MB-231 cells was discarded and co-culture was initiated by seeding 50 μL of NFkB-Luc2P / 4-1BB Jurkat cell suspension on top of the monolayer of ES-2 or MDA-MB-231 cells. Cells were incubated with serial dilutions of antibodies (in-assay concentration range from 0.00128 to 100 μg / mL in 5-fold dilution steps) in RPMI 1640, 10% FBS for 6 hours at 37°C. The assay plate was then removed from the incubator and equilibrated to room temperature (RT) for 10 minutes. Bio-Glo™ Luciferase Reagent (Cat. No. G7941, Promega GmbH, Walldorf, Germany) was reconstituted and pre-warmed to RT. 75 μL of Luciferase Reagent was added per well and incubated in the dark at RT for 10 minutes. Induced luminescence was measured using an Infinite F200 Pro plate reader (Tecan Deutschland GmbH, Crailsheim, Germany).

[0243] When GEN1046 was added to ES-2:Jurkat (Figure 3A) and MDA-MB-231:Jurkat reporter cell co-cultures (Figure 3B), luciferase expression, as a readout of CD137 agonist activation, was effectively induced in a concentration-dependent manner following a bell-shaped dose-response curve. Mid-dose levels of GEN1046 around 0.1 μg / mL produced the most prominent luminescence signal, while low and high dose levels were less effective in inducing luciferase expression. Importantly, luciferase expression was not detectable at very low (0.00128 μg / mL GEN1046) and very high GEN1046 concentrations (100 μg / mL GEN1046). Incubation with b12-FEAL control antibody did not result in luciferase expression in either co-culture analyzed.

[0244] Example 7: Polyclonal T cell proliferation assay to measure the effect of bispecific antibody binding to PD-L1 and CD137 To measure induction of T cell proliferation in polyclonal activated T cells, PBMCs were incubated with a suboptimal concentration of anti-CD3 antibody (clone UCHT1) to activate T cells and combined with bispecific antibody GEN1046 or a control antibody. Within the PBMC population, cells expressing PD-L1 can be bound to the PD-L1-specific arm of the bispecific antibody, while activated T cells within the population can be bound to the CD137-specific arm. In this assay, transactivation of T cells by the CD137-specific arm induced by crosslinking of PD-L1 expressing cells by the bispecific antibody and by blocking the PD-L1:PD-1 interaction is measured as T cell proliferation.

[0245] PBMCs were obtained from buffy coats of healthy donors (Sanquin, Amsterdam, The Netherlands) using a Ficoll gradient (Lonza, Lymphocyte Separation Medium, Cat. No. 17-829E). PBMCs were labeled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life technologies, Cat. No. C34554) in PBS according to the manufacturer's instructions. 75,000 CFSE-labeled PBMCs were seeded per well in a 96-well round-bottom plate (Greiner bio-one, catalog no. 650180) and incubated at 37 °C and 5% CO in 200 μL of IMDM GlutaMAX supplemented with 5% human AB serum and 1% penicillin / streptomycin with suboptimal concentrations of anti-CD3 antibody (Stemcell, clone UCHT1, catalog no. 60011; 0.03 μg / mL final concentration) and bispecific or control antibodies (0.0032–10 μg / mL) previously determined to induce suboptimal T cell proliferation. 2 The mixture was incubated at RT for 4 days.

[0246] The proliferation of different T cell subsets was analyzed by flow cytometry. Cells were washed in PBS and stained with Fixable Viability Stain 510 (50 μL / well; BD Biosciences, Cat. No. 564406) for 20 min at 4° C. to exclude dead cells. After another wash in FACS buffer, cells were stained with PE-CF594-conjugated CD56-specific antibody (BD Biosciences, Cat. No. 564406) to distinguish the various cell subsets. Cells were stained with 100% IgG4-conjugated CD4 specific antibody (BioLegend, catalog no. 564849), Pacific Blue conjugated CD4 specific antibody (BioLegend, catalog no. 300521), AF700 conjugated CD8 specific antibody (BioLegend, catalog no. 301028), BV711 conjugated CD197 specific antibody (CCR7; BioLegend, catalog no. 353228), PE-Cy7 conjugated CD45RO specific antibody (BioLegend, catalog no. 304230), APC conjugated CD274 specific antibody (PD-L1; BioLegend catalog no. 329708) and BV605 conjugated CD137 specific antibody (BioLegend, catalog no. 309822) in FACS buffer for 30 minutes at 4°C. Cells were washed three times in FACS buffer and then measured in 80 μL of FACS buffer on a FACS Fortessa (BD Biosciences). CFSE dilutions were measured for total T cells as well as for different T cell subsets (e.g., CCR7 + CD45RO + Central memory T cells and CCR7 - CD45RO + The expansion index was measured in 100% T cells (effector memory T cells) and 100% T cells (effector memory T cells). Detailed analysis of T cell proliferation based on CFSE peaks indicating cell division was performed by FlowJo 10.4 software, and dose-response curves were plotted in GraphPad Prism version 6.04 (GraphPad Software, Inc) using exported expansion index values. The expansion index determines the fold expansion of the entire culture; an expansion index of 2.0 represents a doubling of the cell count, while an expansion index of 1.0 represents no change in the overall cell count.

[0247] Figure 4A shows that bispecific antibody GEN1046 induced increased T cell expansion compared to CD3 prestimulation alone, isotype control antibody b12-FEAL, and monovalent PD-L1-control antibody PD-L1-547-FEALxb12-FEAR with one irrelevant arm and one corresponding to the bivalent parent antibody PD-L1-547-FEAR. GEN1046-induced T cell proliferation was most optimal at 0.4μg / mL, while at lower and higher concentrations, GEN1046-induced T cell expansion was less obvious. CCR7 + CD45RO + Central memory T cells and CCR7 - CD45RO + A similar pattern emerged when effector memory T cells were analyzed separately (Fig. 4B), in which GEN1046 enhanced T cell proliferation, with T cell proliferation being optimal at 0.4 μg / mL.

[0248] Example 8: Antigen-specific CD8 to measure the effect of bispecific antibody binding to PD-L1 and CD137 + T cell proliferation assay To measure the induction of T cell proliferation by bispecific antibodies targeting PD-L1 and CD137 in antigen-specific assays, dendritic cells (DCs) were transfected with claudin-6 in vitro transcribed RNA (IVT-RNA) to express claudin-6 antigen. T cells were transfected with PD-1 IVT-RNA and harbored claudin-6-specific HLA-A2-restricted T cell receptors (TCRs). The TCRs can recognize claudin-6-derived epitopes presented by HLA-A2 on DCs. The PD-L1xCD137 bispecific antibody GEN1046 can crosslink PD-L1, endogenously expressed on monocyte-derived dendritic cells or tumor cells, with CD137 on T cells, resulting in the inhibition of inhibitory PD-1 / PD-L1 interactions and concomitant clustering of CD137, leading to T cell proliferation. Clustering of CD137 receptors expressed on T cells leads to activation of the CD137 receptor, which delivers a costimulatory signal to the T cell.

[0249] HLA-A2 + Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs by magnetic activated cell sorting (MACS) technique using anti-CD14 MicroBeads (Miltenyi; catalogue no. 130-050-201) according to the manufacturer's instructions. Peripheral blood lymphocytes (PBLs, CD14 negative fraction) were frozen for later T cell isolation. For differentiation into immature DCs (iDCs), 1 × 10 6 Monocytes / 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 1,000 IU / mL interleukin-4 (IL-4; Miltenyi, catalog number 130-093-924). Half of the medium was replaced with fresh medium once during these 5 days. iDCs were harvested by collecting non-adherent cells, and adherent cells were detached by incubation in PBS containing 2 mM EDTA at 37° for 10 min. After washing, iDCs were frozen in RPMI GlutaMAX containing 10% v / v DMSO (AppliChem GmbH, Cat. No. A3672,0050) + 50% v / v human AB serum for subsequent antigen-specific T cell assays.

[0250] Antigen specific CD8 +One day before the start of the T cell proliferation assay, frozen PBLs and iDCs from the same donor were thawed. + T cells were isolated from PBLs by MACS technology using anti-CD8 MicroBeads (Miltenyi, Cat. No. 130-045-201) according to the manufacturer's instructions. Approximately 10–15 × 10 6 CD8 + T cells were electroporated with 10 μg of in vitro translated (IVT)-RNA encoding the alpha chain of claudin-6 specific mouse TCR (HLA-A2 restricted; described in WO2015150327A1) + 10 μg of IVT-RNA encoding the beta chain + 0.4-10 μg of IVT-RNA encoding PD-1 in 250 μL of X-Vivo15 (Biozym Scientific GmbH, Cat. No. 881026) in a 4-mm electroporation cuvette (VWR International GmbH, Cat. No. 732-0023) using a BTX ECM® 830 Electroporation System device (BTX; 500 V, 1 × 3 ms pulse). After electroporation, cells were immediately transferred into fresh IMDM medium (Life Technologies GmbH, Cat. No. 12440-061) supplemented with 5% human AB serum and incubated at 37 °C, 5% CO 2 T cells were labeled with 1.6 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Cat. No. C34564) in PBS according to the manufacturer's instructions and incubated O / N in IMDM medium supplemented with 5% human AB serum.

[0251] 5×10 6 Up to 100 thawed iDCs were electroporated in 250 µL X-Vivo15 medium with 0.3–1 µg of IVT-RNA encoding full-length claudin-6 using the electroporation system described above (300 V, 1 x 12 ms pulse) and incubated O / N in IMDM medium supplemented with 5% human AB serum.

[0252] The next day, cells were harvested. Cell surface expression of claudin-6 and PD-L1 on DCs and TCR and PD-1 on T cells was confirmed by flow cytometry. DCs were stained with Alexa647-conjugated CLDN6-specific antibody (not commercially available; generated in-house) and anti-human CD274 antibody (PD-L1, eBioscienes, catalog number 12-5983), and T cells were stained with anti-mouse TCR β chain antibody (Becton Dickinson GmbH, catalog number 553174) and anti-human CD279 antibody (PD-1, eBioscienes, catalog number 17-2799). 5,000 electroporated DCs were incubated with 50,000 electroporated CFSE-labeled T cells in the presence of bispecific or control antibodies in IMDM GlutaMAX supplemented with 5% human AB serum in 96-well round-bottom plates. T cell proliferation was measured 5 days later by flow cytometry. Detailed analysis of T cell proliferation based on CFSE peaks indicating cell division was performed by FlowJo 10.4 software, and dose-response curves were plotted in GraphPad Prism version 6.04 (GraphPad Software, Inc) using exported expansion index values. The expansion index determines the fold expansion of the entire culture; an expansion index of 2.0 represents a doubling of the cell count, while an expansion index of 1.0 represents no change in the overall cell count.

[0253] Figure 5 shows that GEN1046 dose-dependently enhanced T cell proliferation compared to the isotype control antibody b12-FEAL, reflected by an increased expansion index at concentrations ≥ 0.004 μg / mL. GEN1046-induced T cell proliferation was most optimal at 0.03-0.11 μg / mL and decreased slightly at the highest concentration tested, demonstrating a bell-shaped dose-response curve.

[0254] Example 9: Antigen-specific CD8 to measure cytokine release induced by binding of a bispecific antibody to PD-L1 and CD137 + T cell proliferation assay Induction of cytokine release by the bispecific antibody GEN1046 targeting PD-L1 and CD137 was measured in antigen-specific assays and performed essentially as described in Example 8.

[0255] T cells were electroporated with 10 μg of TCR α-chain-encoding RNA and 10 μg of β-chain-encoding RNA with or without 2 μg of PD-1-encoding IVT RNA. Electroporated T cells were not CFSE-labeled (as above) and were immediately transferred after electroporation into fresh IMDM medium (Life Technologies GmbH, Cat. No. 12440-061) supplemented with 5% human AB serum. iDCs were electroporated as above with 5 μg of claudin-6 (CLDN6)-encoding RNA. After O / N incubation, DCs were stained with Alexa647-conjugated CLDN6-specific antibody and T cells with anti-mouse TCR β-chain and anti-human CD279 antibodies as above.

[0256] 5,000 electroporated DCs were incubated with 50,000 electroporated T cells in the presence of different concentrations of the bispecific antibody GEN1046 or the control antibody b12-FEAL in IMDM GlutaMAX supplemented with 5% human AB serum in 96-well round-bottom plates. After a 48-h incubation period, the plates were centrifuged at 500×g for 5 min and the supernatants were carefully transferred from each well to a new 96-well round-bottom plate and incubated for 1 h at 37°C for 1 h at 4°C. (登録商標) The supernatants collected from the antigen-specific proliferation assays were analyzed for cytokine levels of 10 different cytokines using the MSD V-Plex Human Proinflammatory panel 1 (10-Plex) kit (Meso Scale Diagnostics, LLC., Catalog No. K15049D-2) on a MESO QuickPlex SQ 120 instrument (Meso Scale Diagnostics, LLC., Catalog No. R31QQ-3) according to the manufacturer's instructions.

[0257] The addition of GEN1046 resulted in a dose-dependent increase in the secretion of primarily IFN-γ, TNF-α, IL-13, and IL-8 (Figure 6), which was most optimal at concentrations between 0.04 and 0.33 μg / mL. Lower dose levels as well as the higher dose level of 1 μg / mL were less effective in inducing these cytokines, showing a bell-shaped dose-response curve. When T cell:DC cocultures in which T cells were not electroporated with PD-1 RNA were compared to those in which T cells were electroporated with 2 μg of PD-1 RNA, slightly higher cytokine levels were detectable in the cocultures without PD-1 RNA electroporation. This was observed in both the GEN1046 dose-response curves as well as the b12-FEAL control antibody values.

[0258] Example 10: Ex vivo TIL expansion assay to evaluate the effect of CD137xPD-L1 bispecific antibodies on tumor-infiltrating lymphocytes To evaluate the effect of CD137-009-FEALxPD-L1-547-FEAR on tumor infiltrating lymphocytes (TILs), ex vivo culture of human tumor tissues was performed as follows: Fresh human tumor tissue resection specimens were washed three times by transferring isolated tumor masses from one well to the next well of a 6-well plate (Fisher Scientific Catalog No. 10110151) containing washing medium using a spatula or serological pipette. The washing medium consisted of X-VIVO 15 (Biozym, Catalog No. 881024) supplemented with 1% penicillin / streptomycin (Thermo Fisher, Catalog No. 15140-122) and 1% Fungizone (Thermo Fisher, Catalog No. 15290-026). The tumor was then dissected using a surgical knife (Braun / Roth, Cat. No. 5518091 BA223) and cut into small pieces of approximately 1-2 mm in diameter. Two small pieces were each placed into one well of a 24-well plate (VWR international, Cat. No. 701605) containing 1 mL of TIL medium (X-VIVO 15, 10% human serum albumin (HSA, CSL Behring, Cat. No. PZN-6446518) 1% penicillin / streptomycin, 1% fungizone and supplemented with 10 U / mL of IL-2 (Proleukin® S, Novartis Pharma, Cat. No. 02238131)). CD137-009-FEALxPD-L1-547-FEAR was added at the indicated final concentrations. The culture plates were incubated at 37°C and 5% CO 2 After 72 hours, 1 mL of fresh TIL medium containing the indicated concentrations of bispecific antibodies was added to each well. Wells were monitored every other day by microscopy for the presence of TIL clusters. When more than 25 TIL microclusters were detected in each well, the wells were transferred individually. To split the TIL cultures, cells in the wells of a 24-well plate were resuspended in 2 mL of medium and transferred into the wells of a 6-well plate. Each well was further replenished with another 2 mL of TIL medium.

[0259] After a total culture period of 10–14 days, TILs were harvested and analyzed by flow cytometry. Cells were stained with the following reagents: anti-human CD4-FITC (Miltenyi Biotec, Cat. No. 130-080-501), anti-human CD3-PE-Cy7 (BD Pharmingen, Cat. No. 563423), 7-aminoactinomycin D (7-AAD, Beckman Coulter, Cat. No. A07704), anti-human CD56-APC (eBioscience, Cat. No. 17-0567-42), and anti-human CD8-PE (TONBO, Cat. No. 50-0088), all diluted 1:50 in staining buffer (D-PBS containing 5% FCS and 5 mM EDTA). To allow a quantitative comparison of the resulting cells between the different treatment groups, the cell pellet was resuspended in FACS-buffer supplemented with BD™ CompBeads (BD biosciences, Cat. No. 51-90-9001291) after the final washing step. Flow cytometric analysis was performed on a BD FACSCanto™ II flow cytometer (Becton Dickinson) and the resulting data were analyzed using FlowJo 7.6.5 software. Relative viable TIL counts per 1,000 beads correlated with the corresponding wells of a 6-well plate, CD3 + CD8 + T cell count, CD3 + CD4 + T cell count and CD3 - CD56 + NK cell counts were calculated by normalization of the resulting 7AAD negative cell fraction to the resulting bead counts.

[0260] Figure 7 shows the analysis of TIL expansion from human non-small cell lung cancer tissue specimens, where the following concentrations: 0.01, 0.1 and 1 μg / mL CD137-009-FEALxPD-L1-547-FEAR were added; tissue specimens from the same patient without the addition of antibody were used as negative controls. After 10 days of culture, TILs were harvested and analyzed by flow cytometry. For each antibody concentration, five samples (of the five first wells) from different wells of a 24-well plate were measured. In all samples cultured with bispecific antibodies, the count of viable TILs was increased compared to the control sample without antibody. Overall, a significant (up to 10-fold) increase in viable TILs was observed when 0.1 μg / mL CD137-009-FEALxPD-L1-547-FEAR was added to the cultures (Figure 7A). When analyzed separately, CD3 + CD8 + A strong effect on T cell expansion was observed, which was significant at 0.1 μg / mL CD137-009-FEALxPD-L1-547-FEAR (Figure 7B; 7.4-fold expansion compared to control). + CD4 + T cells were only slightly expanded, and the expansion was not significant compared to cultures without antibody (Figure 7C). The most prominent TIL expansion was CD3 - CD56 + This was seen in NK cells (Figure 7D; up to 64-fold increase compared to control), which was significant at 0.1 μg / mL CD137-009-FEALxPD-L1-547-FEAR.

[0261] Example 11: Pharmacodynamic evaluation of GEN1046 in peripheral blood of patients with advanced solid tumors To investigate the biological activity of GEN1046 at various dose levels in patients with advanced tumors, blood and serum samples were collected at baseline and multiple time points during treatment. Based on the mechanism of action of GEN1046, biologically active dose levels were predicted to modulate circulating levels of interferon-γ (IFN-γ) and interferon-γ-inducible protein 10 (IP-10) and induce proliferation of peripheral CD8 T cells.

[0262] To measure serum levels of IFN-γ and IP-10, serum samples were collected from patients at baseline and at multiple time points following administration of GEN1046 in cycles 1 and 2 (day 1 [2 and 4-6 hours post-dose], days 2, 3, 8, and 15). Serum levels of IFN-□ and IP-10 were measured by Mesoscale Discovery (MSD) multiplex immunoassay (catalog no. K15209G) according to the manufacturer's instructions.

[0263] To measure peripheral regulation of immune cell subsets, peripheral blood immunophenotyping was performed on whole blood collected in EDTA tubes at baseline and multiple time points (days 2, 3, 8, and 15) after GEN1046 administration in cycles 1 and 2. 100 μL of whole blood was added to fluorochrome-conjugated monoclonal antibodies that specifically bind to cell surface antigens: CD45RA-FITC (clone LEU-18, BD Biosciences catalog number 335039), CCR7-BV510 (clone 3D12, BD Biosciences, catalog number 563449), CD8-PerCP-Cy5.5 (clone RPA-T8, BD Biosciences, catalog number 560662). After incubation on ice, stained samples were treated with FACS Lysing Solution (BD Biosciences, catalog number 349202) to lyse red blood cells. Excess antibody and cell debris were removed by washing with Stain Buffer (BD Biosciences, Cat. No. 554656). After lysis / washing, cells were fixed and permeabilized by incubation with Permeabilizing Solution 2 buffer (BD Biosciences, Cat. No. 340973). Cells were then washed, resuspended in Stain Buffer, and incubated on ice with an antibody against Ki67 (BV421 B56, BD Biosciences, Cat. No. 562899) to detect proliferating cells. After incubation, excess antibody was removed by washing with Stain Buffer. Cells were resuspended in Stain Buffer and loaded onto a BD FACSCanto™ II flow cytometer (Becton Dickinson) within 1 hour of staining.

[0264] Administration of GEN1046 to cancer patients resulted in modulation of circulating levels of IFN-γ and IP-10 and proliferating effector memory CD8 T cells (Table 5 and Figure 8). In the preliminary data set shown in Table 5, IFN-γ levels increased more than 2-fold at all dose levels tested in the first treatment cycle. The greatest increases were detected at the 50 mg and 80 mg dose levels, with the majority of patients (75%) in the 80 mg cohort having an increase of >2 (Table 5). GEN1046 also inhibited Ki67 + CD8 + CD45RA - CCR7 - It also induced the proliferation of effector memory CD8+ T cells, as measured by an increase in the proportion of T cells present. Paralleling the changes observed with modulation of circulating levels of IFN-γ, the proliferation of CD8 + The greatest and more consistent modulation of effector memory T cells was observed in patients in the 80 mg cohort. Notably, the magnitude of changes in circulating levels of both IFN-γ and proliferating effector memory CD8 T cells was smaller in the 400 mg cohort compared to the 25-200 mg cohort. These results indicate that GEN1046 induces an immune response characterized by modulation of immune effector cells and soluble factors crucial for generating antitumor immune responses, with the magnitude of the response being greater at the 80 mg dose level.

[0265] In the data set shown in Figure 8, increases in IFN-γ and IP-10 were observed at dose levels ≦200 mg in the first treatment cycle (Figure 8A-B). Increases in IFN-γ and IP-10 were also observed at dose levels ≧400 mg, although the maximum fold change from baseline during the first treatment cycle was significantly lower compared to the lower dose levels. GEN1046 also inhibited Ki67 + CD8 + T cells and Ki67 + CD8 + CD45RA - CCR7 - Total CD8 measured by increasing proportion of T cells +It also induced proliferation of T cells and effector memory CD8+ T cells (Figure 8C-D). Parallel to the changes observed in the modulation of circulating levels of IFN-γ and IP-10, the proliferation of CD8 + The greatest and more consistent modulation of effector memory T cells was observed in patients treated at dose levels ≤200 mg, with the magnitude of expanded effector memory CD8 T cell and total CD8 T cell changes being significantly smaller in the ≥400 mg cohort compared to the 25-200 mg cohort. These results indicate that GEN1046 induces an immune response characterized by modulation of immune effector cells and soluble factors critical for generating antitumor immune responses, with the magnitude of the response being greater at dose levels ≤200 mg.

[0266] Table 5. GEN1046 Modulation of Peripheral Pharmacodynamic Endpoints in Cancer Patients: Peak Fold Change from Baseline by Dose Level During Cycle 1 a TIFF2024523438000011.tif83150Preliminary data as of January 27, 2020. n: number of patients per dose cohort; Min: minimum measured; Q1: 25th percentile; Q3: 75th percentile; Max: maximum measured. a Pharmacodynamic assessments, including changes in circulating levels of interferon-γ and effector memory T cells, were performed using blood samples from patients with advanced solid tumors enrolled in the dose escalation phase of an open-label, multicenter safety trial of GEN1046 (NCT03917381). b Circulating levels of interferon-γ were measured in serum samples at baseline and at multiple time points after administration of GEN1046 (day 1 [2 and 4-6 hours post-dose], days 2, 3, 8, and 15) in cycles 1 and 2. Interferon-γ levels in serum samples were measured by Mesoscale Discovery (MSD) multiplex immunoassay. cPeripheral blood immunophenotyping was performed on whole blood collected at baseline and at multiple time points (days 2, 3, 8, and 15) after administration of GEN1046 in cycles 1 and 2. Proliferation (Ki67 + ) Effector memory CD8 T cells (CD8 + CD45RA - CCR7 - The abundance of erythrocytes (erythrocyte mesenchymal stem cells, erythrocyte endothelial cells, and erythrocyte T cells) was assessed in whole blood samples by flow cytometry.

[0267] Example 12: Clinical Trials Clinical Trial Design: The clinical trial on GCT1046-01 (ClinicalTrials.gov identifier: NCT03917381) was designed as a two-part trial, including an ongoing dose escalation part and a planned expansion part.

[0268] This study was designed as an open-label, multicenter, Phase I / IIa safety study of GEN1046 (DuoBody®-PD-L1x4-1BB). The study consisted of two parts; first-in-human (FIH) dose escalation (Phase I) and expansion (Phase IIa). Figure 9 shows a schematic diagram of the clinical trial design.

[0269] GEN1046 has the following amino acid sequence: CD137 binding arm; heavy and light chain sequences, respectively: CD137-009-HC7LC2-FEAR VH_CD137-009-H7(SEQ ID NO:1), IgG1-FEAR-Fc(SEQ ID NO:34) VL_CD137-009-L2(SEQ ID NO:5), κ-C(SEQ ID NO:21) PD-L1 binding arm; heavy and light chain sequences, respectively: PD-L1-547-FEAL VH-PD-L1-547(SEQ ID NO:8), IgG1-FEAL-Fc(SEQ ID NO:35) VL- PD-L1-547(SEQ ID NO:12), λ-C(SEQ ID NO:22)

[0270] Dose escalation Dose escalation was designed to evaluate GEN1046 in subjects with solid malignancies and to determine the maximum tolerated dose (MTD) or maximum administered dose (MAD) and / or recommended phase II dose (RP2D). In the expansion, the safety, tolerability, PK and antitumor activity of selected doses were further evaluated in selected solid tumors.

[0271] For dose escalation, subjects were required to be male or female, ≥18 years of age, and to have measurable disease per RECIST 1.1.

[0272] Subjects were required to have histologically or cytologically confirmed non-CNS solid tumors that were metastatic or unresectable, for which there was no available standard treatment that could provide clinical benefit or who were not candidates for such available treatments, and who, in the opinion of the investigator, may benefit from experimental treatment with GEN1046.

[0273] In dose escalation, subjects received one infusion of GEN1046 every 3 weeks (1Q3W) until a protocol-defined treatment discontinuation criterion was met; e.g., radiographic disease progression or clinical progression. GEN1046 was administered over a minimum of 60 minutes on day 1 of each 3-week treatment cycle (21 days) using an iv infusion. The conceptual design of the trial is shown in Figure 9.

[0274] The 1Q3W dose escalation was designed to evaluate GEN1046 at seven fixed main dose levels: 25, 80, 200, 400, 800, 1200 and 1600 mg, as well as potentially at six fixed optional intermediate dose levels (depending on data collected during the trial).

[0275] The recommended phase II dose (RP2D) was based on a review of available safety and dosing information and may have been less than the maximum tolerated dose (MTD).

[0276] expansion The purpose of the expansion is to provide further data on safety, tolerability, MoA, PK and antitumor activity of the selected dose / schedule.

[0277] The expansion was designed to enroll subjects with relapsed or refractory advanced and / or metastatic non-small cell lung cancer (NSCLC), endometrial cancer, urothelial carcinoma (UC), triple-negative breast cancer (TNBC), squamous cell carcinoma of the head and neck (SCCHN), or cervical cancer who are no longer candidates for standard of care (if the subject had access to and was eligible for the respective treatment) and who, in the opinion of the investigator, may benefit from experimental treatment with GEN1046. A complete overview of the expansion cohort is shown in Table 6.

[0278] Table 6. Expansion cohort TIFF2024523438000012.tif111150

[0279] The expansion cohort will enroll patients who meet the following inclusion criteria:

[0280] Expansion cohort 1 (NSCLC): PD-1 / L1 pretreated Subjects with NSCLC who have received up to four prior systemic conditioning regimens (maintenance therapy is considered part of one line of treatment) for metastatic disease with radiographic disease progression during or after the last conditioning treatment. ·NSCLC subjects of any histological diagnosis may be enrolled. Subjects with a histological or cytological diagnosis of non-squamous NSCLC must not have epidermal growth factor receptor (EGFR) sensitizing mutations and / or anaplastic lymphoma kinase (ALK) translocations / ROS1 (c-ROS oncogene 1) rearrangements. EGFR sensitizing mutations are those amenable to treatment with approved tyrosine kinase inhibitors (TKIs). Records of EGFR and ALK status should be available per site assessment. If records of EGFR and ALK status are not available, sponsor approval of medical monitoring is required prior to enrollment. Subjects should have received platinum-based therapy (or alternative chemotherapy due to platinum ineligibility, e.g. gemcitabine-containing regimens). Subjects must have received prior treatment with a PD-1 / L1 inhibitor, either alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with best overall response (BOR) of stable disease (SD) or progressive disease (PD) on a checkpoint inhibitor (CPI)-containing regimen with treatment duration up to 16 weeks. A recent in-site PD-L1 test result must be provided prior to enrollment. If in-site PD-L1 test results are not available, enrollment requires sponsor approval.

[0281] Expansion cohort 2 (NSCLC) - PD-1 / L1 untreated Subjects with NSCLC of any histology may be enrolled. Subjects with NSCLC who have received up to four prior systemic conditioning regimens (maintenance therapy is considered part of one line of treatment) for metastatic disease with radiographic disease progression during or after their last conditioning regimen. Subjects with a histologic or cytologic diagnosis of non-squamous NSCLC must not have EGFR sensitizing mutations and / or ALK translocations / ROS1 rearrangements. EGFR sensitizing mutations are those mutations amenable to treatment with an approved TKI. Records of EGFR and ALK status should be available per site assessment. If records of EGFR and ALK status are not available, sponsor approval of medical monitoring is required prior to enrollment. Subjects should have received platinum-based therapy (or alternative chemotherapy due to platinum ineligibility, e.g. gemcitabine-containing regimens). Subjects were required to have received prior treatment with a PD-1 / L1 inhibitor. This should not happen .

[0282] Expansion Cohort 3 (UC): Subjects with UC (of the bladder, ureter, urethra, or renal pelvis) who have received up to four prior systemic conditioning regimens (maintenance therapy is considered part of one line of treatment) for locally advanced / metastatic disease with predominantly transitional cell features on histology and radiographic disease progression during or after the last conditioning regimen. Subjects must have received prior treatment with a PD-1 / L1 inhibitor, either alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with SD or PD best overall response (BOR) on a CPI-containing regimen with treatment duration up to 16 weeks. Prior to enrollment, most recent in-laboratory PD-L1 testing results should be provided (if available). Cohort 3a: For subjects eligible to receive platinum-based therapy: Subjects must have received platinum-based chemotherapy. Cohort 3b: For subjects ineligible to receive platinum-based therapy: Subjects must not be eligible for any platinum-based chemotherapy or any chemotherapy containing cisplatin.

[0283] Expansion Cohort 4 (Endometrial Cancer): Subjects with endometrial cancer who have received up to four prior systemic conditioning regimens (maintenance therapy is considered part of one line of treatment) for advanced / metastatic disease with radiographic disease progression during or after the last conditioning treatment. Subjects must have: a histological diagnosis of endometrial epithelium, including endometrioid, serous, squamous, clear cell carcinoma, or carcinosarcoma. Note: Sarcoma and mesenchymal endometrial carcinoma are excluded. Subjects were not previously treated with PD-1 / L1 inhibitors. This should not happen (Established facility labels / access must be respected). Prior to enrollment, the most recent in-house results of mismatch repair deficient (dMMR) or microsatellite instability (MSI) status from an institutional assessment should be provided (if available).

[0284] Expansion Cohort 5 (TNBC): Human epidermal growth factor receptor 2 (HER2) negative (HER2 is negative by fluorescent in situ hybridization) assay (HER2 to CEP17 ratio <2.0 in non-amplified cases, mean HER2 gene copy number per probe <4 signals / cell) or Alternatively, TNBC defined as HER2 protein expression 1+ negative or IHC 0- negative by institutional assessment and estrogen receptor and progesterone receptor negative status (defined as <1% hormone receptor expressing cells by IHC analysis).Subjects who have received up to four systemic conditioning regimens (maintenance therapy is considered part of one treatment line) for locally advanced / metastatic disease with radiographic disease progression on or after the last conditioning regimen, including but not limited to anthracycline-containing, taxane-containing, antimetabolite-containing, or antimicrotubule-containing regimens.Institutional pathological confirmation of triple negative disease is required prior to study entry. Subjects with a history of breast cancer of a different phenotype must have confirmed TNBC in a biopsy obtained after the subject's last prior systemic therapy. Prior to enrollment, the most recent in-house results of dMMR or MSI status from a site assessment should be provided (if available). Cohort 5a - Subjects who have received prior PD-1 / L1 inhibitor therapy: Subjects must have received prior treatment with a PD-1 / L1 inhibitor, either alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with BOR of SD or PD on a CPI-containing regimen with treatment duration up to 16 weeks. Prior to enrollment, most recent in-laboratory PD-L1 testing results should be provided (if available). Cohort 5b - PD-1 / L1 inhibitor-naïve subjects: Subjects were not previously treated with PD-1 / L1 inhibitors. This should not happen (Established facility labels / access must be respected).

[0285] Expansion Cohort 6 (SCCHN): Subjects with recurrent or metastatic SCCHN (oral cavity, pharynx, larynx) who have received up to four prior systemic conditioning regimens (maintenance therapy is considered part of one line of treatment) for recurrent / metastatic disease with radiographic PD during or after the last conditioning treatment. Subjects must have had disease progression during or after prior treatment with platinum-based chemotherapy (alternative combination chemotherapy is acceptable if the subject's platinum-ineligible status is documented). Cohort 6a - Subjects who have received prior PD-1 / L1 inhibitor therapy: Subjects must have received prior treatment with a PD-1 / L1 inhibitor, either alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with BOR of SD or PD on a CPI-containing regimen with treatment duration up to 16 weeks. Prior to enrollment, most recent in-laboratory PD-L1 testing results should be provided (if available). Cohort 6b - PD-1 / L1 inhibitor-naïve subjects: Subjects were not previously treated with PD-1 / L1 inhibitors. This should not happen (Established facility labels / access must be respected).

[0286] Expansion Cohort 7 (Cervical Cancer): Cervical cancer subjects who have received up to four systemic conditioning regimens for recurrent / metastatic disease with radiographic disease progression during or after the last conditioning regimen, e.g., chemotherapy (per applicable labeling) in combination with bevacizumab (chemotherapy administered in the adjuvant or neoadjuvant setting or in combination with radiation therapy should not be counted as prior lines of treatment) until the subject is ineligible for bevacizumab per institutional criteria. Subjects must have cervical cancer with squamous cell, adenocarcinoma or adenosquamous histology. Subjects were not previously treated with PD-1 / L1 inhibitors. This should not happen (Established facility labels / access must be respected).

[0287] result Dose escalation The following preliminary results were obtained during dose escalation: Table 7 shows the best overall response (RECIST v1.1) at enrollment and by dose level for a total of 30 patients (data extraction date: February 3, 2020).

[0288] Tables 8 and 9 show the objective response rate and confirmed objective response rate (RECIST v1.1), respectively, at enrollment and by dose level administered to a total of 61 patients (data cutoff: October 12, 2020).

[0289] The maximum percent change from baseline in tumor size for all patients is shown in Figure 10. During the dose escalation phase, disease control occurred in 40 / 61 (65.6%) patients. Four patients with triple-negative breast, ovarian, or non-small cell lung cancer (NSCLC) achieved partial responses (PR); 36 patients maintained stable disease.

[0290] Clinical activity observed in patients with NSCLC (best change from baseline in tumor size) is shown in Figure 11 (data cutoff: October 12, 2020). Of six patients with NSCLC, all of whom had received prior checkpoint immunotherapy, two achieved an unconfirmed PR, two maintained stable disease, and two experienced disease progression.

[0291] Table 7. Best overall response by dose level (RECIST v1.1) TIFF2024523438000013.tif19490

[0292] Table 8. Objective Response Rate - Dose Escalation TIFF2024523438000014.tif222125

[0293] Table 9. Confirmed Response Rate - Dose Escalation TIFF2024523438000015.tif225125

[0294] expansion: Expansion Cohort 1: As of January 29, 2021, 39 patients had been dosed in Expansion Cohort 1, including patients with advanced / metastatic PD-1 / L1-pretreated NSCLC. Of the 39 patients, 31 were evaluable for efficacy at the time of data cutoff, i.e., either had at least one post-baseline scan or treatment had been discontinued. Six of the 31 efficacy-evaluable patients achieved a best overall response of either confirmed PR or unconfirmed PR, and seven patients achieved a best overall response of SD (Figure 13).

[0295] Expansion Cohort 2: As of January 29, 2021, 11 patients had been dosed in Expansion Cohort 2, including a patient with advanced / metastatic PD-1 / L1-naïve NSCLC. Of the 11 patients, 10 were evaluable for efficacy at the time of data cutoff, i.e., either had at least one post-baseline scan or treatment had been discontinued. None of the 10 efficacy-evaluable patients achieved a best overall response of PR, while 5 patients achieved a best overall response of SD (Figure 14).

[0296] Expansion Cohort 3: As of January 29, 2021, 13 patients had been dosed in Expansion Cohort 3, including a patient with advanced / metastatic PD-1 / L1-pretreated urothelial carcinoma. Of the 13 patients, 9 were evaluable for efficacy at the time of data cutoff, i.e., either had at least one post-baseline scan or treatment had been discontinued. None of the 10 efficacy-evaluable patients achieved a best overall response of PR, while 4 patients achieved a best overall response of SD (Figure 15).

[0297] Expansion Cohort 4: As of January 29, 2021, 21 patients including those with advanced / metastatic PD-1 / L1-naïve endometrial cancer had been dosed in Expansion Cohort 4. Of the 21 patients, 17 were evaluable for efficacy at the time of data cutoff, i.e., either had at least one post-baseline scan or treatment had been discontinued. Of the 17 efficacy-evaluable patients, one achieved a best overall response of PR, while seven patients achieved a best overall response of SD (Figure 16).

[0298] Expansion Cohort 5: As of January 29, 2021, 20 patients had been dosed in Expansion Cohort 5, including patients with advanced / metastatic TNBC. Of the 20 patients, 15 were evaluable for efficacy at the time of data cutoff, i.e., either had at least one post-baseline scan or treatment had been discontinued. Of the 15 efficacy-evaluable patients, one achieved a best overall response of PR, while five patients achieved a best overall response of SD (Figure 17).

[0299] Expansion Cohort 6: As of January 29, 2021, 22 patients had been dosed in Expansion Cohort 6, including patients with advanced / metastatic SCCHN. Of the 22 patients, 18 were evaluable for efficacy at the time of data cutoff, i.e., either had at least one post-baseline scan or treatment had been discontinued. Two of the 18 efficacy-evaluable patients achieved a best overall response of PR, while six patients achieved a best overall response of SD (Figure 18).

[0300] Expansion Cohort 7: As of January 29, 2021, 16 patients had been dosed in expansion cohort 7, including a patient with advanced / metastatic PD-1 / L1-naïve cervical cancer. Of the 16 patients, 11 were evaluable for efficacy at the time of data cutoff, i.e., either had at least one post-baseline scan or treatment had been discontinued. One of the 11 efficacy-evaluable patients achieved a best overall response of PR, while seven patients achieved a best overall response of SD (Figure 19).

[0301] In the entire expansion cohort, progression-free survival (PFS) was longer in subjects who had received prior checkpoint inhibitor treatment (Figure 20).

[0302] In the combined CPI-pretreated expansion cohort of patients, clinical response to GEN1046 therapy in checkpoint inhibitor-pretreated NSCLC subjects is associated with time from last prior anti-PD-1 therapy (Figure 21). NSCLC subjects who benefited from GEN1046 therapy tended to have been treated more recently with their last anti-PD-1 agent. The short time from anti-PD-1 agent-containing treatment may suggest that residual anti-PD-1 activity promotes response to GEN1046. In support of this, patients treated with anti-PD-1 agents in the clinic have shown long-term PD-1 receptor occupancy by therapeutic antibodies, which may last for more than 200 days (Brahmer et al., JCO 2010;28(19):3167-3175). The fact that the therapeutic a-PD-1 agent is still bound to the PD-1 receptor may further result in a large number of free PD-L1 molecules available for binding with GEN1046. The presence of residual a-PD-1 activity may also allow for a more complete blockade of the PD-1 pathway (blocking PD-1 interactions with both PD-L1 and PD-L2), which may be important for the biological activity of GEN1046 in the post-CPI setting. The more recent anti-PD-1 treatment may have a direct impact on the tumor microenvironment, for example, by eliciting an anti-tumor immune response that may be enhanced by GEN1046 when administered immediately or shortly after progression on anti-PD-1-containing therapy.

[0303] The responders described as having a "low" PD-1+ CD8 T cell abundance may reflect receptor occupancy (RO) due to prior a-PD-1 treatment.

[0304] Conversely, non-responders, who were generally shown to have a higher proportion of PD-1+ CD8 T cells, may represent a more exhausted phenotype.

[0305] Conclusion: GEN1046 is a next-generation, first-in-class PD-L1x4-1BB bispecific antibody that, unlike existing 4-1BB agonists, has a tolerable safety profile and encourages early clinical activity.

[0306] In the dose-escalation phase of this Phase I / IIa trial, GEN1046 demonstrated a manageable safety profile and preliminary clinical activity in a heavily pre-treated population with advanced solid tumors.

[0307] Most adverse events were mild to moderate; treatment-related grade 3 transaminase elevations resolved with corticosteroids. No treatment-related bilirubin increases or grade 4 transaminase elevations were observed. Six patients had dose-limiting toxicities (DLTs); the maximum tolerated dose (MTD) was not reached.

[0308] Clinical benefit was observed at a range of dose levels in patients, including those refractory to prior immunotherapy and those with tumors that are typically not very sensitive to immune checkpoint inhibitors (ICIs).

[0309] Disease control, including partial responses in triple-negative breast cancer (1), ovarian cancer (1), and ICI-pretreated NSCLC (2), was achieved in 65.6% of patients.

[0310] Modulation of pharmacodynamic endpoints was observed across a wide range of dose levels indicative of biological activity.

[0311] Example 13: Pharmacokinetic / Pharmacodynamic Model An integrated semi-mechanistic PK / PD (pharmacokinetic / pharmacodynamic) model was developed that assumes the distribution of GEN1046 in the central and peripheral PK compartments as well as the partitioning into the tumor and lymphatic compartments. The model utilizes literature PK and pharmacodynamic data and physiological parameters to parameterize the expression of PD-L1 and 4-1BB and T cell trafficking into these compartments. The model compartments consist of well-mixed 2- and 3-dimensional space with free drug transfer between all compartments. The model also incorporates the dynamic binding of GEN1046 with PD-L1 and 4-1BB to predict the formation of trimers (crosslinked with PD-L1 and 4-1BB) in tumors as well as the receptor occupancy (RO) of PD-L1 and 4-1BB.

[0312] Semi-mechanistic PK / pharmacodynamic modeling demonstrated that intratumoral trimer formation peaked with the 100 mg Q3W GEN1046 regimen, which is expected to result in sustained 4-1BB activation and was selected as the activation dose for the first two cycles. Based on available pharmacodynamic clinical data, greater and more consistent modulation of peripheral pharmacodynamic endpoints (IFNγ and expanded Ki67+ effector memory CD8+ T cells) was also seen at dose levels ≤200 mg. In the GCT1046-01 trial, clinical data from the expansion cohort demonstrated that a dose of 100 mg Q3W resulted in responses within the first two cycles.

[0313] Based on PK / pharmacodynamic modeling predictions and available clinical data, the dose of GEN1046 100 mg 1Q3W was selected as the activation dose to be administered in the first two cycles that could result in maximal trimer formation and mean RO(%) of PD-L1 at reasonable levels.

[0314] A maintenance regimen of GEN1046 500 mg 1Q6W was used after the first two cycles, and this dosing cycle is expected to result in higher PD-L1 receptor occupancy and intermittent 4-1BB activation due to a lower degree of trimer engagement compared to 100 mg Q3W (Figure 12). This dose is expected to result in improved duration of response. In addition, GEN1046 at 500 mg Q6W is expected to result in less trimer engagement in the liver compared to 100 mg Q3W, and therefore may have a better safety profile.

[0315] Example 14: Additional expansion cohorts; activation / maintenance dosing An integrated semi-mechanistic physiologically-based pharmacokinetic / pharmacodynamic model, shown in Example 13, was used to predict the formation of trimers (crosslinked with PD-L1 and 4-1BB) in tumors and the receptor occupancy (RO) of PD-L1. The model was then used to explore the predicted in vivo trimer formation and PD-L1 RO under different dosing regimens. The model showed that trimer formation in tumors peaked at a dose of GEN1046 100 mg once every 3 weeks (1Q3W), which was selected as the activation dose for two cycles. This was followed by a maintenance dose of GEN1046 500 mg 1Q6W, which was predicted to result in higher PD-L1 receptor occupancy (RO) and intermittent 4-1BB activation in dosing cycles due to trimer engagement compared to 100 mg Q3W.

[0316] Two further expansion cohorts were designed to evaluate administration of GEN1046 at a "activation dose" of 100 mg 1Q3W followed by a "maintenance dose" of 500 mg GEN1046 1Q6W for two cycles.

[0317] The first expansion cohort will enroll checkpoint inhibitor (CPI)-pretreated metastatic non-small cell lung cancer (NSCLC) patients with the following inclusion criteria: Subjects with NSCLC who have received up to four prior systemic conditioning regimens (maintenance therapy is considered part of one line of treatment) for metastatic disease with radiographic disease progression during or after the last conditioning treatment. ·NSCLC subjects of any histological diagnosis may be enrolled. Subjects with a histological or cytological diagnosis of non-squamous NSCLC must not have epidermal growth factor receptor (EGFR) sensitizing mutations and / or anaplastic lymphoma kinase (ALK) translocations / ROS1 (c-ROS oncogene 1) rearrangements. EGFR sensitizing mutations are those amenable to treatment with approved tyrosine kinase inhibitors (TKIs). Records of EGFR and ALK status should be available per site assessment. If records of EGFR and ALK status are not available, sponsor approval of medical monitoring is required prior to enrollment. Subjects should have received platinum-based therapy (or alternative chemotherapy due to platinum ineligibility, e.g. gemcitabine-containing regimens). Subjects must have received prior treatment with a PD-1 / L1 inhibitor, either alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with best overall response (BOR) of stable disease (SD) or progressive disease (PD) on a checkpoint inhibitor (CPI)-containing regimen with treatment duration up to 16 weeks. A recent in-site PD-L1 test result must be provided prior to enrollment. If in-site PD-L1 test results are not available, enrollment requires sponsor approval.

[0318] The second expansion cohort will enroll treatment-naïve metastatic NSCLC patients with the following inclusion criteria: Subjects with metastatic NSCLC who have not received a systemic conditioning regimen for metastatic disease. Subjects must not have received prior treatment with a PD-1 / L1 inhibitor. Subjects must have had radiographic disease progression on or after their last conditioning treatment. This is not required for subjects with newly diagnosed disease. Subjects with any histological diagnosis of NSCLC may be enrolled. Subjects with a histological or cytological diagnosis of non-squamous NSCLC must not have EGFR sensitizing mutations and / or ALK translocations / ROS1 rearrangements. EGFR sensitizing mutations are those mutations suitable for treatment with an approved TKI. Records of EGFR and ALK status should be available per site assessment. If records of EGFR and ALK status are not available, sponsor approval of medical monitoring is required prior to enrollment. Subjects must have PD-L1 expression results from a central clinical laboratory available prior to C1D1 from fresh tumor specimens obtained by core needle or excision biopsy, or from tumor tissue resected at the time metastatic disease was diagnosed. The following specimens are not acceptable for this study: Endobronchial Ultrasound (EBUS)-guided specimens, fine needle aspirates, cell blocks, cell pellets, blood clots, bone marrow, and cytological specimens. The tumor has PD-L1 expression on ≥ 1% of tumor cells (TPS ≥ 1%) as assessed by IHC as measured by testing in a central clinical laboratory.

[0319] Example 15: Phase 2, multicenter, randomized, open-label study of GEN1046 in subjects with recurrent / refractory metastatic non-small cell lung cancer after standard of care treatment with immune checkpoint inhibitors This is a randomized, open-label trial evaluating the safety and efficacy of GEN1046 in adult subjects with relapsed / refractory metastatic NSCLC following treatment with a CPI-containing therapy. The trial includes a study arm A in which patients are treated with GEN1046 monotherapy, and the primary objective is to evaluate the antitumor activity (ORR) of GEN1046 as a monotherapy. ORR is a well-established efficacy parameter for assessing antitumor activity in proof-of-concept trials of NSCLC.

[0320] Arm A will be studied with an activation dose of GEN1046 (100 mg Q3W for two cycles) followed by a higher maintenance dose of GEN1046 (500 mg Q6W in subsequent cycles) based on the following: · Semi-mechanistic PK / pharmacodynamic modeling demonstrated that intratumoral trimer formation peaked with the 100 mg Q3W GEN1046 regimen, which is expected to result in sustained 4-1BB activation and was selected as the activation dose for the first two cycles. In the GCT1046-01 trial, clinical data from the expansion cohort demonstrated that a dose of 100 mg Q3W resulted in responses within the first two cycles. A maintenance regimen of GEN1046 500 mg Q6W was used after the first two cycles, which is estimated to result in higher PD-L1 RO and intermittent 4-1BB activation in dosing cycles due to a lower degree of trimer engagement compared to 100 mg Q3W. This dose is expected to result in improved duration of response (DOR). GEN1046 500 mg Q6W also showed a significantly improved maximum concentration (C) after 500 mg Q6W. max ) is expected to have an acceptable benefit / risk profile compared with GEN1046 1200 mg Q3W, the highest dose tested in the dose escalation phase. Doses from 25 to 1200 mg Q3W evaluated in the dose escalation phase of the FIH trial were safe and generally well tolerated, and the MTD was not achieved.

[0321] In Arm A, GEN1046 100 mg Q3W will be administered as a 30-minute IV infusion on day 1 of the first two treatment cycles; then, GEN1046 500 mg Q6W will be administered as a 30-minute IV infusion on day 1 of the subsequent 6-week treatment cycle. No dose reductions of GEN1046 will be permitted.

[0322] Important inclusion criteria: Subjects must be at least 18 years old. Subjects must have a histologically or cytologically confirmed diagnosis of stage 4 NSCLC with at least one prior systemic treatment line containing an anti-PD-1 / PD-L1 mAb for metastatic disease as described below. Subjects must have demonstrated progressive disease (PD) as defined by RECIST v1.1. For subjects whose most recent anticancer therapy included an anti-PD-1 / PD-L1 mAb, recent evidence of PD must be confirmed by a second assessment no earlier than 4 weeks from the date of first documented PD. Note : Subjects must have received at least two doses of an approved anti-PD-1 / PD-L1 mAb approved in NSCLC. Subjects had progressed during or after treatment with one anti-PD-1 / PD-L1 mAb administered either as monotherapy or in combination with SOC (subjects who had only received anti-PD-1 / PD-L1 mAb monotherapy as first-line treatment were eligible for the study if the investigator determined that treatment with platinum-containing chemotherapy was not appropriate according to institutional treatment guidelines), or o Subjects have progressed during or after platinum doublet chemotherapy following anti-PD-1 / PD-L1 mAb, or; * Subjects have progressed during or after platinum doublet chemotherapy followed by anti-PD-1 / PD-L1 mAb. Subjects must have tumor PD-L1 expression results available prior to C1D1 with ≥1% of tumor cells demonstrating PD-L1 expression as assessed by a sponsor-designed central clinical laboratory using the Dako PD-L1 IHC 22C3 pharmDx assay (TPS ≥1%) or by local site assessment per manufacturer's instructions using the Dako PD-L1 IHC 22C3 pharmDx assay (TPS ≥1%) or VENTANA PD-L1 (SP263) assay (TC ≥1%). Note: In-house PD-L1 results must be performed on fresh tumor tissue (obtained within 3 months prior to enrollment and after failure / discontinuation of last prior therapy) or, if not feasible, on archival tissue (obtained within 12 months prior to enrollment). Subjects must have measurable disease per RECIST v1.1 as assessed by the investigator. Subjects must have an Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤ 1. Subjects must have a life expectancy of at least 3 months. Subjects must have adequate organ and bone marrow function as described in the protocol.

[0323] Important exclusion criteria: Documented known EGFR susceptibility mutations, KRAS, RET, ROS1, BRAF mutations, NTRK infusions, RET rearrangements, ALK rearrangements, high-level MET amplifications or METex 14 skipping. If documented mutation status is not available, for subjects with a non-squamous or mixed non-squamous and squamous histology diagnosis, formalin-fixed paraffin-embedded tumor tissue should be tested for biomarker panel analysis (which may include, but is not limited to, EGFR, ALK, ROS1, BRAF, KRAS mutations, RET rearrangements or NTRK infusions). Subjects should not be randomized until biomarker status is available in the local source documentation records. Note Subjects with tumors harboring such targetable mutations, gene rearrangements, or gene amplifications as described above may be enrolled in the clinical trial, provided that such subjects have also received an approved targeted therapy for this indication (specifically, at least one prior systemic treatment line containing an anti-PD-1 / PD-L1 mAb for metastatic NSCLC disease) and satisfactorily meet all other eligibility criteria. Subjects were treated with the following: - Pretreatment with docetaxel for NSCLC, pretreatment with a 4-1BB (CD137) targeting agent, any type of antitumor vaccine or autologous cellular immunotherapy, Treatment with anticancer drugs within 28 days prior to administration of GEN1046 have received any of the following: Subjects who discontinued treatment due to disease progression within the first 6 weeks of immune CPI-containing treatment.

Claims

1. A conjugate substance comprising a first antigen-binding region that binds to human CD137 such as human CD137 consisting of the amino acid sequence shown in SEQ ID NO:24 and a second antigen-binding region that binds to human PD-L1 such as human PD-L1 consisting of the amino acid sequence shown in SEQ ID NO:26, for use in a method for reducing or suppressing the progression of a tumor in the human subject or treating cancer in the human subject, which comprises administering the conjugate substance to a human subject, wherein, in the method, the conjugate substance is administered to the human subject according to a dosing schedule including administration of dose A in one or more treatment cycles and administration of dose B in one or more treatment cycles, the amount of the conjugate substance in dose A is a) about 0.3 to 2.5 mg / kg body weight, or about 25 to 200 mg in total; and / or b) Approximately 2.1×10 -9 ~1.7×10 -8 mol / kg body weight, or approximately 1.7×10 -7 ~1.4×10 -6 mol and the amount of the conjugate substance in dose B is c) about 3.8 to 7.5 mg / kg body weight, or about 300 to 600 mg in total; and / or d) about 2.6×10 -8 to 5.1×10 -8 mol / kg body weight, or about 2.0 to 4.1×10 -6 mol in total and the pharmaceutical composition comprising the conjugate substance.

2. The amount of the conjugate substance in dose A is 0.4 to 2.3 mg / kg body weight, or 30 to 180 mg in total and / or 2.56×10 -9 ~1.53×10 -8 mol / kg body weight, or 2.04×10 -7 ~1.23×10 -6 mol; 0.5 to 2.0 mg / kg body weight, or 40 to 160 mg in total and / or 3.41×10 -9 ~1.36×10 -8 mol / kg body weight, or 2.73×10 -7 ~1.09×10 -6 mol; 0.6 to 1.9 mg / kg body weight, or 50 to 150 mg in total and / or 4.26×10 -9 ~1.28×10 -8 mol / kg body weight, or 3.41×10 -7 ~1.02×10 -6 mol; 0.8 to 1.8 mg / kg body weight, or 60 to 140 mg in total and / or 5.11×10 -9 ~1.19×10 -8 mol / kg body weight, or 4.09×10 -7 ~9.54×10 -7 mol; 0.9 to 1.6 mg / kg body weight, or 70 to 130 mg in total and / or 5.96×10 -9 ~1.11×10 -8 mol / kg body weight, or 4.77×10 -7 ~8.86×10 -7 mol; 1 to 1.5 mg / kg body weight, or 80 to 120 mg in total and / or 6.81×10 -9 ~1.02×10 -8 mol / kg body weight, or 5.45×10 -7 ~8.18×10 -7 mol; 1.1 to 1.4 mg / kg body weight, or 90 to 110 mg in total and / or 7.67×10 -9 ~9.37×10 -9 mol / kg body weight, or 6.13×10 -7 ~7.49×10 -7 mol; or 1.2 to 1.3 mg / kg body weight, or 95 to 105 mg in total and / or 8.09×10 -9 ~8.94×10 -9 mol / kg body weight, or 6.47×10 -7 ~7.16×10 -7 mol and is the pharmaceutical composition according to Claim 1.

3. The amount of the conjugate substance in dose A is a) about 1.25 mg / kg body weight, or about 100 mg in total; and / or b) about 8.5×10 -9 mol / kg body weight, or about 6.8×10 -7 mol in total and is the pharmaceutical composition according to Claim 1.

4. The amount of the conjugate substance in dose B is 4.4 to 7.4 mg / kg body weight, or 350 to 590 mg in total and / or 2.98×10 -8 ~5.03×10 -8 mol / kg body weight, or 2.39×10 -6 ~4.02×10 -6 mol; 5.0 to 7.25 mg / kg body weight, or 400 to 580 mg in total and / or 3.41×10 -8 ~4.94×10 -8 mol / kg body weight, or 2.73×10 -6 ~3.95×10 -6 mol; 5.3 to 7.1 mg / kg body weight, or a total amount of 420 to 570 mg and / or 3.58×10 -8 ~4.86×10 -8 mol / kg body weight, or 2.86×10 -6 ~3.88×10 -6 mol; 5.4 to 7.0 mg / kg body weight, or a total amount of 430 to 560 mg and / or 3.66×10 -8 ~4.77×10 -8 mol / kg body weight, or 2.93×10 -6 ~3.82×10 -6 mol; 5.5 to 6.9 mg / kg body weight, or a total amount of 440 to 550 mg and / or 3.75×10 -8 ~4.69×10 -8 mol / kg body weight, or 3.00×10 -6 ~3.75×10 -6 mol; 5.6 to 6.8 mg / kg body weight, or a total amount of 450 to 540 mg and / or 3.83×10 -8 ~4.60×10 -8 mol / kg body weight, or 3.07×10 -6 ~3.68×10 -6 mol; 5.8 to 6.6 mg / kg body weight, or a total amount of 460 to 530 mg and / or 3.92×10 -8 ~4.51×10 -8 mol / kg body weight, or 3.13×10 -6 ~3.61×10 -6 mol; 5.9 to 6.5 mg / kg body weight, or a total amount of 470 to 520 mg and / or 4.00×10 -8 ~4.43×10 -8 mol / kg body weight, or 3.20×10 -6 ~3.54×10 -6 mol; 6.0 to 6.4 mg / kg body weight, or a total amount of 480 to 515 mg and / or 4.09×10 -8 ~4.39×10 -8 mol / kg body weight, or 3.27×10 -6 ~3.51×10 -6 mol; 6.1 to 6.4 mg / kg body weight, or a total amount of 490 to 510 mg and / or 4.17×10 -8 ~4.34×10 -8 mol / kg body weight, or 3.34×10 -6 ~3.48×10 -6 mol; or 6.2 to 6.3 mg / kg body weight, or a total amount of 495 to 505 mg and / or 4.22×10 -8 ~4.30×10 -8 mol / kg body weight, or 3.37×10 -6 ~3.44×10 -6 mol is the medicament according to claim 1.

5. The amount of the conjugate substance in dose B is a) about 6.25 mg / kg body weight, or about 500 mg in total; and / or b) about 4.3×10 -8 mol / kg body weight, or about 3.4×10 -6 mol in total is the medicament according to claim 1.

6. The medicament according to claim 1, wherein the dosing schedule includes administration of dose A in one or more treatment cycles, followed by administration of dose B in one or more treatment cycles.

7. Dose A is administered once in each treatment cycle, for example, on the first day of each treatment cycle, and / or Dose B is administered once in each treatment cycle, for example, on the first day of each treatment cycle, is the medicament according to claim 1.

8. Dose A is administered in one or more 3-week / 21-day treatment cycles, for example, 2, 3, 4, or 5 3-week / 21-day treatment cycles. Preferably, dose A is administered once (Q3W) in each 3-week / 21-day treatment cycle. The medicament according to claim 1.

9. Dose B is administered in one or more 6-week / 42-day treatment cycles. Preferably, dose B is administered once (Q6W) in each of the one or more 6-week / 42-day treatment cycles. The medicament according to claim 1.

10. The medicament according to claim 1, wherein the dosing schedule includes administration of dose A in two treatment cycles, followed by administration of dose B in one or more treatment cycles.

11. The pharmaceutical according to claim 1, wherein the dosing schedule comprises administration of dose A, followed by administration of dose B until complete tumor regression or disease progression.

12. The pharmaceutical according to claim 1, wherein the conjugate substance is administered by systemic administration, preferably by intravenous injection or infusion.

13. The pharmaceutical according to claim 1, wherein each dose is infused over at least 30 minutes, for example, over at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 240 minutes.

14. a) The first antigen-binding region comprises a heavy-chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 2, 3, and 4, respectively, and a light-chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 6, GAS, SEQ ID NO: 7, respectively; and b) The second antigen-binding region comprises a heavy-chain variable region (VH) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 9, 10, and 11, respectively, and a light-chain variable region (VL) comprising the sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 13, DDN, SEQ ID NO: 14, respectively, The pharmaceutical according to claim 1.

15. a) The first antigen-binding region comprises a heavy-chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 1 and a light-chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 5; and b) The second antigen-binding region comprises a heavy-chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 8 and a light-chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 12, The pharmaceutical according to claim 1.

16. The pharmaceutical according to claim 1, wherein the conjugate substance is an antibody, preferably a multispecific antibody, such as a bispecific antibody.

17. The pharmaceutical according to claim 1, wherein the conjugate substance is in the form of a full-length antibody or an antibody fragment.

18. The conjugate substance is (i)A first binding arm comprising a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first VH comprises a first HCDR1 sequence, a first HCDR2 sequence, and a first HCDR3 sequence, the first HCDR1 sequence is shown in SEQ ID NO:2, the first HCDR2 sequence is shown in SEQ ID NO:3, and the first HCDR3 sequence comprises and is shown in SEQ ID NO:4; and the first VL comprises a first LCDR1 sequence, a first LCDR2 sequence, and a first LCDR3 sequence, the first LCDR1 sequence is shown in SEQ ID NO:6, the first LCDR2 sequence is GAS, and the first LCDR3 sequence is shown in SEQ ID NO:7, the first binding arm; and (ii)A second binding arm comprising a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second VH comprises a second HCDR1 sequence, a second HCDR2 sequence, and a second HCDR3 sequence, the second HCDR1 sequence is shown in SEQ ID NO:9, the second HCDR2 sequence is shown in SEQ ID NO:10, and the second HCDR3 sequence is shown in SEQ ID NO:11; and the second VL comprises a second LCDR1 sequence, a second LCDR2 sequence, and a second LCDR3 sequence, the second LCDR1 sequence is shown in SEQ ID NO:13, the second LCDR2 sequence is DDN, and the second LCDR3 sequence is shown in SEQ ID NO:14, the second binding arm comprising the first binding arm comprises a first heavy chain constant region (CH) and the second binding arm comprises a second CH, and the positions of L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A in the first CH and the second CH, respectively, and The amino acid at the position corresponding to F405 according to the EU numbering in the human IgG1 heavy chain is L in the first CH, and the amino acid at the position corresponding to K409 according to the EU numbering in the human IgG1 heavy chain is R in the second CH, or the amino acid at the position corresponding to K409 according to the EU numbering in the human IgG1 heavy chain is R in the first CH, and the amino acid at the position corresponding to F405 according to the EU numbering in the human IgG1 heavy chain is L in the second CH, The medicament according to claim 1.

19. The conjugate substance is (i) a first binding arm comprising a first heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:1 and a first light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:5; and (ii) a second binding arm comprising a second heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:8 and a second light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:12 and the first binding arm comprises a first heavy chain constant region (CH) and the second binding arm comprises a second CH, the positions of L234, L235, and D265 according to the EU numbering in the human IgG1 heavy chain are F, E, and A respectively in the first CH and the second CH, and the amino acid at the position corresponding to K409 according to the EU numbering in the human IgG1 heavy chain is R in the first CH, and the amino acid at the position corresponding to F405 according to the EU numbering in the human IgG1 heavy chain is L in the second CH, or the amino acid at the position corresponding to F405 according to the EU numbering in the human IgG1 heavy chain is L in the first CH, and the amino acid at the position corresponding to K409 according to the EU numbering in the human IgG1 heavy chain is R in the second CH, The medicament according to claim 1.

20. The conjugate substance is i) a first heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO:36, and a first light chain comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO:37; and ii) A second heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO:38, and a second light chain comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO:39 The medicament according to claim 1, comprising the same.

21. The medicament according to claim 1, wherein the conjugate substance is acasunlimab or a biosimilar thereof.

22. The medicament according to claim 1, wherein the tumor or cancer is a solid tumor.

23. The medicament according to claim 1, wherein the tumor is a PD-L1 positive tumor.

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

25. The medicament according to claim 1, wherein the tumor or cancer is lung cancer.

26. The lung cancer is non-small cell lung cancer (NSCLC), e.g., squamous or non-squamous NSCLC, and preferably, the NSCLC does not have an epidermal growth factor (EGFR) sensitive mutation and / or an anaplastic lymphoma kinase (ALK) gene translocation / ROS1 rearrangement. The medicament according to claim 25.

27. The human subject has, for example, received one, two, three, or four systemic pretreatment regimens for advanced / metastatic disease and has experienced disease progression, e.g., disease progression determined by radiography, during or after the last systemic pretreatment. The medicament according to claim 1.

28. whether the human subject has received platinum-based chemotherapy, or the human subject is not eligible for platinum-based treatment and has received treatment with an alternative chemotherapy, such as a regimen containing gemcitabine, The medicament according to claim 27.

29. The medicament according to claim 1, wherein the human subject has been pre-treated with a checkpoint inhibitor, such as an agent targeting PD-1 / PD-L, such as a PD-1 / PD-L1 inhibitor.

30. The medicament according to claim 1, wherein the human subject has experienced disease progression during or after treatment with a checkpoint inhibitor, such as an agent targeting PD-1 / PD-L, such as a PD-1 / PD-L1 inhibitor.

31. The medicament according to claim 1, wherein the human subject has experienced disease progression during or after the last pre-treatment with a checkpoint inhibitor, such as an agent targeting PD-1 / PD-L, such as a PD-1 / PD-L1 inhibitor.

32. The medicament according to claim 1, wherein the human subject has experienced disease progression, such as disease progression determined by radiography, during or after the last systemic pre-treatment.

33. The last pre-treatment of the human subject is pre-treatment with a PD1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, wherein the PD-1 inhibitor or PD-L1 inhibitor is administered as monotherapy or as part of a combination therapy, The medicament according to claim 1.

34. The period from progression during the last treatment with a PD1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, is 8 months or less, such as 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less or such as 2 weeks or less, The medicament according to claim 1.

35. The period from the last dosing of a PD1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, as part of the final pretreatment is 8 months or less, for example, 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less or for example 2 weeks or less, the medicament according to claim 1.

36. The medicament according to claim 1, wherein the human subject has not been pretreated with a checkpoint inhibitor, such as an agent targeting PD-1 / PD-L, such as a PD-1 / PD-L1 inhibitor.

37. The medicament according to claim 1, wherein the method is for first-line treatment of the tumor or cancer or for second-line treatment of the tumor or cancer.

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