An agent having the ability to bind to CD27 in combination therapy

Abstract

The present invention provides a combination therapy for reducing or preventing tumor progression or treating cancer, which uses a first binder comprising at least one binding region that binds to CD27 in combination with a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137. TIFF2025516633000020.tif66170

Description

Technical Field

[0001] The present invention relates to a combination therapy for reducing or preventing tumor progression or treating cancer, which uses a first binder comprising at least one binding region that binds to CD27 in combination with a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

Background Art

[0002] Background of the Invention Cluster of differentiation (CD) 27 (TNFRSF7) is a 55 kDa type I transmembrane protein member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) that co-stimulates T cell activation after binding to its ligand CD70. It is expressed on the cell membranes of T, B, natural killer (NK) cells, and their direct progenitor cells, all of which are part of the lymphoid lineage, in humans. On human T cells, CD27 is expressed on resting αβ CD4 + (Treg and conventional T cells), CD8 + T cells, stem cell memory cells, and central memory-like cells. On human B cells, CD27 is a memory B cell marker, and CD27 signaling promotes the differentiation of B cells into plasma cells.

[0003] The only known ligand for CD27 is the type II transmembrane protein CD70 (tumor necrosis factor superfamily member 7, TNFSF7; CD27 ligand, CD27L), which is expressed only quite restrictively and transiently on activated immune cells, including T, B, NK, and dendritic cells (DC).

[0004] CD27 plays a role in the early generation of the primary immune response and is required for the generation and long-term maintenance of T cell immunity. The binding of CD27 - CD70 leads to the activation of the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and the mitogen-activated protein kinase (MAPK) 8 / Jun N-terminal kinase (JNK) pathways. The adapter proteins TNF receptor-associated protein (TRAF) 2 and TRAF5 have been shown to mediate the signaling resulting from CD27 engagement.

[0005] T cells require T cell antigen receptor-mediated recognition of their cognate antigens in the context of major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) and activation of co-stimulatory receptors to unleash their effector functions. CD27 and CD28 are considered the most important co-stimulatory receptors expressed on T cells.

[0006] In mice, CD27 stimulation during the priming phase of T cell activation has been found to promote the clonal expansion of antigen-specific CD4 + and CD8 + T cells by interleukin (IL)-2-independent survival signaling (Carr JM et al, Proc Natl Acad Sci USA 2006 Dec 19;130(51):19454 - 9 (Non-Patent Document 1)). CD27 also counteracts the apoptosis of T cells activated through continuous division and has also been shown to play an important role in the memory differentiation of mouse CD8 + T cells (Van de Ven K, Borst J. Immunotherapy 2015;7(6):655 - 67 (Non-Patent Document 2)). As a result, CD27 stimulation promotes the generation of effector T cells in lymphoid organs and broadens the repertoire of responder T cells. In human naive T cells, CD27 stimulation is associated with CD4 +Promote the Th1 differentiation of T cells and support the effector differentiation of cytotoxic T lymphocytes (Oosterwijk et al, Int Immunol. 2007 Jun;19(6):713-8 (Non-Patent Document 3)).

[0007] In contrast to its presence on tumor cells in some hematological malignancies, CD27 expression has not been detected on tumor cells in solid malignancies. However, CD27-expressing lymphoid cells have been reported in the tumor microenvironment (TME) of both hematological malignancies and solid cancers.

[0008] In cancer treatment, the involvement and stimulation of the immune response have been shown to induce and / or enhance anti-tumor immunity and result in a clinical response, as exemplified by the clinical success of immune checkpoint inhibitors (CPIs). Active immune responses and / or existing anti-tumor immunity can be increased by providing co-stimulatory signaling, such as CD27 co-stimulatory signaling.

[0009] In mouse tumor models, T cell function and thus anti-tumor immunity can be enhanced by agonist CD27 antibodies. In a human CD27 (hCD27) transgenic lymphoma mouse model, CD27 activation using agonist antibodies results in potent anti-tumor activity, as well as CD4 + and CD8 +Induction of protective immunity dependent on T cells was shown (He LZ et al. J Immunol. 2013 Oct 15;191(8):4174 - 83 (Non - Patent Document 4)). Furthermore, CD27 activation using monoclonal antibodies has prevented tumor growth in mouse xenografts, including models derived from leukemia (Vitale et al, Keler T. Clin Cancer Res. 2012 Jul 15;18(14):3812 - 21 (Non - Patent Document 5)), melanoma (Roberts DJ, et al., J Immunother. 2010 Oct;33(8):769 - 79 (Non - Patent Document 6)), colon cancer, and thymoma (He LZ, et al., J Immunol. 2013 Oct 15;191(8):4174 - 83 (Non - Patent Document 4)).

[0010] Monoclonal immunoglobulin G (IgG)1 agonist antibodies against human CD27 have been disclosed in the prior art.

[0011] WO2012 / 004367 (Patent Document 1) describes a humanized anti - human CD27 agonist antibody (designated hCD27.15). It has been reported that hCD27.15 does not require cross - linking by crystallizable fragment (Fc) gamma receptor (FcyR) - expressing cells to activate CD27 - mediated costimulation of the immune response. However, this antibody does not bind to a single nucleotide polymorphism (SNP) (A59T) that is frequently present in hCD27 and does not bind to cynomolgus monkey CD27.

[0012] WO2011 / 130434 (Patent Document 2) discloses a human agonist anti - human CD27 antibody designated 1F5. 1F5 activates CD27 by cross - linking with FcyR - expressing cells and further blocks the binding of soluble CD70 (sCD70) ligand. 1F5 has been reported to have Fc - mediated effector function activity, including complement - dependent cytotoxicity (CDC) and antibody - dependent cell - mediated cytotoxicity (ADCC) in target cells, as well as enhancing the immune response and having antitumor activity in mouse models.

[0013] WO2018 / 058022 (Patent Document 3) discloses agonist mouse anti-human CD27 antibody 131A and its humanized version. 131A is disclosed to bind to the frequently occurring hCD27 SNP A59T and cynomolgus monkey CD27. WO2018 / 058022 (Patent Document 3) further discloses that in a mouse tumor model, antibody 131A had a stronger antitumor response compared to antibody 1F5.

[0014] WO2019 / 195452 (Patent Document 4) discloses a non-ligand-blocking agonist anti-human CD27 antibody designated BMS-986215, and it has been reported that BMS-986215 has a higher affinity for human and cynomolgus monkey CD27 than the above-described CD27 antibody 1F5. It is disclosed that in the presence of BMS-986215, CD27 co-stimulation of T cells occurs by binding to its ligand CD70. BMS-986215 reduces the suppression of responder T cells by regulatory T cells (Tregs), and it is further disclosed that BMS-986215 binds to C1q and induces CDC, moderate ADCC, and low levels of antibody-dependent cell phagocytosis (ADCP). It is further disclosed that BMS-986215 has only weak agonist activity in the absence of FcyR and in the absence of sCD70. + It further reduces the suppression of responder T cells by regulatory T cells (Tregs), and it is further disclosed that BMS-986215 binds to C1q and induces CDC, moderate ADCC, and low levels of antibody-dependent cell phagocytosis (ADCP). It is further disclosed that BMS-986215 has only weak agonist activity in the absence of FcyR and in the absence of sCD70.

[0015] An anti-CD27 antibody must induce clustering of CD27 on the plasma membrane to induce CD27 agonism. In the case of wild-type IgG1 antibodies, clustering of CD27 can be achieved through interaction of membrane-bound CD27 antibodies with FcγR-bearing cells such as monocytes, macrophages, B cells, and other immune cells. As a result, anti-CD27 IgG1 molecules can be of lower efficiency when the number of FcγR-expressing cells is limited. Optimization of effector functions by modification of the Fc region of the antibody can improve the efficacy of therapeutic antibodies for treating cancer or other diseases and, for example, improve the ability of the antibody to induce an immune response against antigen-expressing cells.Such efforts are described, for example, in WO 2013 / 004842 A2 (Patent Document 5); WO 2014 / 108198 A1 (Patent Document 6); WO2018 / 146317 (Patent Document 7); WO2018 / 083126 (Patent Document 8); WO 2018 / 031258 A1 (Patent Document 9); Dall'Acqua, Cook et al. J Immunol 2006, 177(2):1129-1138 (Non-Patent Document 7); Moore, Chen et al. MAbs 2010 2(2):181-189 (Non-Patent Document 8); Desjarlais and Lazar, Exp Cell Res 2011, 317(9):1278-1285 (Non-Patent Document 9); Kaneko and Niwa, BioDrugs 2011, 25(1):1-11 (Non-Patent Document 10); Song, Myojo et al., Antiviral Res 2014, 111:60-68 (Non-Patent Document 11); Brezski and Georgiou, Curr Opin Immunol 2016, 40:62-69 (Non-Patent Document 12); Sondermann and Szymkowski, Curr Opin Immunol 2016, 40:78-87 (Non-Patent Document 13); Zhang, Armstrong et al. MAbs 2017, 9(7):1129-1142. (Non-Patent Document 14); Wang, Mathieu et al. Protein&Cell 2018, 9(1):63-73 (Non-Patent Document 15); Diebolder FJ et al., Science.2014 Mar 14;343(6176):1260-3 (Non-Patent Document 16)).

[0016] Among others, Garber et al. discussed the opportunity of combination therapies consisting of agonist antibodies targeting co-stimulatory receptors on T cells, such as 4-1BB (CD137), OX40, glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR), and inducible co-stimulator (ICOS), as well as monoclonal antibodies that block the PD-1 / PD-L1 system (Garber et al. Nat Rev Drug Discov. 2020 Jan;19(1):3-5 (Non-Patent Document 17)). Azpilikueta et al. (J Thorac Oncol 2016;11:524-36) (Non-Patent Document 18) published preclinical data from a combination therapy including a PD-1 blocking antibody and a 4-1BB targeting antibody in a mouse lung cancer model, showing that the combination therapy outperformed monotherapy. Also, Diggs et al. reported improved antitumor activity by a combination therapy of a PD-1 blocking antibody and an anti-CD40 antibody in a mouse tumor hepatocellular carcinoma model (Diggs et al. J Hepatol. 2021 May;74(5):1145-1154 (Non-Patent Document 19)).

[0017] WO2008 / 051424A2 (Patent Document 10) provides methods comprising the administration of a CD27 targeting agonist antibody alone or in combination with other immunomodulatory agents, such as antibodies targeting CD40, OX40, 4-1BB, or CTLA-4.

[0018] However, despite these and other efforts in the art, there remains a need for improved antibody-based immunotherapies with increased agonism and / or increased potency for engaging CD27, provided together as combination therapies with other immunomodulatory antibodies.

Prior Art Documents

Patent Documents

[0019]

Patent Document 1

[0020] [Non-Patent Document 1] Carr JM et al,Proc Natl Acad Sci USA 2006 Dec 19;130(51):19454-9 [Non-Patent Document 2] Van de Ven K,Borst J.Immunotherapy 2015;7(6):655-67 [Non-Patent Document 3] Oosterwijk et al,Int Immunol.2007 Jun;19(6):713-8 [Non-Patent Document 4] He LZ et al.J Immunol.2013 Oct 15;191(8):4174-83 [Non-Patent Document 5] Vitale et al,Keler T.Clin Cancer Res.2012 Jul 15;18(14):3812-21 [Non-Patent Document 6] Roberts DJ, et al., J Immunother. 2010 Oct; 33(8): 769 - 79

Non - Patent Document 7

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Non - Patent Document 14

Non - Patent Document 15

Non - Patent Document 16

Non - Patent Document 17

Non - Patent Document 18

Non-Patent Document 19

Summary of the Invention

[0021] The present invention relates to a binder having the ability to bind to CD27 in combination therapy.

[0022] In a first aspect, the present disclosure provides a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0023] In a second aspect, the present disclosure provides a kit comprising i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0024] In a third aspect, the present disclosure provides a kit for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the kit comprising i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0025] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising i) a first binder comprising at least one binding region that binds to CD27; ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137; and iii) optionally, a pharmaceutically acceptable carrier.

[0026] In a fifth aspect, the present disclosure provides a pharmaceutical composition for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the pharmaceutical composition comprising: i) a first binder comprising at least one binding region that binds to CD27; ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137; and iii) optionally, a pharmaceutically acceptable carrier.

[0027] In a sixth aspect, the present disclosure provides a first binder for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising the step of administering to the subject: i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0028] In a seventh aspect, the present disclosure provides a second binder for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising the step of administering to the subject: i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137. **DETAILED DESCRIPTION OF THE INVENTION**

[0029] Detailed Description of the Invention Definitions As used in the context of the present invention, the term "antibody" (Ab) 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. The antibodies of the present invention include the Fc domain of an immunoglobulin and an antigen-binding region. Antibodies generally contain two CH2-CH3 regions and a connecting region, such as a hinge region, for example at least the Fc domain. Thus, the antibodies of the present invention may include an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with an antigen. The constant or "Fc" region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. As used herein, unless the context dictates otherwise, the Fc region of an immunoglobulin typically contains at least the CH2 and CH3 domains of the immunoglobulin CH and may include a connecting region, such as a hinge region. The Fc region is typically in a dimeric form, for example, via disulfide bridges connecting two hinge regions and / or non-covalent interactions between two CH3 regions. The dimer may be a homodimer (the amino acid sequences of the two Fc region monomers are identical) or a heterodimer (the amino acid sequences of the two Fc region monomers differ in one or more amino acids). As is well known in the art, fragments of the Fc region of a full-length antibody can be generated, for example, by digestion of the full-length antibody with papain. The antibodies defined herein may further include one or both of the immunoglobulin CH1 region and the CL region, in addition to the Fc region and the antigen-binding region. Antibodies may also be multispecific antibodies, such as bispecific antibodies or similar molecules. The term "bispecific antibody" refers to an antibody that has specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. When the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. As indicated above, unless otherwise described,Or, if not clearly inconsistent with the context, the term "antibody" as used herein includes antibodies and antibody fragments that include at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the terms "Ab" or "antibody" are monovalent antibodies (described in WO2007059782 by Genmab); heavy chain antibodies consisting only of two heavy chains, such as those that occur naturally in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMab, Roche, WO2011069104); strand-exchange engineered domain (SEED or Seed-body), an asymmetric and bispecific antibody-like molecule (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792); Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768); mAb-Fv (Xencor, WO2011 / 028952); Xmab (Xencor); dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); dual domain dual head antibody (Unilever; Sanofi Aventis, WO20100226923); Di-diabody (ImClone / Eli Lilly); knob-into-hole antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545); DuetMab (MedImmune, US2014 / 0348839); electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004; Chugai,US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545); CrossMAb (Roche, WO2011117329); LUZ-Y (Genentech); Biclonic (Merus, WO2013157953); Dual targeting domain antibody (GSK / Domantis); Two-in-one antibody or bispecific Fab recognizing two targets (Genentech, NovImmune, Adimab); Cross-linked Mab (Karmanos Cancer Center); Covalently fused mAb (AIMM); CovX-body (CovX / Pfizer); FynomAb (Covagen / Janssen ilag); DutaMab (Dutalys / Roche); iMab (MedImmune); IgG-like bispecifics (ImClone / Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74); TIG-body, DIG-body and PIG-body (Pharmabcine); Bispecific affinity retargeting molecules (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538); BEAT (Glenmark); Zybodies (Zyngenia); In addition to approaches using a common light chain (Crucell / Merus, US7262028) or a common heavy chain (κλBodies by NovImmune, WO2012023053), fusion proteins containing a polypeptide sequence fused to an antibody fragment containing an Fc region-like scFv, e.g., BsAb by ZymoGenetics / BMS,HERCULES by Biogen Idec (US007951918); SCORPIONS (Emergent BioSolutions / Trubion and Zymogenetics / BMS); Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009.393(3):p.672-92); scFv fusions (Genentech / Roche); scFv fusions (Novartis); scFv fusions (Immunomedics); scFv fusions (Changzhou Adam Biotech Inc, CN 102250246); TvAb (Roche, WO 2012025525, WO 2012025530); mAb2 (f-Star, WO2008 / 003116); and bispecific scFv fusions, including but not limited to. The term antibody, unless otherwise specified, refers to monoclonal antibodies (e.g., human monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, monospecific antibodies (e.g., bivalent monospecific antibodies), bispecific antibodies, antibodies of any isotype and / or allotype; e.g., antibody mixtures (recombinant polyclonal) generated by techniques exploited by Symphogen and Merus (Oligoclonics), multimeric Fc proteins described in WO2015 / 158867, and fusion proteins described in WO2014 / 031646. These different antibody fragments and formats are generally included within the meaning of antibody, but they are distinct features of the present invention both collectively and each independently, exhibiting different biological properties and utilities.,

[0030] An "agonist antibody" for a native receptor is a compound that binds to the receptor to form a receptor-antibody complex and activates the receptor, thereby initiating pathway signaling and further biological processes.

[0031] The terms "agonism" and "agonist(s)" are used interchangeably herein and refer to or describe an antibody having the ability to substantially induce, promote, or enhance the biological activity or activation of CD27, either directly or indirectly. Optionally, an "agonist CD27 antibody" results in the activation of one or more intracellular signaling pathways, which may include the activation of the NF-KB and MAPK8 / JNK pathways, and has the ability to activate the CD27 receptor by a mechanism similar to that of the ligand for CD27, known as CD70 (tumor necrosis factor superfamily member 7, TNFSF7; CD27 ligand, CD27L). "Agonism" as defined herein may be determined according to Example 2 herein.

[0032] As used herein, a "CD27 antibody" or "anti-CD27 antibody" is an antibody that specifically binds to the protein CD27, particularly human CD27.

[0033] "Variant", as used herein, refers to a protein or polypeptide sequence that differs from a parent or reference sequence at one or more amino acid residues. A variant may have, for example, at least 80%, such as 90%, or 95%, or 97%, or 98%, or 99% sequence identity to the parent or reference sequence. Additionally, or alternatively, a variant may differ from the parent or reference sequence by 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, such as an amino acid residue substitution, insertion, or deletion. Thus, "variant antibody" or "antibody variant", used interchangeably herein, refers to an antibody that differs from a parent or reference antibody at one or more amino acid residues, for example, in the antigen-binding region, the Fc region, or both. Similarly, "variant Fc region" or "Fc region variant" refers to an Fc region that differs from a parent or reference Fc region at one or more amino acid residues, optionally by 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, such as an amino acid residue substitution, insertion, or deletion, from the parent or reference Fc region amino acid sequence. The parent or reference Fc region is typically the Fc region of a human wild-type antibody, which may be a particular isotype depending on the context. A variant Fc region may be in a dimerized form, which may be a homodimer or a heterodimer, for example, where one of the amino acid sequences of the dimerized Fc region contains a mutation and the other is identical to the parent or reference wild-type amino acid sequence. Examples of wild-type (typically parent or reference sequence) IgG CH and variant IgG constant region amino acid sequences, including the Fc region amino acid sequence, are shown in Table 3.

[0034] The term "immunoglobulin heavy chain" or "heavy chain of immunoglobulin", as used herein, is intended to refer to one of the heavy chains of an immunoglobulin. A heavy chain typically includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the isotype of the immunoglobulin. The heavy chain constant region typically includes three domains, CH1, CH2, and CH3. The term "immunoglobulin", as used herein, is a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four of which are potentially interconnected by disulfide bonds. The structure of immunoglobulins is well characterized (see, e.g., Fundamental Immunology Ch.7 Paul, W., 2nd ed. Raven Press, N.Y. 1989). Within the structure of an immunoglobulin, the two heavy chains are interconnected via disulfide bonds in the so-called "hinge region". Similar to the heavy chain, each light chain typically includes several regions; a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically includes one domain, CL. Further, the VH and VL regions can be further divided into hypervariable regions (also referred to as complementarity determining regions (CDRs)) that are separated by more conserved regions referred to as framework regions (FRs), which may be hypervariable in the sequence and / or conformation of a structurally defined loop. Each VH and VL is typically composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences herein are defined according to IMGT (see Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)).

[0035] As used herein, the terms "half molecule", "Fab arm", and "arm" refer to one of the heavy chain-light chain pairs. 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 resulting bispecific antibody. In this context, "recombine" is not intended to be limited by any particular method of recombination, and thus, for example, in addition to recombination by "Fab arm exchange" and "half molecule exchange" as also described in the art as the DuoBody® method, it includes all of the methods for producing bispecific antibodies described herein below, including recombination at the nucleic acid level and / or co-expression of two half molecules in the same cell.

[0036] The term "antigen binding region" or "binding region" or antigen binding domain, as used herein, refers to the region of an antibody that has the ability to bind to an antigen. This binding region is typically defined by the VH and VL domains of an antibody that can be further divided into hypervariable regions (or hypervariable regions that may be hypervariable in the sequence and / or conformation of structurally defined loops) also referred to as complementarity determining regions (CDRs) flanked by more conserved regions referred to as framework regions (FRs). The antigen can be any molecule, such as a polypeptide, present on, for example, a cell, bacterium, or virion. The terms "antigen binding region" and "antigen binding site" and "antigen binding domain" may be used interchangeably in the context of the present invention, provided there is no contradiction in the context.

[0037] The terms "antigen" and "target" may be used interchangeably in the context of the present invention, provided there is no contradiction in the context.

[0038] As used herein, the term "binding", when determined by biolayer interferometry using an antibody as a ligand and an antigen as an analyte, is typically 1E 6 M or less, such as 5E 7 M or less, 1E 7 M or less, such as 5E 8 M or less, such as 1E 8 M or less, such as 5E 9 M or less, or such as 1E 9 M or less of K D and refers to the binding of an antibody to a predetermined antigen or target having a binding affinity corresponding thereto, which is at least 10-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower than its affinity for binding to non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). D It binds to the predetermined antigen with an affinity corresponding to K

[0039] The term "K D "(M), as used herein, refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is obtained by dividing k d by k a .

[0040] The term "k d "(sec -1 ), as used herein, refers to the dissociation rate constant of a specific antibody-antigen interaction. The said value is also referred to as the k off value or off-rate.

[0041] The term "k a "(M -1 ×sec -1 ), as used herein, refers to the association rate constant of a specific antibody-antigen interaction. The said value is also referred to as the k on value or on-rate.

[0042] As used herein, the term "CD27" refers to the human protein designated CD27, also known as tumor necrosis factor receptor superfamily member 7 (TNFRSF7). In the amino acid sequence shown in SEQ ID NO:1 (Uniprot ID P26842), amino acid residues 1-19 are the signal peptide and amino acid residues 20-240 are the mature polypeptide. Where the context is not inconsistent, CD27 may also refer to variants, isoforms and orthologs of CD27. A naturally occurring variant of human CD27 containing the A59T mutation is shown in SEQ ID NO:2.

[0043] In cynomolgus monkeys (Macaca fascicularis), the CD27 protein has the amino acid sequence shown in SEQ ID NO:3 (Genbank XP_005569963). In the 240 amino acid sequence shown in SEQ ID NO:3, the signal peptide is not defined.

[0044] The term "antibody binding region" refers to the region of an antigen that contains the epitope to which the antibody binds. The antibody binding region may be determined by epitope binding using biolayer interferometry, by alanine scanning, or by a shuffle assay (using an antigen construct in which the region of the antigen has been exchanged with that of another species and determining whether the antibody still binds to the antigen). Amino acids within the antibody binding region involved in interaction with the antibody may be determined by hydrogen / deuterium exchange mass spectrometry and by X-ray crystallography of the antibody bound to its antigen.

[0045] The term "epitope" means an antigenic determinant that is specifically bound by an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids, sugar side chains, or combinations thereof, and usually have specific charge characteristics in addition to specific three-dimensional structural features. Conformational and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. 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 an antibody when the antibody is bound to an antigen (in other words, the amino acid residues are within or adjacent to the footprint of a particular antibody).

[0046] Terms such as "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", or "mAb", as used herein, refer 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 having a single binding specificity and having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies may be produced by hybridomas containing B cells obtained from transgenic or transchromosomal non-human animals, such as transgenic mice or rats, having a genome containing a human heavy chain transgene and a light chain transgene, which have been fused to immortalized cells. Monoclonal antibodies may also be produced from recombinant modified host cells, or from a system using a cell extract that supports in vitro transcription and / or translation of a nucleic acid sequence encoding the antibody.

[0047] As used herein, the term "isotype" refers to an immunoglobulin class (e.g., IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f) encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

[0048] As used herein, the term "full-length antibody" refers to an antibody that contains all of the domains of a particular isotype that are normally found in nature for that isotype, rather than as a fragment, e.g., for an IgG1 antibody, contains VH, CH1, CH2, CH3, hinge, VL, and CL domains. In a full-length variant antibody, the heavy and light chain constant and variable domains may particularly contain amino acid substitutions that improve the functional properties of the antibody when compared to the full-length parent or wild-type antibody. The full-length antibodies according to the present invention may be produced by a method comprising (i) a step of cloning a CDR sequence into a suitable vector containing a complete heavy chain sequence and a complete light chain sequence, and (ii) a step of expressing the complete heavy and light chain sequences in a suitable expression system. It is within the knowledge of those skilled in the art to produce a full-length antibody starting from either a CDR sequence or an entire variable region sequence. Thus, those skilled in the art know how to generate the full-length antibodies according to the present invention.

[0049] As used herein, the term "human antibody" is intended to include antibodies that contain variable and framework regions derived from human germline immunoglobulin sequences as well as human immunoglobulin constant domains. The human antibodies of the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by in vitro random or site-directed mutagenesis or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, are grafted onto human framework sequences.

[0050] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody that has been modified to contain a non-human variable domain that contains a high level of sequence homology to a human antibody constant domain and a human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen-binding site onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). Substitution of framework residues from the parental antibody (i.e., non-human antibody) into the human framework region (back mutations) may be required to fully reconstruct the binding affinity and specificity of the parental antibody. Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, a human framework region optionally containing one or more amino acid back mutations to a non-human amino acid sequence, and a fully human constant region. Optionally, additional amino acid modifications, which are not necessarily back mutations, may be applied to obtain a humanized antibody with desirable characteristics such as affinity and biochemical properties.

[0051] As used herein, the terms "Fc region" or "Fc domain" may be used interchangeably and refer to the region of the heavy chain constant region that includes at least the hinge region, CH2 region, and CH3 region in the direction from the N-terminus to the C-terminus of the antibody. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0052] Unless otherwise stated or clearly inconsistent with the context, the terms "parent polypeptide" or "parent antibody" should be understood to be a polypeptide or antibody that is identical to the polypeptide or antibody according to the present invention, except that the parent polypeptide or parent antibody has no mutations. For example, the antibody IgG1-CD27-A of the present invention is the parent antibody of IgG1-CD27-A-P329R-E345R.

[0053] As used herein, the term "hinge region" refers to the hinge region of the immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the Eu numbering (Eu index) shown in Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991). However, the hinge region may also be any of the other subtypes described herein.

[0054] As used herein, the terms "CH1 region" or "CH1 domain" refer to the CH1 region of the immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering shown in Kabat (supra). However, the CH1 region may also be any of the other subtypes described herein.

[0055] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering shown in Kabat (supra). However, the CH2 region may also be any of the other subtypes described herein.

[0056] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering shown in Kabat (supra). However, the CH3 region may also be any of the other subtypes described herein.

[0057] As used herein, the terms "Fc-mediated effector function" or "Fc effector function" are used interchangeably and are intended to refer to functions that are the consequence of the binding of a polypeptide or antibody to a target or antigen on a cell membrane, where the Fc-mediated effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc gamma receptor (FcγR) binding, (vi) antibody-dependent, FcγR-mediated antigen bridging, (vii) antibody-dependent cell phagocytosis (ADCP), (viii) complement-dependent cell cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x) binding of an antibody-opsonized antibody to a complement receptor, (xi) opsonization, and (xii) any combination of (i)-(xi).

[0058] The terms "reduced Fc effector function" or "reduced Fc-mediated effector function", as used herein, are used interchangeably and are intended to refer to an Fc effector function that is reduced for an antibody when directly compared to the Fc effector function of a parental polypeptide or antibody in the same assay.

[0059] The terms "inertness", "inert", or "non-activating", as used herein, refer to an Fc region that is unable to bind to any FcγR, unable to induce Fc-mediated cross-linking of FcγR, or unable to induce FcγR-mediated cross-linking of a target antigen via the two Fc regions of an individual antibody, or unable to bind to C1q. Thus, in certain embodiments of the invention, the Fc region is inert. Accordingly, in certain embodiments, some or all of the Fc-mediated effector function is attenuated or completely absent.

[0060] The term "oligomerization", as used herein, is intended to refer to the process of converting monomers to a finite degree of polymerization. Antibodies according to the invention can form oligomers, such as hexamers, for example, via non-covalent association of the Fc regions after target binding on the cell surface. Oligomerization of anti-CD27 antibodies in cell surface binding through Fc:Fc interactions may increase CD27 clustering, which results in activation of CD27 intracellular signaling. The ability of antibodies containing the E345R or E430G mutations to form oligomers, such as hexamers, in cell surface binding can be evaluated as described in de Jong RN et al, PLoS Biol. 2016 Jan 6;14(1):e1002344. Antibody Fc-Fc-mediated oligomerization occurs after target binding on the (cell) surface through intermolecular association of the Fc regions between adjacent antibodies and is increased by the introduction of the E345R or E430G mutations (numbering according to the Eu index).

[0061] The term "clustering", as used herein, refers to the oligomerization of antibodies through non-covalent interactions.

[0062] The term "Fc-Fc enhancing", as used herein, is intended to refer to increasing the binding strength between the Fc regions of two Fc region-containing antibodies such that the antibody forms an oligomer, e.g., hexamer, on the cell surface, or stabilizing the interaction between the Fc regions. This enhancement can be obtained by certain amino acid mutations in the Fc region of the antibody, e.g., E345R or E430G. The term "monovalent antibody" refers, in the context of the present invention, to an antibody molecule that can interact with a specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm). In the context of a bispecific antibody, "monovalent antibody binding" refers to the binding of a bispecific antibody to one specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm).

[0063] The term "monospecific antibody" refers, in the context of the present invention, to an antibody that has binding specificity for only one epitope. The antibody may be a monospecific, monovalent antibody (i.e., having only one antigen-binding region) or a monospecific, bivalent antibody (i.e., an antibody having two identical antigen-binding regions).

[0064] The term "bispecific antibody" refers to an antibody that has two non-identical antigen-binding domains, such as two non-identical Fab arms or two Fab arms having non-identical CDR regions. In the context of the present invention, a bispecific antibody has specificity for at least two different epitopes. Such epitopes may be on the same or different antigens or targets. When the epitopes are on different antigens, such antigens may be on the same cell or different cells, cell types or structures, such as the extracellular matrix or vesicles and soluble proteins. A bispecific antibody may therefore have the ability to crosslink multiple antigens, such as two different cells. Certain bispecific antibodies of the present invention have the ability to bind to CD27 and a second target.

[0065] The term "bivalent antibody" refers to an antibody that has two antigen-binding regions that bind to an epitope on one or two targets or antigens, or to one or two epitopes on the same antigen. Thus, a bivalent antibody may be a monospecific bivalent antibody or a bispecific bivalent antibody.

[0066] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as being limiting. An amino acid is an organic compound that contains an amine (-NH 2 ) and a carboxyl (-COOH) functional group, along with a side chain (R group) that is specific to each amino acid. In the context of the present invention, amino acids can be classified based on their structure and chemical characteristics. Thus, the classes of amino acids can be reflected in one or both of the following tables.

[0067] (Table 5) Main classification based on the structure of the R group and general chemical characterization TIFF2025516633000002.tif47128

[0068] (Table 6) Alternative physical and functional classification of amino acid residues TIFF2025516633000003.tif106128

[0069] The substitution of one amino acid for another may be classified as a conservative or non-conservative substitution. In the context of the present invention, a "conservative substitution" is the substitution of one amino acid for another having similar structural and / or chemical characteristics, e.g., the substitution of one amino acid residue for another amino acid residue of the same class as defined in either of the two tables above: for example, leucine may be substituted with isoleucine, since they are both aliphatic, branched hydrophobic substances. Similarly, aspartic acid may be substituted with glutamic acid, because they are both small, negatively charged residues.

[0070] In the context of the present invention, a substitution in an antibody is designated as the original amino acid-position-substituted amino acid ; Reference is made to the well-recognized notations, three-letter codes, or one-letter codes for amino acids, including the codes "Xaa" or "X" to denote any amino acid residue. Thus, Xaa or X can typically represent any of the 20 naturally occurring amino acids. The term "naturally occurring" as used herein refers to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Thus, the notation "K409R" or "Lys409Arg" means that the antibody contains a substitution of arginine for lysine at amino acid position 409.

[0071] The substitution of an amino acid at a given position with any other amino acid is designated as the original amino acid-position; or e.g. "K409" ;

[0072] For modifications where the original amino acid and / or substituted amino acid may contain more than one, but not all, amino acids, the more than one amino acids may be separated by "," or " / ". For example, the substitution of lysine at position 409 with arginine, alanine, or phenylalanine is "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409→R, A, or F".

[0073] Such designations can be used interchangeably in the context of the present invention and can have the same meaning and purpose.

[0074] Furthermore, the term "substitution" includes substitution with any one or the other 19 natural amino acids, or with other amino acids, such as non-natural amino acids. For example, the substitution of amino acid K at position 409 includes the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. Note that this is equivalent to the designation 409X, where X designates any amino acid other than the original amino acid. These substitutions may also be designated as K409A, K409C, etc., or K409A,C, etc., or K409A / C / , etc. The same applies to each and every position mentioned herein, and any such substitutions are specifically included herein.

[0075] The antibodies according to the present invention may also contain deletions of amino acid residues. Such deletions may be denoted as "del", for example, including the description K409del. Thus, in such an embodiment, the lysine at position 409 is deleted from the amino acid sequence.

[0076] As used herein, the term "host cell" is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such term is intended to refer not only to a particular subject cell but also to the progeny of such cell. Since certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytic cells, as well as prokaryotic cells such as Escherichia coli (E. coli) and other eukaryotic hosts such as plant cells and fungi.

[0077] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express an antibody or a target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungal cells including yeast cells.

[0078] For the purposes of the present invention, the sequence identity between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) version 5.0.0 or later versions. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled as "longest identity" (obtained using the -nobrief option) is used as the percent identity and is as follows: (The same residue × 100) / (the length of the alignment - the total number of gaps in the alignment) is calculated as follows.

[0079] Retention of similar residues may additionally or alternatively be measured by a similarity score as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through NCBI using standard settings BLOSUM62, open gap = 11 and extend gap = 1). Preferred variants typically exhibit at least about 45%, such as at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or higher % (e.g., about 99%) similarity to the parental sequence.

[0080] The term “internalized” or “internalization,” as used herein, refers to the biological process by which a molecule, e.g., an antibody according to the invention, is engulfed by a cell membrane and introduced into the interior of the cell. Internalization may also be referred to as “endocytosis.”

[0081] As used herein, the term "effector cell" refers to immune cells involved in the effector phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., T cells including B cells and cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcgRs) or complement receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, etc., have the ability to induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells that express FcgR are involved in the specific killing of target cells and / or the presentation of antigens to other components of the immune system, or the binding to antigen-presenting cells. In some embodiments, ADCC can be further enhanced by antibody-driven classical complement activation, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CRs) expressed on myeloid cells, such as CR3. Recognition of complement fragments by CRs on effector cells can promote the enhancement of Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products can promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells may phagocytose target antigens, target particles, or target cells, which can be antibody-binding dependent and mediated by FcγRs expressed by the effector cells. The expression of specific FcRs or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, the expression of FcγRI has been found to be upregulated by interferon γ (IFN γ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells.These C3 cleavage products can promote phagocytosis either directly by effector cells or indirectly by enhancing antibody-mediated phagocytosis. In certain embodiments herein where the antibody has an inactive Fc region, the antibody does not induce Fc-mediated effector functions.

[0082] "Effector T cells" or "Teffs" or "Teff", as used herein, refer to T lymphocytes that perform the activation of an anti-tumor immune response that can result in functions of the immune response, such as the killing of tumor cells and / or the clearance of tumor cells from the body. Examples of the Teff phenotype are CD3 + CD4 + and CD3 + CD8 + Teff can secrete, contain, or express markers such as IFNγ, granzyme B, and ICOS. It is understood that Teff may not be completely restricted to these phenotypes.

[0083] "Memory T cells", as used herein, refer to T lymphocytes that remain in the body over a long time period after an infection has been cleared. Examples of memory T cells include central memory T cells (CD45RA-CCR7+) and effector memory T cells (CD45RA-CCR7-). It is understood that memory T cells may not be completely restricted to these phenotypes.

[0084] "Regulatory T cells" or "Tregs" or "Treg", as used herein, refer to T lymphocytes that regulate the activity of other T cells and / or other immune cells, usually by suppressing their activity. Examples of the Treg phenotype are CD3 + CD4 + CD25 + CD127dim. Treg can further express Foxp3. It is understood that Treg may not be completely restricted to this phenotype.

[0085] As used herein, the term "complement activation" refers to the activation of the classical complement pathway initiated by a large macromolecular complex called C1 that binds to antibody-antigen complexes on a surface. C1 is a complex consisting of six recognition proteins C1q and a heterotetramer of serine proteases, C1r2C1s2. C1 is the first protein complex in the early events of the classical complement cascade, which involves a series of cleavage reactions initiated with the cleavage of C4 into C4a and C4b and of C2 into C2a and C2b. C4b is deposited and together with C2a forms an enzymatic active convertase called C3 convertase, which cleaves complement component C3 into C3b and C3a, which in turn forms C5 convertase. This C5 convertase cleaves C5 into C5a and C5b, and the final components are deposited on the membrane and then trigger the late events of complement activation, in which the terminal complement components C5b, C6, C7, C8 and C9 assemble into a membrane attack complex (MAC). The complement cascade results in the formation of pores in the cell membrane that cause cell lysis, also known as complement-dependent cytotoxicity (CDC). In certain embodiments herein where the antibody has an inactive Fc region, the antibody does not induce complement activation.

[0086] Complement activation can be evaluated by using C1q binding efficacy, CDC kinetics CDC assay (described in WO2013 / 004842, WO2014 / 108198), or by the method of cell deposition of C3b and C4b described in Beurskens et al., J Immunol April 1, 2012 vol.188 no.7, 3532-3541.

[0087] As used herein, the term "C1q binding" is intended to refer to the binding of C1q in the context of its binding to an antibody bound to an antigen. The antibody bound to the antigen should be understood to occur both in vivo and in vitro in the context described herein. As described in Example 8 herein, C1q binding can be evaluated, for example, by using an antibody immobilized on an artificial surface or an antibody bound to a predetermined antigen on the surface of a cell or virion. The binding of C1q to an antibody oligomer should be understood herein as a multivalent interaction resulting in high affinity binding. A decrease in C1q binding, such as that resulting from the introduction of a mutation in an antibody of the invention, may be measured by comparing the C1q binding of the mutated antibody to the C1q binding of its parental antibody (an antibody of the invention without the mutation within the same assay).

[0088] The term "treatment" refers to the administration of an effective amount of a therapeutically active antibody of the invention with the aim of alleviating, ameliorating, halting, or eradicating (curing) a symptom or disease state.

[0089] The term "effective amount" or "therapeutically effective amount" refers to an amount effective with respect to dosage and time period to achieve a desired therapeutic result. The therapeutically effective amount of an antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody variant are outweighed by the therapeutically beneficial effects.

[0090] As used herein, the term "pharmacokinetic profile" can be determined as plasma IgG levels over time as described in Example 12 herein.

[0091] As used herein, the term "CD40" refers to CD40, also referred to as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), which is the receptor for the ligand TNFSF5 / CD40L. CD40 is known to transmit TRAF6- and MAP3K8-mediated signals that activate ERK in macrophages and B cells, leading to induction of immunoglobulin secretion by B cells. Other synonyms used for CD40 include, but are not limited to, B cell surface antigen CD40, Bp50, CD40L receptor, and CDw40. In one embodiment, CD40 is human CD40 having the UniProt accession number P25942. The sequence of human CD40 is also shown in SEQ ID NO:68. Amino acids 1-20 of SEQ ID NO:68 correspond to the signal peptide of human CD40; amino acids 21-193 of SEQ ID NO:68 correspond to the extracellular domain of human CD40; the remaining portions of the protein, i.e., amino acids 194-215 and 216-277 of SEQ ID NO:68 are the transmembrane and cytoplasmic domains, respectively.

[0092] As used herein, the term "CD137" refers to CD137 (4-1BB), also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is a receptor for the ligand TNFSF9 / 4-1BBL. CD137 (4-1BB) is thought to be involved in T cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDw137, T cell antigen 4-1BB homolog, and T cell antigen ILA. In one embodiment, CD137 (4-1BB) is human CD137 (4-1BB) having UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO:70. Amino acids 1-23 of SEQ ID NO:70 correspond to the signal peptide of human CD137; amino acids 24-186 of SEQ ID NO:70 correspond to the extracellular domain of human CD137; and the remaining portions of the protein, namely amino acids 187-213 and 214-255 of SEQ ID NO:70, are the transmembrane and cytoplasmic domains, respectively.

[0093] The Fc region may have a lysine at its C-terminus. The origin of this lysine is the naturally occurring sequence found in humans from which these Fc regions are derived. During the cell culture production of recombinant antibodies, this terminal lysine can be cleaved off by proteolysis by endogenous carboxypeptidase, resulting in a constant region having the same sequence but lacking the C-terminal lysine. For the purpose of antibody production, the DNA encoding this terminal lysine can be omitted from the sequence so that the antibody is produced without the lysine. Antibodies produced from either the nucleic acid sequence encoding the terminal lysine or the nucleic acid sequence that does not encode it are substantially identical in sequence and function because, for example, when using antibodies produced in a CHO-based production system, the degree of processing of the terminal lysine is typically high (Dick, L. W. et al. Biotechnol. Bioeng. 2008;100:1132 - 1143). Therefore, it is understood that the proteins according to the present invention, such as antibodies, can be produced with or without encoding or having a terminal lysine. It is also understood according to the present invention that a sequence having a terminal lysine, such as a constant region sequence having a terminal lysine, can be understood as the corresponding sequence without a terminal lysine, and that a sequence without a terminal lysine can also be understood as the corresponding sequence having a terminal lysine.

[0094] Aspects and embodiments of the present disclosure In a first aspect, the present disclosure provides a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject: i) a first binding agent comprising at least one binding region that binds to CD27; and ii) a second binding agent comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0095] A first binder that binds to CD27 In one aspect, the first binder comprises at least one antigen-binding region having the ability to bind to human CD27, and the first binder comprises a heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0096] In a further aspect, the first binder comprises two of the antigen-binding regions comprising a VH region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and a VL region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively. The present invention provides an anti-CD27 antibody that can bind to human CD27 and further to a variant of human CD27 comprising the A59T mutation.

[0097] In one aspect of the present invention, the first binder binds to CD27 on, for example, T cells and is agonistic in its binding to its target. The present invention provides a first binder that stimulates the activation and proliferation of T cells. The first binder may further stimulate the memory formation and survival of T cells. Such a first binder is useful, for example, in the treatment of cancer. The first binder further has the ability to bind to cynomolgus CD27, which is useful for toxicology studies of the first binder.

[0098] In one aspect, the first binder of the present invention is an isolated antibody.

[0099] In one aspect, the first binder is an antibody. In another aspect, the first binder is a human antibody. In another aspect, the first binder is a humanized antibody. In another aspect, the first binder is a chimeric antibody.

[0100] The first binder of the present invention is, in a preferred embodiment, a full-length antibody. Thus, the first binder of the present invention may further comprise a light chain constant region (CL) and a heavy chain constant region (CH). CH preferably includes a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0101] Mutations in the VH and VL of an antibody can be made, for example, to increase the affinity of the antibody for its target antigen, to reduce its potential immunogenicity, and / or to increase the yield of the antibody expressed by the host cell, which is well known in the art. Thus, in some embodiments, variants of the CDR, VH, and / or VL sequences of the first binder according to the present invention are also envisioned, particularly functional variants of the VH and / or VL regions shown in SEQ ID NO:4 and SEQ ID NO:8, respectively. A functional variant may differ in one or more amino acids in one or more CDRs, for example, compared to the parental VH and / or VL sequences, but the antigen-binding region still retains at least a substantial proportion (at least about 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, or higher) of the affinity and / or specificity of the parental antibody, or retains all of it. Typically, such functional variants retain significant sequence identity to the parental sequence. Exemplary variants include variants that differ from the respective parental VH or VL regions by 12 or fewer, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, such as an amino acid residue substitution, insertion, or deletion. Exemplary variants include variants that differ from the VH and / or VL and / or CDR regions of the parental sequence mainly by conservative amino acid substitutions; for example, 12, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the amino acid substitutions in the variant can be conservative. In a further embodiment of the invention, the first binder may contain up to 1, 2, or 3 mutations in the VH CDR region and / or the VL CDR region, respectively. Such mutations may be substitutions. Such substitutions preferably do not significantly alter the binding affinity and / or binding specificity of the first binder of the present invention.Thus, the present invention encompasses variants of the first binder of the present invention that have the same functional characteristics as the first binder comprising the VH region CDR sequences shown in SEQ ID NOs: 5, 6, and 7, and the VL region CDR sequences shown in SEQ ID NOs: 9, 10, and 11.

[0102] In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 80% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 85% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 90% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 95% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 96% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 97% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 98% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 99% identical to the VH region shown in SEQ ID NO: 4. In another aspect of the present invention, the first binder comprises a VH region comprising the sequence shown in SEQ ID NO: 4.

[0103] In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 80% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 85% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 90% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 95% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 96% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 97% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 98% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising a sequence that is at least 99% identical to the VH region shown in SEQ ID NO:8. In another aspect of the present invention, the first binder comprises a VH region comprising the sequence shown in SEQ ID NO:8.

[0104] In another aspect of the present invention, the first binder comprises a VH region and a VL region comprising the sequences shown in SEQ ID NO:4 and SEQ ID NO:8, respectively.

[0105] The first binder may comprise a light chain constant region that is a human kappa light chain. In another aspect, it may comprise a human lambda light chain constant region.

[0106] The first binder may preferably further comprise the heavy chain constant region which is a human IgG isotype. Optionally, it may comprise a modified human IgG constant region. Such human IgG comprises an Fc region containing CH2 and CH3 regions. By modifying the IgG constant region in the Fc region, it is possible, for example, to regulate the Fc effector function of the antibody or to increase the tendency of the antibody to form Fc-Fc interactions and thereby clusters, such as hexamers. In one aspect of the invention, the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4. In one aspect, it is IgG1. In another aspect, it is IgG2. In yet another aspect, it is IgG3. In a further aspect, it is IgG4. In one particular aspect, the IgG is a modified human IgG comprising one or more amino acid substitutions in the Fc region. In one aspect, it may be a modified human IgG1 comprising one or more amino acid substitutions in the Fc region. In a further aspect of the invention, the IgG1 comprises two or more amino acid substitutions in the Fc region. In one aspect, the IgG1 Fc region has two amino acid substitutions.

[0107] In a further aspect of the invention, the modified human IgG heavy chain constant region comprises up to 10 amino acid substitutions in the Fc region. In another aspect, it comprises up to 9 amino acid substitutions. In another aspect, it comprises up to 8 amino acid substitutions. In another aspect, it comprises up to 7 amino acid substitutions. In another aspect, it comprises up to 6 amino acid substitutions. In another aspect, it comprises up to 5 amino acid substitutions. In another aspect, it comprises up to 4 amino acid substitutions. In another aspect, it comprises up to 3 amino acid substitutions. In another aspect, it comprises up to 2 amino acid substitutions in the Fc region.

[0108] Mutations in the amino acid residues at positions corresponding to E430, E345, and S440 in the human IgG1 heavy chain, numbered according to the EU index, can improve the ability of the antibody to induce CDC. Without being bound by theory, substitution of one or more amino acids at these positions stimulates oligomerization of the antibody, thereby modulating Fc-mediated effector functions and increasing other related functions that can provide, for example, C1q binding, complement activation, CDC, ADCP, internalization, or in vivo efficacy.

[0109] In a further aspect of the invention, the first binder is a variant antibody comprising an antigen-binding region and a variant Fc region.

[0110] In certain embodiments, an antibody variant that binds human CD27 (a) a heavy chain comprising a VH region comprising VH CDR1 comprising the sequence shown in SEQ ID NO:5, VH CDR2 comprising the sequence shown in SEQ ID NO:6, VH CDR3 comprising the sequence shown in SEQ ID NO:7, and a human IgG1 CH region comprising a mutation in one or more of E430, E345, and S440, wherein the amino acid residues are numbered according to the EU index; (b) a light chain comprising a VL region comprising VL CDR1 comprising the sequence shown in SEQ ID NO:9, VL CDR2 comprising the sequence shown in SEQ ID NO:10, and VL CDR3 comprising the sequence shown in SEQ ID NO:11 and comprising.

[0111] In certain other embodiments, an antibody variant that binds human CD27 (a) a heavy chain comprising a VH region comprising SEQ ID NO:4 and a human IgG1 CH region comprising a mutation in one or more of E430, E345, and S440, wherein the amino acid residues are numbered according to the EU index, and (b) Light chain containing a VL region comprising SEQ ID NO:8 comprises.

[0112] The variant antibodies used according to the present invention comprise a variant Fc region or a variant human IgG1 CH region containing a mutation in one or more of P329, E430 and E345. In the following, reference to a mutation in the Fc region may equally apply to a mutation in the human IgG1 CH region, and vice versa.

[0113] As described herein, the position of the amino acid to be mutated in the Fc region can be given with respect to its position in the naturally occurring (wild-type) human IgG1 heavy chain (i.e., “corresponding” to that position) when numbered according to the Eu index. Thus, when the parental Fc region already contains one or more mutations and / or the parental Fc region is, for example, an IgG2, IgG3 or IgG4 Fc region, the position of the amino acid corresponding to an amino acid residue, such as E430, etc., in the human IgG1 heavy chain numbered according to the Eu index can be determined by alignment. In particular, the parental Fc region is aligned with the wild-type human IgG1 heavy chain sequence to identify the residue at the position corresponding to E430 in the human IgG1 heavy chain sequence. Any wild-type human IgG1 constant region amino acid sequence comprising any one of the different human IgG1 allotypes shown in Table 3 may be useful for this purpose.

[0114] In one aspect of the invention, the modification in the IgG Fc region induces increased CD27 agonism compared to the same antibody except that it contains the wild-type IgG Fc region of the same isotype, e.g., IgG1. This may be obtained, for example, by introducing an amino acid other than E at the amino acid position corresponding to position E345 and / or E430 in the human IgG1 heavy chain according to the Eu numbering. In one aspect of the invention, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to the Eu numbering is selected from the group consisting of A, C, D, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W and Y. In another aspect of the invention, the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain according to the Eu numbering is selected from the group consisting of A, C, D, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W.

[0115] In a preferred aspect, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to the Eu numbering is R. Thus, the first binder of the invention may comprise an E345R substitution in the Fc region. In another aspect of the invention, the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain according to the Eu numbering is G. Thus, the first binder of the invention may comprise an E430G substitution in the Fc region. In another aspect, the first binder comprises an amino acid substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y.

[0116] The present invention provides an antibody having enhanced Fc-Fc interaction that can lead to antibody-dependent clustering of CD27 on the cell surface upon antibody binding, thereby increasing the agonism of the antibody of the present invention.

[0117] In another aspect of the first binder of the present invention, the amino acid residue at the position corresponding to position P329 in the human IgG1 heavy chain according to Eu numbering is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y. Thus, the first binder of the present invention may further contain a mutation at position 329.

[0118] In a further aspect of the present invention, the first binder has an amino acid residue R at the position corresponding to position P329 in the human IgG1 heavy chain according to Eu numbering. Thus, the first binder of the present invention may have a P329R substitution in the Fc region. Without being bound by theory, it is believed that the first binder of the present invention containing the E345R mutation in the Fc region (as shown, for example, in SEQ ID NO: 13) has increased serum clearance. The inventors have found that introducing a further mutation at position 329, for example P329R (as shown, for example, in SEQ ID NO: 15), restores the clearance of the first binder of the present invention to the level of the first binder containing wt IgG1 as shown, for example, in SEQ ID NO: 12.

[0119] In another preferred aspect, the amino acid residues at the positions corresponding to positions P329 and E345 in the human IgG1 heavy chain according to Eu numbering are both R. The present invention provides a first binder having increased CD27 receptor agonism and equivalent pharmacokinetic properties, such as serum clearance, when compared to a first binder containing the same VH and VL regions and the same IgG1 heavy chain constant region with the exception of containing the wild-type amino acid P at position 329 and the wild-type amino acid E at position 345.

[0120] Accordingly, in one aspect, the present invention provides a first binder that has increased receptor agonism in binding to CD27 and that has pharmacokinetic properties that are equivalent, e.g., similar or identical, to those of a first binder that includes the same VH and VL regions but includes a wild-type IgG1 heavy-chain constant region such as that shown in SEQ ID NO:12, when compared to the pharmacokinetic properties of the first binder. In other words, the present invention provides a first binder that has pharmacokinetic properties that do not significantly differ from those of the same first binder except for including a wild-type IgG1 heavy-chain constant region.

[0121] In another aspect of the invention, the first binder includes a variant Fc region according to any one of the preceding sections, the variant Fc region being a variant of a human IgG Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 Fc regions. That is, the mutation(s) in one or more of the amino acid residues corresponding to E430 and E345 and P329 are made in a parental Fc region that is a human IgG Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. Preferably, the parental Fc region is a naturally occurring (wild-type) human IgG Fc region, e.g., a human wild-type IgG1, IgG2, IgG3, or IgG4 Fc region, or a mixed isotype thereof. Thus, the variant Fc region may be a human IgG1, IgG2, IgG3, or IgG4 isotype, or a mixed isotype thereof, except for the described mutation(s) (in one or more of the amino acid residues selected from E430 and E345 and P329).

[0122] In one aspect, the parental Fc region and / or the human IgG1 CH region is of the wild-type human IgG1 isotype.

[0123] Thus, the variant Fc region may be a human IgG1 Fc region except for the described mutation (at E430 or E345 or P329).

[0124] In certain embodiments, the parental Fc region and / or the human IgG1 CH region is the human wild-type IgG1m(f) isotype.

[0125] In certain embodiments, the parental Fc region and / or the human IgG1 CH region is the human wild-type IgG1m(z) isotype.

[0126] In certain embodiments, the parental Fc region and / or the human IgG1 CH region is the human wild-type IgG1m(a) isotype.

[0127] In certain embodiments, the parental Fc region and / or the human IgG1 CH region is the human wild-type IgG1m(x) isotype.

[0128] In certain embodiments, the parental Fc region and / or the human IgG1 CH region is a mixed allotype of human wild-type IgG1, such as IgG1m(za), IgG1m(zax), or IgG1m(fa).

[0129] Thus, the variant Fc region and / or the human IgG1 CH region may be the human IgG1m(f), IgG1m(a), IgG1m(x), IgG1m(z) allotype or a mixed allotype of any two or more thereof, except for the mutations described at (E430 or E345 or P329).

[0130] In certain embodiments, the parental Fc region and / or the human IgG1 CH region is the human wild-type IgG1m(za) isotype.

[0131] In certain embodiments, the parental Fc region is the human wild-type IgG2 isotype.

[0132] In certain embodiments, the parental Fc region is the human wild-type IgG3 isotype.

[0133] In certain embodiments, the parental Fc region is the human wild-type IgG4 isotype.

[0134] The CH region amino acid sequences of specific examples of wild-type human IgG isotypes and IgG1 allotypes are shown in Table 3.

[0135] In another aspect, the present invention provides a first binder comprising a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34, and 36. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 12. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 13. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 14. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 15. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 18. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 19. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 20. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 21. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 22. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 23. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 27. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 28. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 29. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 30. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 31. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 32. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 33. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 34. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 36.

[0136] In one aspect, the first binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:15 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 It contains.

[0137] In another aspect, the first binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:12 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 It contains.

[0138] In another aspect, the first binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:13 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 It contains.

[0139] In another aspect, the first binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:14 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 It contains.

[0140] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:18 and d. a CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprises.

[0141] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:19 and d. a CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprises.

[0142] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:20 and d. a CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprises.

[0143] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:21 and d. a CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprises.

[0144] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:22 and d. a CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprises.

[0145] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:23 and d. a CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprises.

[0146] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:27 and d. a CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprises.

[0147] In another aspect, the first binder comprises a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence shown in SEQ ID NO:28 and d. The CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0148] In another aspect, the first binder is a. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. The CH region comprising the amino acid sequence shown in SEQ ID NO:29 and d. The CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0149] In another aspect, the first binder is a. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. The CH region comprising the amino acid sequence shown in SEQ ID NO:30 and d. The CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0150] In another aspect, the first binder is a. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. The CH region comprising the amino acid sequence shown in SEQ ID NO:31 and d. The CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0151] In another aspect, the first binder is a. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:32 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0152] In another aspect, the first binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:33 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0153] In another aspect, the first binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:34 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0154] In another aspect, the first binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. A CH region comprising the amino acid sequence shown in SEQ ID NO:36 and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:16 and comprising.

[0155] In an alternative aspect of the first binder described above, the CL region may be the amino acid sequence shown in SEQ ID NO:17.

[0156] In one aspect, the first binder comprises e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:15 and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprises.

[0157] In another aspect, the first binder comprises e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:12 and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprises.

[0158] In another aspect, the first binder comprises e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:13 and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprises.

[0159] In another aspect, the first binder comprises e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:14 and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 includes

[0160] In another aspect, the first binder e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:18 and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 includes

[0161] In another aspect, the first binder e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:19 and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 includes

[0162] In another aspect, the first binder e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:20 and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 includes

[0163] In another aspect, the first binder e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence shown in SEQ ID NO:21 and h. The CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0164] In another aspect, the first binder is e. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. The CH region comprising the amino acid sequence shown in SEQ ID NO:22 and h. The CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0165] In another aspect, the first binder is e. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. The CH region comprising the amino acid sequence shown in SEQ ID NO:23 and h. The CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0166] In another aspect, the first binder is e. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. The CH region comprising the amino acid sequence shown in SEQ ID NO:27 and h. The CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0167] In another aspect, the first binder is e. The VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. The VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:28 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0168] In another aspect, the first binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:29 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0169] In another aspect, the first binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:30 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0170] In another aspect, the first binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:31 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 and comprising.

[0171] In another aspect, the first binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:32 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 comprising.

[0172] In another aspect, the first binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:33 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 comprising.

[0173] In another aspect, the first binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:34 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 comprising.

[0174] In another aspect, the first binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. A CH region comprising the amino acid sequence shown in SEQ ID NO:36 and h. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 comprising.

[0175] In another aspect, the first binder comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:24 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0176] In another aspect, the first binder comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:35 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0177] In yet another aspect, the first binder comprises a heavy chain constant region that is modified such that the first binder induces Fc-mediated effector function to a lesser extent compared to the same first binder except for the modification. Examples thereof are the CD27-binding antibodies of the present invention that include P329R and E345R substitutions. Such antibodies induce one or more Fc-mediated effector functions to a lesser extent compared to an antibody that includes the same sequence except for not including the P329R substitution, and also compared to the same antibody that includes the same sequence except for not including the P329R and E345R substitutions, e.g., a wild-type IgG1 heavy chain. In one aspect, the Fc-mediated effector function is reduced by at least 20%. In another aspect, the Fc-mediated effector function is reduced by at least 30%. In another aspect, the Fc-mediated effector function is reduced by at least 40%. In another aspect, the Fc-mediated effector function is reduced by at least 50%. In another aspect, the Fc-mediated effector function is reduced by at least 60%. In another aspect, the Fc-mediated effector function is reduced by at least 70%. In another aspect, the Fc-mediated effector function is reduced by at least 80%. In another aspect, the Fc-mediated effector function is reduced by at least 90%. In another aspect, the first binder does not induce one or more Fc-mediated effector functions. One or more Fc effector functions that are reduced or not induced at all are selected from the following group: complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding. Thus, in one aspect, the first binder induces CDC to a lesser extent, e.g., by at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% compared to the same first binder except for the wild-type IgG1 HC constant region. In another aspect, the first binder does not induce CDC.

[0178] In another aspect, the first binder induces CDCC to a level that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80% decreased, or decreased by at least 90% compared to the same first binder except that it has a wild-type IgG1 HC constant region. In another aspect, the first binder of the present invention does not induce CDCC.

[0179] In another aspect, the first binder induces ADCC to a level that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80% decreased, or decreased by at least 90% compared to the same first binder except that it has a wild-type IgG1 HC constant region. In another aspect, the first binder does not induce ADCC.

[0180] In another aspect, the first binder induces ADCP to a level that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80% decreased, or decreased by at least 90% compared to the same first binder except that it has a wild-type IgG1 HC constant region. In another aspect, the first binder does not induce ADCP.

[0181] In another aspect, the first binder induces C1q binding to a level that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80% decreased, or decreased by at least 90% compared to the same first binder except that it has a wild-type IgG1 HC constant region. In another aspect, the first binder does not induce C1q binding. Preferably, C1q binding is determined as in Example 8.

[0182] In another aspect, the first binder induces FcγR binding to a degree that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% decreased compared to the same first binder except having a wild-type IgG1 HC constant region. In another aspect, the first binder of the present invention does not induce FcγR binding. Preferably, FcγR binding is determined as in Example 9.

[0183] In one aspect, the first binder has reduced C1q binding and reduced FcγR binding compared to a first binder having the same amino acid sequence except containing a P329R substitution.

[0184] In one aspect, the first binder is a human antibody except for the described mutations.

[0185] In one aspect of the present invention, the first binder is a monovalent antibody.

[0186] In another aspect, the first binder is a bivalent antibody.

[0187] Furthermore, the first binder may be a monospecific antibody.

[0188] In one aspect, the first binder is a monoclonal antibody, such as a human monoclonal antibody, such as a human bivalent monoclonal antibody, such as a human bivalent full-length monoclonal antibody.

[0189] In a preferred aspect, the first binder is an IgG1 antibody, such as a full-length IgG1 antibody, such as a human full-length IgG1 antibody, optionally a human monoclonal full-length bivalent IgG1,κ antibody, such as a human monoclonal full-length bivalent IgG1m(f),κ antibody, except for any described mutations in the Fc region.

[0190] The first binder according to the present invention is advantageously of a bivalent monospecific format, comprising two antigen-binding regions that bind to the same epitope. However, a bispecific format in which one of the antigen-binding regions binds to a different epitope is also envisioned. Thus, the first binder according to any aspect or embodiment herein can be either a monospecific antibody or a bispecific antibody, provided it is not inconsistent with the context.

[0191] Thus, in another aspect, the first binder is a bispecific antibody comprising a first antigen-binding region having the ability to bind to human CD27 as described herein and a second antigen-binding region having the ability to bind to a different epitope on human CD27. In another aspect, the first binder is a bispecific antibody comprising a first antigen-binding region having the ability to bind to human CD27 as described herein and a second antigen-binding region having the ability to bind to a different target. Such a target may be on a different cell or the same cell as CD27.

[0192] In one aspect of the present invention, the first binder has the ability to bind to human CD27 having the sequence shown in SEQ ID NO:1. However, human CD27 can be expressed as its variant in some individuals. Thus, in another aspect, the first binder of the present invention further has the ability to bind to a human CD27 variant, such as the human CD27 variant shown in SEQ ID NO:2. In another aspect, the first binder of the present invention further has the ability to bind to cynomolgus CD27, such as that shown in SEQ ID NO:3.

[0193] In a further aspect of the present invention, the first binder has the ability to bind to CD27-expressing human T cells.

[0194] In another aspect of the present invention, the first binder has the ability to bind to CD27-expressing cynomolgus T cells.

[0195] In one aspect of the present invention, the full-length IgG1 antibody has the C-terminal lysine of the HC cleaved. Such an antibody may also be considered a "full-length antibody".

[0196] In another aspect of the present invention, the first binder has the ability to induce the proliferation of human T cells, such as CD4 + and CD8 + T cells, such as helper T cells and cytotoxic T cells. Such activity may be assayed as described in Examples 6 or 7 herein.

[0197] In another aspect of the present invention, the first binder has the ability to induce the activation of human CD27-expressing Jurkat reporter T cells, such as those described in Example 2 herein.

[0198] In another aspect of the present invention, the first binder has the ability to induce the activation of human CD27-expressing Jurkat reporter T cells in the absence of Fcγ receptor IIb cross-linking, such as those described in Example 11 herein.

[0199] In another aspect of the present invention, the first binder has the ability to induce the proliferation of CD4+ and CD8 + T cells having a central memory T cell phenotype.

[0200] In another aspect of the present invention, the first binder has the ability to induce the production of IFN gamma.

[0201] In another aspect of the present invention, the first binder is in a composition or formulation containing acetate, sorbitol, polysorbate 80 and has a pH of 5 to 6, preferably 5.5.

[0202] A second binder that binds to CD40 and CD137 In one embodiment, CD40 is human CD40, particularly human CD40 comprising the sequence shown in SEQ ID NO:62. In one embodiment, CD137 is human CD137, particularly human CD137 comprising the sequence shown in SEQ ID NO:63.

[0203] In one embodiment of the second binding agent, the second binding agent comprises a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0204] In one embodiment of the second binding agent, a) the first binding region of the second binding agent comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 44, 45, and 46, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 47, YTS, and SEQ ID NO: 48, respectively; and b) the second binding region of the second binding agent comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 51, 52, and 53, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 54, GAS, and SEQ ID NO: 55, respectively.

[0205] In one embodiment of the second binding agent, a) the first binding region of the second binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:49 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO:50; and b) the second binding region of the second binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:56 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO:57.

[0206] In one embodiment of the second binding agent, the second binding agent is a multispecific antibody, such as a bispecific antibody.

[0207] In one embodiment of the second binder, the second binder is in the form of a full-length antibody or an antibody fragment.

[0208] In one embodiment of the second binder, the second binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL) and the second binding arm comprises and the second binding arm comprises iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL) In one embodiment of the second binder, the second binder is

[0209] i) a first heavy chain and light chain comprising the first binding region having the ability to bind to CD40, the first heavy chain comprising a first heavy chain constant region, and the first light chain comprising a first light chain constant region; and ii) a second heavy chain and light chain comprising the second binding region having the ability to bind to CD137, the second heavy chain comprising a second heavy chain constant region, and the second light chain comprising a second light chain constant region In one embodiment of the second binder, the second binder is In one embodiment of the second binder, the second binder is

[0210] In one embodiment of the second binder, (i) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

[0211] In one embodiment of the second binder, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, in the first and second heavy chains.

[0212] In one embodiment of the second binder, the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in the first and second heavy chain constant regions (HC).

[0213] In one embodiment of the second binder, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R, and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0214] In one embodiment of the second binder, the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain is R, and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L.

[0215] In one embodiment of the second binder, the first and / or second heavy chains, such as the constant region of the second heavy chain, a) the sequence [IgG1-Fc_FEAL] shown in SEQ ID NO:58 or 60; b) a subsequence of the sequence in a), for example, a subsequence starting from the N-terminus or C-terminus of the sequence defined in a) and having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids deleted; and c) a sequence having up to 6 substitutions, such as 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). comprises an amino acid sequence selected from the group consisting of, consists essentially of, or consists of the amino acid sequence.

[0216] In one embodiment of the second binder, the first and / or second heavy chains, such as the constant region of the first heavy chain, a) the sequence [IgG1-Fc_FEAR] shown in SEQ ID NO:59 or 61; b) a subsequence of the sequence in a), for example, a subsequence starting from the N-terminus or C-terminus of the sequence defined in a) and having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids deleted; and c) an amino acid sequence having up to 6 substitutions, such as up to 5 substitutions, up to 4, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in a) or b) comprising an amino acid sequence selected from the group consisting of, consisting essentially of, or consisting of the amino acid sequence

[0217] In one embodiment of the second binder, the second binder comprises a kappa (κ) light chain constant region.

[0218] In one embodiment of the second binder, the second binder comprises a lambda (λ) light chain constant region.

[0219] In one embodiment of the second binder, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0220] In one embodiment of the second binder, the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0221] In one embodiment of the second binder, the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region, or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0222] In one embodiment of the second binder, the kappa (κ) light chain is a) the sequence shown in SEQ ID NO:16 b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) an amino acid sequence having up to 10 substitutions, such as up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in a) or b) comprising an amino acid sequence selected from the group consisting of

[0223] In one embodiment of the second binder according to the first aspect, the lambda (λ) light chain is a) the sequence shown in SEQ ID NO:17 b) a subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) an amino acid sequence having up to 10 substitutions, such as up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution, compared to the amino acid sequence defined in a) or b) comprising an amino acid sequence selected from the group consisting of

[0224] In one embodiment of the second binder according to the first aspect, the second binder is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4

[0225] In one embodiment of the second binder according to the first aspect, the second binder is a full-length IgG1 antibody

[0226] In one embodiment of the second binder according to the first aspect, the second binder is an antibody of the IgG1m(f) allotype

[0227] In one aspect, the second binder is a bispecific antibody that binds to CD40 and CD137, and the bispecific antibody has: i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 64 and a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 65, and ii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 66 and a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 67.

[0228] A subject and a tumor or cancer to be treated The subject to be treated in accordance with the present disclosure is preferably a human subject.

[0229] In one aspect, the tumor or cancer is a solid tumor.

[0230] In one aspect, the tumor is a PD-L1 positive tumor.

[0231] In one aspect, the tumor or cancer is head and neck squamous cell carcinoma (HNSCC), such as HNSCC of the oral cavity, pharynx or larynx.

[0232] In one aspect, the HNSCC is recurrent, unresectable or metastatic.

[0233] In one aspect, the tumor or cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

[0234] In one aspect, the NSCLC is recurrent, unresectable or metastatic.

[0235] In one aspect, the NSCLC does not have an epidermal growth factor (EGFR) sensitive mutation and / or an anaplastic lymphoma kinase (ALK) translocation and / or a ROS1 rearrangement.

[0236] In one aspect, NSCLC is NTRK1 / 2 / 3 (neurotrophic tyrosine kinase receptor 1 / 2 / 3) fusion positive and / or has a mutation in the KRAS (KRAS proto-oncogene, GTPase), BRAF (B-Raf proto-oncogene, serine / threonine kinase), or MET (MET proto-oncogene, receptor tyrosine kinase) gene and / or has a RET (ret proto-oncogene) gene rearrangement, and the subject has received prior treatment with each respective targeted therapy.

[0237] In one aspect, the subject has received prior treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, preferably at least 2 doses of a PD-1 inhibitor or a PD-L1 inhibitor.

[0238] In one aspect, the subject has received prior treatment with a platinum-based therapy or, if platinum is ineligible, an alternative chemotherapy, such as a gemcitabine-containing regimen.

[0239] In one aspect, the tumor or cancer has recurred and / or progressed after treatment, such as systemic treatment with a checkpoint inhibitor.

[0240] In one aspect, the subject has received at least one prior line of systemic therapy, such as a systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0241] In one aspect, the cancer or tumor has recurred and / or is refractory, or the subject has progressed after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

[0242] In one aspect, the previous last treatment was with a PD1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of combination therapy.

[0243] In one aspect, the time from progression in the last treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, is 6 months or less.

[0244] In one aspect, the time from the last dosing of a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, as part of the previous last treatment is 6 months or less.

[0245] In one aspect, the cancer or tumor is recurrent and / or refractory, or the subject has progressed during or after i) platinum doublet chemotherapy after treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody, or ii) treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody after platinum doublet chemotherapy.

[0246] In a second aspect, the present disclosure provides a kit comprising i) a first binder comprising at least one binding region that binds to CD27 and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0247] In one aspect of the kit according to the second aspect, the first binder is as defined in any aspect or embodiment of the present disclosure.

[0248] In one aspect of the kit according to the second aspect, the second binder is as defined in any aspect or embodiment of the present disclosure.

[0249] In one aspect of the kit according to the second aspect, the first binder, the second binder, and, if present, one or more additional therapeutic agents are for systemic administration, particularly for injection or infusion, such as intravenous injection or infusion.

[0250] In a third aspect, the present disclosure provides a kit for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the kit comprising: i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0251] In one aspect of the kit for use according to the third aspect, the kit is as defined in any aspect or embodiment of the present disclosure.

[0252] In one aspect of the kit for use according to the third aspect, the tumor or cancer is as defined in any aspect or embodiment of the present disclosure.

[0253] In one aspect of the kit for use according to the third aspect, the subject is as defined in any aspect or embodiment of the present disclosure.

[0254] In one aspect of the kit for use according to the third aspect, the method is as defined in any aspect or embodiment of the present disclosure.

[0255] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising: i) a first binder comprising at least one binding region that binds to CD27; ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137; and iii) optionally, a pharmaceutically acceptable carrier.

[0256] In one aspect of the pharmaceutical composition according to the fourth aspect, the first binder is as defined in any aspect or embodiment of the present disclosure.

[0257] In one embodiment of the pharmaceutical composition according to the fourth aspect, the second binder is as defined in any aspect or embodiment of the present disclosure.

[0258] In a fifth aspect, the present disclosure provides a pharmaceutical composition for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the pharmaceutical composition comprising: i) a first binder comprising at least one binding region that binds to CD27; ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137; and iii) optionally, a pharmaceutically acceptable carrier.

[0259] In one embodiment of the pharmaceutical composition for use according to the fifth aspect, the pharmaceutical composition is as defined in any aspect or embodiment of the present disclosure.

[0260] In one embodiment of the pharmaceutical composition for use according to the fifth aspect, the tumor or cancer is as defined in any aspect or embodiment of the present disclosure.

[0261] In one embodiment of the pharmaceutical composition for use according to the fifth aspect, the subject is as defined in any aspect or embodiment of the present disclosure.

[0262] In one embodiment of the pharmaceutical composition for use according to the fifth aspect, the method is as defined in any aspect or embodiment of the present disclosure.

[0263] In a sixth aspect, the present disclosure provides a first binder for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising the steps of administering to the subject: i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0264] In one embodiment of the first binder for use according to the sixth aspect, the method is as defined in any aspect or embodiment of the present disclosure.

[0265] In one embodiment of the first binder for use according to the sixth aspect, the first binder is as defined in any aspect or embodiment of the present disclosure.

[0266] In one embodiment of the first binder for use according to the sixth aspect, the second binder is as defined in any aspect or embodiment of the present disclosure.

[0267] In the seventh aspect, the present disclosure provides a second binder for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject: i) a first binder comprising at least one binding region that binds to CD27; and ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0268] In one embodiment of the second binder for use according to the seventh aspect, the method is as defined in any aspect or embodiment of the present disclosure.

[0269] In one embodiment of the second binder for use according to the seventh aspect, the first binder is as defined in any aspect or embodiment of the present disclosure.

[0270] In one embodiment of the second binder for use according to the seventh aspect, the second binder is as defined in any aspect or embodiment of the present disclosure.

[0271] References to documents and studies cited herein are not intended as an admission that any of the above are relevant prior art. All statements regarding the content of these documents are based on information available to the applicant and do not constitute any admission as to the accuracy of the content of these documents.

[0272] The descriptions (including the examples below) are presented to enable one of ordinary skill in the art to make and use the various aspects. The descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to one of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various aspects. Accordingly, the various aspects are not intended to be limited to the examples described and shown herein and are commensurate with the scope consistent with the claims.

[0273] Items of the present disclosure 1. (i) A first binder comprising at least one binding region that binds to CD27; and (ii) A second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 A method for reducing or preventing tumor progression or treating cancer in a subject, comprising the step of administering to the subject.

[0274] 2. The method of item 1, wherein the first binder comprises a heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0275] 3. The method according to item 1 or 2, wherein the first binder comprises two binding regions having the ability to bind to human CD27, and the first binder comprises a heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0276] 4. The method according to any of the preceding items, wherein the first binder comprises a VH region comprising the sequence shown in SEQ ID NO: 4.

[0277] 5. The method according to any of the preceding items, wherein the first binder comprises a VL region comprising the sequence shown in SEQ ID NO: 8.

[0278] 6. The method according to any of the preceding items, wherein the first binder comprises a VH region and a VL region comprising the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

[0279] 7. The method according to any of the preceding items, wherein the first binder is an antibody, preferably a human antibody or a humanized antibody.

[0280] 8. The method according to any of the preceding items, wherein the antibody is a full-length antibody further comprising a light chain constant region (CL) and a heavy chain constant region (CH).

[0281] 9. The method according to item 8, wherein the light chain constant region is human kappa.

[0282] 10. The method according to item 8, wherein the light chain constant region is human lambda.

[0283] 11. The method according to any of the preceding items, wherein the first binder further comprises a heavy chain constant region, and the heavy chain constant region is a human IgG isotype and optionally a modified human IgG.

[0284] 12. The method of item 11, wherein the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4, such as human IgG1.

[0285] 13. The method of item 11 or 12, wherein the IgG is a modified human IgG comprising one or more amino acid substitutions.

[0286] 14. The method according to any one of items 11 to 13, wherein the modified human IgG is a modified human IgG1 comprising one or more amino acid substitutions, such as two or more amino acid substitutions.

[0287] 15. The method according to any one of items 11 to 14, wherein the modified human IgG heavy chain constant region comprises up to 10 amino acid substitutions, such as up to 9, such as up to 8, such as up to 7, such as up to 6, such as up to 5, such as up to 4, such as up to 3, such as up to 2 amino acid substitutions.

[0288] 16. The method according to any one of items 11 to 15, wherein the substitution in the heavy chain constant region induces an increase in CD27 agonism compared to the same antibody except that it comprises the wild-type IgG1 antibody heavy chain constant region.

[0289] 17. The method according to any one of items 11 to 16, wherein the amino acid residue at the position corresponding to position E345 or E430 in the human IgG1 heavy chain according to Eu numbering is selected from the group comprising A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

[0290] 18. The method according to any one of items 11 to 17, wherein the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to Eu numbering is R.

[0291] 19. The method according to any one of items 11 to 18, wherein the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain according to Eu numbering is G.

[0292] 20. A method according to any one of items 11 to 19, wherein the amino acid residue at the position corresponding to position P329 in the human IgG1 heavy chain according to Eu numbering is R.

[0293] 21. A method according to any one of items 11 to 20, wherein the amino acid residues at the positions corresponding to positions E345 and P329 in the human IgG1 heavy chain according to Eu numbering are both R.

[0294] 22. A method according to any one of items 11 to 21, wherein the first binder has a pharmacokinetic profile similar to that of a parental antibody comprising a wild-type IgG1 heavy chain constant region.

[0295] 23. A method according to any one of the preceding items, wherein the first binder comprises a heavy chain constant region comprising a sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and 36.

[0296] 24. A method according to any one of the preceding items, wherein the first binder comprises a heavy chain constant region comprising the sequence shown in SEQ ID NO: 15.

[0297] 25. A method according to any one of the preceding items, wherein the first binder comprises a heavy chain constant region, and the heavy chain constant region is modified such that the first binder induces one or more Fc-mediated effector functions to a lower extent compared to the parental antibody.

[0298] 26. The method of item 25, wherein the one or more Fc-mediated effector functions are reduced by at least 20%, such as at least 30% or at least 40%, or at least 50% or at least 60% or at least 70%, or at least 80% or at least 90%.

[0299] 27. The method of item 25 or 26, wherein the first binder does not induce one or more Fc-mediated effector functions.

[0300] 28. The one or more Fc-mediated effector functions are from the following group: complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding The method according to any one of items 25 to 27, selected from the above.

[0301] 29. The method according to any one of items 25 to 28, wherein the first binder does not induce C1q binding when measured by the method of Example 8.

[0302] 30. The method according to any one of the above items, wherein the first binder is a monovalent antibody.

[0303] 31. The method according to any one of the above items, wherein the first binder is a bivalent antibody.

[0304] 32. The method according to any one of the above items, wherein the first binder is a monospecific antibody.

[0305] 33. The method according to any one of the above items, wherein the first binder is a bispecific antibody comprising a first antigen-binding region having the ability to bind to human CD27 according to any one of the above items and a second antigen-binding region having the ability to bind to a different epitope on human CD27 or having the ability to bind to a different target.

[0306] 34. CD27 is human CD27, and in particular, the human CD27 comprises the sequence shown in SEQ ID NO:1 or the human CD27 variant shown in SEQ ID NO:2. The method according to any one of the above items.

[0307] 35. The first binder is a. a VH region comprising the amino acid sequence shown in SEQ ID NO:4; b. a VL region comprising the amino acid sequence shown in SEQ ID NO:8; c. A CH region comprising the amino acid sequence shown in SEQ ID NO:15; and d. A CL region comprising the amino acid sequence shown in SEQ ID NO:17 The method according to any one of the preceding items, comprising:

[0308] 36. The method according to any one of the preceding items, wherein the first binder comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:35 and a light chain comprising the amino acid sequence shown in SEQ ID NO:25.

[0309] 37. The method according to any one of the preceding items, wherein the first binder is in a composition or formulation comprising acetate, sorbitol, polysorbate 80 and has a pH of 5 to 6, preferably 5.5.

[0310] 38. The method according to any one of the preceding items, wherein CD40 is human CD40, particularly human CD40 comprising the sequence shown in SEQ ID NO:62, and / or CD137 is human CD137, particularly human CD137 comprising the sequence shown in SEQ ID NO:63.

[0311] 39. (a) The first binding region of the second binder comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:44, 45, and 46, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:47, YTS, and SEQ ID NO:48, respectively; and (b) the second binding region of the second binder comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:51, 52, and 53, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:54, GAS, and SEQ ID NO:55, respectively. The method according to any one of the preceding items.

[0312] 40. (a) The first binding region of the second binder comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 49 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO: 50; and (b) The second binding region of the second binder comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO: 57, The method according to any of the preceding items.

[0313] 41. The method according to any of the preceding items, wherein the second binder is a multispecific antibody, such as a bispecific antibody.

[0314] 42. The method according to any of the preceding items, wherein the second binder is in the form of a full-length antibody or an antibody fragment.

[0315] 43. The second binder is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL) and; and the second binding arm comprises (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL) and, The method according to any of the preceding items.

[0316] 44. The second binder is (i) A first heavy chain and light chain comprising the first binding region having the ability to bind to CD40, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and (ii) A second heavy chain and light chain comprising the second binding region having the ability to bind to CD137, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region The method according to any of the preceding items, comprising

[0317] 45. The method of item 43 or 44, wherein (i) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

[0318] 46. The method according to any of items 43 to 45, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, in the first and second heavy chains.

[0319] 47. The method according to any of items 43 to 46, wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in the first and second heavy chain constant regions (HC).

[0320] 48. The method according to any one of items 43 to 47, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU numbering of both the first and second heavy chain constant regions are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L.

[0321] 49. The method according to any one of items 43 to 48, wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain is R, and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L.

[0322] 50. The constant region of the first and / or second heavy chain, for example the second heavy chain, is (a) the sequence shown in SEQ ID NO:58 or 60 [IgG1-Fc_FEAL]; (b) a subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) a sequence having up to 6 substitutions, for example 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). A method according to any one of items 43 to 49, comprising an amino acid sequence selected from the group consisting of, consisting essentially of, or consisting of said amino acid sequence.

[0323] 51. The constant region of the first and / or second heavy chain, for example the first heavy chain, is (a) The sequence shown in SEQ ID NO:59 or 61 [IgG1-Fc_FEAR]; (b) A subsequence of the sequence in (a), for example, starting from the N-terminus or C-terminus of the sequence defined in (a), a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted; and (c) A sequence having up to 6 substitutions, for example 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) A method according to any one of items 43 to 50, comprising an amino acid sequence selected from the group consisting of, consisting essentially of, or consisting of said amino acid sequence.

[0324] 52. A method according to any one of items 43 to 51, wherein the second binder comprises a kappa (κ) light chain constant region.

[0325] 53. A method according to any one of items 43 to 52, wherein the second binder comprises a lambda (λ) light chain constant region.

[0326] 54. A method according to any one of items 43 to 53, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0327] 55. A method according to any one of items 43 to 54, wherein the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0328] 56. The method according to any one of items 43 to 55, wherein the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region, or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0329] 57. The kappa (κ) light chain is (a) the sequence shown in SEQ ID NO: 16, (b) a subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) a sequence having up to 10 substitutions, for example up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in (a) or (b) The method according to any one of items 52 to 56, comprising an amino acid sequence selected from the group consisting of

[0330] 58. The lambda (λ) light chain is (a) the sequence shown in SEQ ID NO: 17, (b) a subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) a sequence having up to 10 substitutions, for example up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in (a) or (b) The method according to any one of items 53 to 57, comprising an amino acid sequence selected from the group consisting of

[0331] 59. The method according to any of the preceding items, wherein the second binder is an isotype binder selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0332] 60. The method according to any of the preceding items, wherein the second binder is a full-length IgG1 antibody.

[0333] 61. The method according to any of the preceding items, wherein the second binder is an antibody of the IgG1m(f) allotype.

[0334] 62. The method according to any of the preceding items, wherein the second binder is a bispecific antibody that binds to CD40 and CD137, and the bispecific antibody has (i) a first heavy chain comprising the amino acid sequence shown in SEQ ID NO: 64 and a first light chain comprising the amino acid sequence shown in SEQ ID NO: 65, and (ii) a second heavy chain comprising the amino acid sequence shown in SEQ ID NO: 66 and a second light chain comprising the amino acid sequence shown in SEQ ID NO: 67.

[0335] 63. (a) The first binder comprises a heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively; (b) The first binding region of the second binder comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 44, 45, and 46, respectively, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 47, YTS, and SEQ ID NO: 48, respectively; and (c) The second binding region of the second binder comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 51, 52, and 53, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 54, GAS, and SEQ ID NO: 55, respectively. The method according to any of the preceding items.

[0336] 64. (a) The first binder comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 4 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 8; (b) The first binding region of the second binder comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 49 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO: 50; and (c) The second binding region of the second binder comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO: 57. The method according to any of the preceding items.

[0337] 65. (a) The first binder is an antibody comprising a VH region comprising the amino acid sequence shown in SEQ ID NO: 4, a VL region comprising the amino acid sequence shown in SEQ ID NO: 8, a CH region comprising the amino acid sequence shown in SEQ ID NO: 15, and a CL region comprising the amino acid sequence shown in SEQ ID NO: 17; (b) The second binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising the first binding region and the second binding arm comprising the second binding region; (c) The first binding arm of the second binder comprises a VH region comprising the amino acid sequence shown in SEQ ID NO:49, a VL region comprising the amino acid sequence shown in SEQ ID NO:50; a CH region comprising the amino acid sequence shown in SEQ ID NO:60, and a CL region comprising the amino acid sequence shown in SEQ ID NO:16; and (d) The second binding arm of the second binder comprises a VH region comprising the amino acid sequence shown in SEQ ID NO:56, a VL region comprising the amino acid sequence shown in SEQ ID NO:57, a CH region comprising the amino acid sequence shown in SEQ ID NO:61, and a CL region comprising the amino acid sequence shown in SEQ ID NO:16, The method according to any of the preceding items.

[0338] 66. (a) The first binder comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:35 and a light chain comprising the amino acid sequence shown in SEQ ID NO:25; (b) The second binder is a bispecific antibody that binds to CD40 and CD137, and the bispecific antibody has (i) a first heavy chain comprising the amino acid sequence shown in SEQ ID NO:64 and a first light chain comprising the amino acid sequence shown in SEQ ID NO:65, and (ii) a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:66 and a second light chain comprising the amino acid sequence shown in SEQ ID NO:67, The method according to any of the preceding items.

[0339] 67. The method according to any of the preceding items, wherein the subject is a human subject.

[0340] 68. The method according to any of the preceding items, wherein the tumor or cancer is a solid tumor.

[0341] 69. The method according to any of the preceding items, wherein the tumor is a PD-L1 positive tumor.

[0342] 70. The method according to any of the preceding items, wherein the tumor or cancer is head and neck squamous cell carcinoma (HNSCC), such as HNSCC of the oral cavity, pharynx or larynx.

[0343] 71. The method according to item 70, wherein the HNSCC is recurrent, unresectable or metastatic.

[0344] 72. The method according to any of items 1 to 69, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

[0345] 73. The method according to item 72, wherein the NSCLC is recurrent, unresectable or metastatic.

[0346] 74. The method according to item 72 or 73, wherein the NSCLC does not have an epidermal growth factor (EGFR) - sensitive mutation and / or anaplastic lymphoma kinase (ALK) translocation and / or ROS1 rearrangement.

[0347] 75. The method according to any of items 72 to 74, wherein the NSCLC is NTRK1 / 2 / 3 (neurotrophic tyrosine kinase receptor 1 / 2 / 3) fusion positive and / or has a mutation in the KRAS (KRAS proto-oncogene, GTPase), BRAF (B-Raf proto-oncogene, serine / threonine kinase), or MET (MET proto-oncogene, receptor tyrosine kinase) gene and / or has a RET (ret proto-oncogene) gene rearrangement, and the subject has received previous treatment with each respective targeted therapy.

[0348] 76. The method according to any of the preceding items, wherein the subject has received previous treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, preferably at least 2 doses of a PD-1 inhibitor or a PD-L1 inhibitor.

[0349] 77. The method of any of the preceding items, wherein the subject has received prior treatment with a platinum-based therapy or, if platinum is ineligible, an alternative chemotherapy, such as a previous treatment with a gemcitabine-containing regimen.

[0350] 78. The method of any of the preceding items, wherein the tumor or cancer has recurred and / or progressed after treatment, such as systemic treatment with a checkpoint inhibitor.

[0351] 79. The method of any of the preceding items, wherein the subject has received at least one previous line of systemic therapy, such as a systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0352] 80. The method of any of the preceding items, wherein the cancer or tumor has recurred and / or is refractory, or the subject has progressed after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

[0353] 81. The method of any of the preceding items, wherein the previous last treatment was with a PD1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

[0354] 82. The method of any of the preceding items, wherein the time from progression in the last treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, is 6 months or less.

[0355] 83. The method of any of the preceding items, wherein the time from the last dose of a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, as part of the previous last treatment is 6 months or less.

[0356] 84. The cancer or tumor has recurred and / or is refractory, or the subject has (i) Treatment with platinum doublet chemotherapy after treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody, or (ii) Treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody after platinum doublet chemotherapy Any method of the above items that progresses during or after.

[0357] 85. (i) A first binder comprising at least one binding region that binds to CD27, and (ii) A second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 A kit comprising.

[0358] 86. The kit of item 85, wherein the first binder is as defined in any one of items 1 to 84, and / or the second binder is as defined in any one of items 1 to 84.

[0359] 87. The kit of item 85 or 86, wherein the first binder, the second binder, and, if present, one or more additional therapeutic agents are for systemic administration, particularly for injection or infusion, such as for intravenous injection or infusion.

[0360] 88. A kit according to any of items 85 to 87 for use in a method for reducing or preventing tumor progression or treating cancer in a subject.

[0361] 89. The kit for use according to item 88, wherein the tumor or cancer is as defined in any one of items 1 to 84, and / or the subject is as defined in any one of items 1 to 84, and / or the method is as defined in any one of items 1 to 84.

[0362] 90. (i) A first binder comprising at least one binding region that binds to CD27; (ii) A second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137; and (iii) Optionally, a pharmaceutically acceptable carrier A pharmaceutical composition comprising.

[0363] 91. The pharmaceutical composition of item 90, wherein the first binder is as defined in any one of items 1 to 84 and / or the second binder is as defined in any one of items 1 to 84.

[0364] 92. The pharmaceutical composition of item 90 or 91 for use in a method for reducing or preventing tumor progression or treating cancer in a subject.

[0365] 93. The pharmaceutical composition for use of item 92, wherein the tumor or cancer is as defined in any one of items 1 to 84 and / or the subject is as defined in any one of items 1 to 84 and / or the method is as defined in any one of items 1 to 84.

[0366] 94. A first binder for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising (i) The first binder comprising at least one binding region that binds to CD27; and (ii) A second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 The first binder for said use, comprising the step of administering to the subject.

[0367] 95. The first binder for use of item 94, wherein the method is as defined in any one of items 1 to 84 and / or the first binder is as defined in any one of items 1 to 84 and / or the second binder is as defined in any one of items 1 to 84.

[0368] 96. A second binder for use in a method for reducing or preventing tumor progression or treating cancer in a subject, said method comprising: (i) a first binder comprising at least one binding region that binds to CD27; and (ii) said second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 administering to the subject. A second binder for said use.

[0369] 97. The second binder for use according to item 96, wherein said method is as defined in any one of items 1 to 84, and / or said first binder is as defined in any one of items 1 to 84, and / or said second binder is as defined in any one of items 1 to 84.

[0370] A further aspect of the present disclosure is disclosed herein.

Examples

[0371] Example 1: Generation of DuoBody-CD40x4-1BB and anti-human CD27 antibodies and their Fc variants The generation of anti-human CD27 antibodies through immunization and hybridoma production was performed at Aldevron GmbH (Freiburg, Germany). The cDNA encoding human CD27 (full length and ECD) was cloned into Aldevron's proprietary expression plasmid. Immunization of OmniRat animals (transgenic rats expressing a diversified repertoire of antibodies with fully human idiotypes; Ligand Pharmaceuticals Inc.) was performed using intradermal application of human CD27 cDNA-coated gold particles using a portable device for particle bombardment ("gene gun") to generate anti-CD27 antibodies. Serum samples were collected after a series of immunizations and tested by flow cytometry in HEK cells transiently transfected with the above-described expression plasmid for full-length human CD27 expression. Antibody-producing cells were isolated from rat spleens and fused with mouse myeloma cells (Ag8) according to standard procedures. RNA from hybridomas producing CD27-specific antibodies was extracted for sequencing.

[0372] From a pool of 71 CD27 antibodies, six antibodies were selected for further characterization based on their binding to primary T cells and diversity in an in vitro CD27 binding competition assay. These six antibodies are named herein IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E and IgG1-CD27-F. Variable regions with single-point mutations that may remove amino acid residues considered disadvantageous for the production of the heavy and light chains of interest (e.g., free cysteines or glycosylation sites) were gene synthesized and cloned into expression vectors containing the framework sequences for the human antibody light chain and the human IgG1 heavy chain.

[0373] Fc variants of six different antibodies were generated by introduction of one or more of the following amino acid mutations according to the EU numbering: E345R, E430G, P329R, G237A, K326A, E333A (see Tables 1 and 3 below). After the in vitro functional characterization described below, CD27-specific IgG1-CD27-A (VH SEQ ID NO: 4; VL SEQ ID NO: 8) was considered to have the most optimal biological properties. The sequences of prior art CD27-targeting antibodies used herein as benchmarks were obtained as follows: IgG1-CD27-15 (WO2012004367; SEQ ID NO:3 and 4), IgG1-CD27-131A (WO2018 / 058022; SEQ ID NO:10 and 15), IgG1-CD27-CDX1127 (WO2016145085; SEQ ID NO:1 and 2), and IgG1-CD27-BMS986215 (WO2019195452A1; SEQ ID NO:8 and 9). The VH and VL sequences of type I anti-human CD20 antibodies were previously described in WO2019 / 145455A1 (SEQ ID NO:35 and 39).

[0374] DuoBody-CD40x4-1BB is a bispecific antibody based on the DuoBody technology platform (WO2011131746A2) that binds to CD40 with one arm and to 4-1BB with the other arm. DuoBody-CD40x4-1BB was generated using the parental clones IgG1-CD40-001 (HC SEQ ID NO:49; LC SEQ ID NO:50; HCDR1 SEQ ID NO:44, HCDR2 SEQ ID NO:45, HCDR3 SEQ ID NO:46, LCDR1 SEQ ID NO:47, LCDR2:YTS, LCDR3 SEQ ID NO:48) and IgG1-CD137-009 (HC SEQ ID NO:56; LC SEQ ID NO:57; HCDR1 SEQ ID NO:51, HCDR2 SEQ ID NO:52, HCDR3 SEQ ID NO:53, LCDR1 SEQ ID NO:54, LCDR2:GAS, LCDR3 SEQ ID NO:55). As a control antibody, the anti-HIV gp120 antibody IgG1-b12 was used in this application (Barbas et al., J Mol Biol 1993 230:812-823; VH: SEQ ID NO 37 of this application, VL: SEQ ID NO 41).

[0375] (Table 1) List of amino acid sequences TIFF2025516633000004.tif83159TIFF2025516633000005.tif222159TIFF2025516633000006.tif225159TIFF2025516633000007.tif221159TIFF2025516633000008.tif227159TIFF2025516633000009.tif227159TIFF2025516633000010.tif225159TIFF2025516633000011.tif225159TIFF2025516633000012.tif242159TIFF2025516633000013.tif31159

[0376] Example 2: Agonist Activity of Anti-CD27 Antibodies in a CD27 Activation Reporter Cell Assay The CD27 agonist activities of different anti-CD27 antibodies with and without the E345R or E430G hexamerization-enhancing Fc mutations were measured using the CD27 Thaw and Use Bioassay kit (Promega, Custom Assay Services, CAS# CS1979A25). The kit contains NF-κB reporter-Jurkat recombinant cells that express the firefly luciferase gene under the control of an NF-κB response element along with constitutive expression of human CD27, and this was used essentially according to the manufacturer's instructions. Briefly, Thaw-and-Use GloResponse NFκB-luc2 / CD27 cells were thawed and incubated with antibody dilution series (final concentration range 0.04 - 20 μg / mL) in Bio-Glo Luciferase Assay Buffer in a 96-well flat-bottom culture plate (PerkinElmer, catalog #6005680) at 37 °C, 5% CO 2 for 6 h. The anti-CD27 antibodies were wild-type (WT *)They were each of the variants harboring the IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E, IgG1-CD27-F, and E430G or E345R mutations. The anti-CD27 benchmark antibodies were IgG1-CD27-131A (WT and E430G variants) and non-hexamerizing IgG1-CD27-15 (IgG1-CD27-15-P329R-E345R-K439E; having a combination of Fc mutations that prevent hexamerization and thus the mutations are not functionally relevant in the context of this experiment and are thus designated as WT in the figure) and the hexamerizing variant of IgG1-CD27-15 containing the E345R mutation. The anti-HIV gp120 human antibody, IgG1-b12-E345R, was used as an unbound negative control antibody (control). After antibody incubation, Bio-Glo Luciferase Assay Reagent (equilibrated to RT) was added to each well and incubated at RT for 5 - 10 minutes. Luminescence was measured using an EnVision Multilabel Reader (PerkinElmer) and presented as relative light units (RLU) in bar charts generated using GraphPad Prism software.

[0377] The introduction of the hexamerization-enhancing Fc mutations (E345R or E430G) resulted in enhanced CD27 agonism for the antibody clones IgG1-CD27-A to -E as well as for the benchmark antibodies IgG1-CD27-131A (tested with E430G) and IgG1-CD27-15 (tested with E345R) compared to the corresponding WT antibodies (Figure 1).

[0378] IgG-CD27-A, B, and C demonstrated enhanced CD27 agonist activity after the introduction of E430G or E345R at all tested concentrations, while IgG1-CD27-D and E variants containing hexamerization-enhancing mutations did not show an increase in agonism at the lowest antibody concentrations. The IgG1-CD27-F variant with the E430G or E345R mutation showed enhanced CD27 agonism only at the highest antibody concentration tested. For variants IgG1-CD27-A to -E, the introduction of the E345R mutation resulted in stronger CD27 activation than the E430G mutation. Antibodies IgG1-CD27-A to -E with the E345R mutation showed higher or similar CD27 activation levels compared to IgG1-CD27-131A with the E430G mutation or CD27-15 with the E345R mutation, respectively. * The WT antibodies for IgG1-CD27-B and IgG1-CD27-F had the F405L mutation in the IgG Fc domain that was not functionally relevant in the context of this experiment.

[0379] Example 3: Binding Affinity of Anti-Human CD27 Antibodies to Recombinant Human, Mouse, and Cynomolgus CD27 The binding affinities of five anti-human CD27 IgG1 antibodies (IgG1-CD27-A, -B, -C, -D, and -E) to recombinant human, cynomolgus, and mouse CD27 proteins were determined using label-free biolayer interferometry on an Octet HTX instrument (ForteBio, Portsmouth, UK). The experiments were performed using bispecific antibodies containing one CD27-specific Fab arm and a non-binding Fab arm such that the antibody was monovalent for CD27. These bispecific antibodies were generated by controlled Fab arm exchange between the CD27 antibody and a non-binding antibody (as described in Labrijn AF et al., Nat Protoc. 2014 Oct;9(10):2450-63).

[0380] To determine the affinity of CD27 antibodies for human and mouse CD27, 100 nM of recombinant His-tagged mouse or human CD27 protein (Sino Biological, Catalog #10039-H08B1 [human], Catalog #50110-M08H [mouse]) was loaded onto a pre-conditioned anti-Penta-HIS (HIS1K) biosensor (ForteBio, Catalog #18-5120) for 600 seconds.

[0381] To evaluate the affinity of CD27 antibodies for cynomolgus monkey CD27, 5 μg / mL of recombinant cynomolgus monkey CD27-Fc fusion protein (R&D systems, Catalog #9904-CD-100) was loaded onto an activated Amine Reactive 2nd Generation (AR2G) biosensor (ForteBio, Catalog #18-5092).

[0382] After a 300-second baseline measurement in Sample Diluent (ForteBio, Catalog #18-1104), the association (200 seconds) and dissociation (1,000 seconds) of the CD27 antibodies were determined for an antibody concentration series of 0.78 - 800 nM with 2-fold dilution steps in Sample Diluent. An antibody molecular mass of 150 kDa was used for the calculations. The reference sensor was incubated with Sample Diluent.

[0383] Data was acquired using Data Acquisition Software v11.1.1.19 (ForteBio) and analyzed using Data Analysis Software v9.0.0.14 (ForteBio). Data traces were corrected for each antibody by subtraction of the reference sensor. The Y-axis was aligned to the last 10 seconds of the baseline, and Interstep Correction alignment and Savitzky-Golay filtering were applied for dissociation. Data traces were excluded from the analysis if the response was <0.05 nm and the calculated equilibrium was near saturation (Req / Rmax > 95% using a 50-second dissociation time). Data was fit to a 1:1 model using the window of interest for association set at 200 seconds and dissociation time set at 50 seconds. The dissociation time was selected based on the coefficient of determination (R 2 )(preferably >0.98), visual inspection of the curve, and at least 5% signal decay during the association step.

[0384] The affinity for human CD27 could be accurately determined for three CD27 antibodies (IgG1-CD27-A, -B, -C) with K D values in the nanomolar concentration range (Table 2). For IgG1-CD27-D and -E, binding to human CD27 with a similar range of affinity was confirmed by biolayer interferometry experiments, but accurate K D value calculation was not possible due to suboptimal curve fitting (shown in Table 2).

[0385] IgG1-CD27-A and -B also showed binding to recombinant cynomolgus CD27 with K D values in the same range as human CD27. Results obtained with IgG1-CD27-C, -D, and -E also confirmed binding to cynomolgus CD27 with a similar range of affinity, but accurate K D value calculation was not possible due to suboptimal curve fitting (shown in Table 2).

[0386] Binding to recombinant mouse CD27 was observed only for antibody IgG1-CD27-C.

[0387] (Table 2) Binding affinities of IgG1-CD27-A to -E antibodies for CD27 from the species indicated TIFF2025516633000014.tif72166 * : Binding was observed, but KD, k on and k dis are of low reliability values due to suboptimal curve fitting that results in uninterpretable results using a 1:1 model. n.b.: Binding was not observed.

[0388] Example 4: Binding of anti-CD27 antibodies to human and cynomolgus monkey CD27 expressed on the cell surface Anti-CD27 antibodies IgG1-CD27-A to -E against human and cynomolgus monkey CD27 expressed on the cell surface * and the prior art IgG1-CD27-131A * were analyzed by flow cytometry using transiently transfected HEK293F cells and primary T cells that endogenously express CD27. The non-binding control antibody IgG1-b12-FEAR was used as a negative control antibody.

[0389] FreeStyle 293-F suspension cells (HEK293F; ThermoFisher, catalog #R79007) were transiently transfected using 293fectin Transfection Reagent (ThermoFisher, catalog #12347019) according to the manufacturer's instructions with a mammalian expression vector pSB encoding full-length human or cynomolgus monkey CD27.

[0390] PBMCs from humans and cynomolgus monkeys were purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM; Corning, catalog #25-072CV) according to the manufacturer's instructions from buffy coats obtained from healthy human donors (Sanquin Blood Bank, the Netherlands) or cynomolgus monkeys (BPRC, the Netherlands, catalog #S-1135).

[0391] Cells were seeded in 96-well plates (100,000 cells / well; Greiner Bio-one, catalog #650180) for sequential incubations involving a washing step with FACS buffer consisting of PBS (Lonza, catalog #BE17-517Q) + 1% BSA (Roche, catalog #10735086001) + 0.02% sodium azide (Bio-World, catalog #41920044-3). The following incubations were applied: antibody concentration series (final concentrations of 0.0001 - 10 μg / mL) for 30 min at 4°C; live / dead marker FVS510 (BD, catalog #564406, diluted 1:1,000 in PBS) for 20 min at RT; PE-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, catalog #109-116-098, diluted 1:500) for 30 min at 4°C; and anti-CD3 antibodies for T cell identification (anti-human CD3: BD, catalog #555335, diluted 1:10; anti-cynomolgus monkey CD3: Miltenyi, catalog #130-091-998, diluted 1:10) for 30 min at 4°C. All samples were analyzed on a FACSCelesta flow cytometer (BD) and FlowJo software. Data were processed and visualized using GraphPad Prism.

[0392] All tested antibodies showed dose-dependent binding to human CD27 in both human T cells and transfected HEK293F cells (Figure 2A, Figure 2B). The highest maximal binding was observed for IgG1-CD27-B and IgG1-CD27-C, with intermediate binding for IgG1-CD27-A and IgG1-CD27-131A, and low binding for IgG1-CD27-D and IgG1-CD27-E, and the differences were most pronounced using human T cells. For binding to cynomolgus CD27 T cells, the highest binding was observed for IgG1-CD27-B, followed by Ig1-CD27-131A and IgG1-CD27-A. Lower binding was observed for IgG1-CD27-D and -E, while IgG1-CD27-C showed the lowest binding to cynomolgus T cells. All CD27 antibodies showed dose-dependent binding to HEK cells transfected with cynomolgus CD27. The highest maximal binding was observed for IgG1-CD27-B and IgG1-CD27-131-A, with somewhat lower binding observed for IgG1-CD27-A, -D, and -E. IgG1-CD27-C showed the lowest binding to HEK cells transfected with cynomolgus CD27 (Figure 2C, Figure 2D).

[0393] In conclusion, IgG1-CD27-A and IgG1-CD27-B showed dose-dependent binding to human and cynomolgus CD27 that is endogenously expressed on human or cynomolgus T cells and transiently expressed in transfected HEK cells. IgG1-CD27-A and IgG-CD27-131A showed comparable binding to human T cells, while IgG1-CD27-B showed higher maximal binding. *Note: IgG1-CD27-A, -B, -C, -D, and -E had the variant F405L-L234F-L235E-D265A in the IgG Fc domain that was not functionally relevant in the context of this experiment. IgG1-CD27-131A had the non-functional F405L mutation in the IgG1 Fc domain.

[0394] Example 5: Binding of Anti-CD27 Antibodies to Native Human CD27-A59T Variant Approximately 19% of the human population expresses a native CD27 variant harboring the A59T mutation in the extracellular domain (SEQ ID NO.2). Binding to human CD27-A59T was tested by flow cytometry for anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C * and the benchmark IgG1-CD27-131A. The non-binding antibody IgG1-b12-FEAL was used as a negative control antibody. Transiently transfected HEK293F cells (15,000 cells / well) expressing human CD27-A59T were incubated with primary test antibodies IgG1-CD27-A-C, non-binding control antibody IgG1-b12 (control), and a concentration series (0.0001 - 10 μg / mL using 10-fold dilution steps) of the prior art benchmark IgG-CD27-131A that has been previously described (WO2018 / 058022) to bind CD27-A59T. After incubation, the antibodies were PE-labeled using polyclonal goat anti-human IgG. Binding was analyzed on a FACSCelesta flow cytometer (BD) and in FlowJo software. Data were processed and visualized using GraphPad Prism v.8.

[0395] The anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, and IgG1-CD27-131A tested showed dose-dependent binding to CD27-A59T transfected HEK293F cells with similar binding curves among the different antibodies (Figure 3). *Note: IgG1-CD27-A, -B, and -C had the mutations F405L-L234F-L235E-D265A in the IgG Fc domain that were not functionally related in the context of this experiment. IgG1-CD27-131A had the non-functionally related F405L mutation in the IgG1 Fc domain.

[0396] Example 6: Induction of human T cell proliferation by anti-CD27 antibodies Enhancement of IgG hexamerization through Fc-Fc interactions upon introduction of the E345R or E430G mutation enhanced the CD27 agonist activity of the anti-CD27 antibodies (Example 2). Thus, the ability of the IgG1-CD27-A, IgG1-CD27-B, and IgG1-CD27-C antibody variants with the E430G or E345R mutation to increase the proliferation of TCR-activated T cells was tested in vitro.

[0397] Additionally, Fc mutations that have been reported to reduce binding to C1q and FcγR (G237A or P329R) or enhance binding to C1q (K326A / E333A double mutation) were introduced to test their potential effects on the CD27 agonist activity of CD27 antibodies with the E345R or E430G mutation. The K326A / E333A double mutation has previously been shown to enhance C1q binding and contribute to the enhancement of agonist activity of a DR5-specific humanized IgG1 antibody containing an Fc-Fc interaction enhancing mutation (WO2018 / 146317A1). In addition to E430G or E345R, the mutations G237A, P329R, or K326A / E333A were introduced into IgG1-CD27-A, IgG1-CD27-B, and IgG1-C (Table 3), and their effects on T cell proliferation were determined using human PBMC obtained from healthy donors (Sanquin Blood Bank, the Netherlands).

[0398] (Table 3) Mutations in the Fc domain of antibody IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C and their biological effects TIFF2025516633000015.tif93166 * In IgG1-CD27-X, X refers to the IgG1-CD27 clones IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C.

[0399] PBMCs were resuspended in PBS at a density of 5×10 6 cells / mL and labeled with CFSE using the CellTrace CFSE Cell Proliferation Kit (Invitrogen, catalog #C34564; 1:10,000) according to the manufacturer's instructions. CFSE-labeled PBMCs (100,000 cells / well) were incubated with 0.1 μg / mL of anti-CD3 antibody clone UCHT1 (Stemcell Technologies, catalog #60011) and CD27 antibody (final concentration of 1 μg / mL) for T cell activation in T-cell Activation Medium (ATCC, catalog #80528190) supplemented with 5% Normal Human Serum (NHS; Sanquin, product #B0625) in a 96-well round-bottom plate (Greiner Bio-one, catalog #650180) at 37°C / 5% CO 2 for 96 h. CD4 + and CD8 +For the identification of viable cells in T cell subsets, cells were sequentially incubated for 20 minutes at RT with the live / dead marker FVS510 (1:1,000), and for 30 minutes at 4°C in the dark with a staining mix for lymphocyte markers containing APC-eFluor780-labeled anti-human CD4 antibody (Invitrogen, catalog #47-0048-42, 1:50), AlexaFluor700-labeled anti-human CD8a antibody (BioLegend, catalog #301028; 1:100), PE-Cy7-labeled mouse anti-human CD14 antibody (BD Biosciences, catalog #557742; 1:50), and BV785-labeled anti-human CD19 antibody (BioLegend, catalog #363028; 1:50). Samples were measured on a FACSCelesta (BD Biosciences) flow cytometer, and viable CD4 + and CD8 + T cell subsets (FVS510 - CD14 - CD19 - CD4 + and FVS510 - CD14 - CD19 - CD8 + ) CFSE dilution peaks were analyzed as a readout for T cell proliferation using FlowJo 10 software. T cell proliferation was represented as the percentage of cell proliferation or the division index, both calculated by using FlowJo software (version 10). The percentage of cell proliferation (division) was determined by gating on cells that had undergone CFSE dilution (CFSE low peaks ). The division index is the average number of divisions the cells have undergone. Heatmaps were generated using GraphPad Prism version 8. Proliferation assays were performed using PBMCs from four different healthy donors.

[0400] Variants of IgG1-CD27-A, -B, and -C with the E430G or E345R mutations were CD8 compared to the control antibody in two of the four donors tested +Induced a small increase in T cell proliferation. Introduction of additional mutations (P329R, G237A, or K326A / E333A) into IgG1-CD27-A, -B, or -C variants with the E430G mutation showed variable effects on CD8 + T cell proliferation across four PBMC donors. In contrast, introduction of the P329R mutation into IgG1-CD27-A and IgG1-CD27-C variants with the E345R mutation consistently increased the ability to enhance activated CD8 + T cell proliferation. This was particularly true for IgG1-CD27-A, and the measured CD8 + T cell proliferation was equivalent for IgG-CD27-A-E345R, IgG1-CD27-B-E345R, and IgG1-CD27-C-E345R in each of the donors, while introduction of the additional P329R mutation led to a consistently higher increase in CD8 + T cell proliferation for the cloned IgG1-CD27-A-E345R compared to IgG1-CD27-B-E345R or IgG1-CD27-C-E345R. Thus, the effect of the E345R mutation combined with the P329R mutation on TCR-activated CD8 + T cell proliferation was consistently greater for the cloned IgG1-CD27-A than for IgG1-CD27-B and IgG1-CD27-C. Across all antibody variants tested, IgG1-CD27-A-E345R-P329R induced the greatest increase in CD8 + T cell proliferation in all donors (Figure 4A).

[0401] Addition of the mutations G237A or K326A-E333A to CD27 antibody variants with the E345R mutation did not increase or only minimally increased CD8 + T cell proliferation in any of the clones tested compared to antibodies containing the single mutation E345R (Figure 4A).

[0402] CD4 +Even in T cells, the highest and most consistent increase in T cell proliferation was observed in the presence of IgG1-CD27-A-E345R-P329R (Figure 4B). CD4 + T cell proliferation was generally equivalent among the IgG1-CD27-A, -B, and -C variants having only the E430G or E345R mutation, while the introduction of the additional P329R mutation led to a greater increase in CD4 + T cell proliferation for the IgG1-CD27-A variant having the E345R variant compared to the IgG1-CD27-B or -C variants having either the E430G or E430G or E345R mutation. This effect was observed in 3 out of the 4 donors tested. In donor 1, CD4 + The effect of additional mutations added to E430G or E345R on T cell proliferation was generally small, and the effect observed in this donor was not reproduced in the other 3 donors.

[0403] The combination of E345R with the P329R mutation also led to a consistent increase in CD4 + T cell proliferation for IgG1-CD27-C, although the difference between the E345R mutation alone and the combination of E345R and P329R was smaller for clone IgG1-CD27-C than for clone -A. For clone IgG1-CD27-B, a modest increase in CD4 + T cell proliferation was observed for IgG1-CD27-B-E345R-P329R compared to IgG1-CD27-B-E345R in 2 out of the 4 donors.

[0404] The introduction of the P329R, G327A or K326A / E333A mutation into the IgG1-CD27-A, -B, or -C variant having the E430G mutation led to CD4 +did not induce an effect, or the induction was inconsistent, in T cell proliferation. Similarly, no effect was observed, or an inconsistent effect was observed, after introduction of G327A or K326A / E333A in IgG1-CD27-A, -B or -C variants having the E345R mutation.

[0405] In summary, IgG1-CD27-A-E345R-P329R consistently induced the highest increase in the proliferation of activated CD8 + and CD4 + T cells, demonstrating that IgG1-CD27-A-E345R-P329R induces the most efficient CD27 agonism. A DR5-specific, hexamerization-enhanced antibody having the P329R mutation was previously shown to have a reduced ability to induce DR5 agonism compared to a DR5-specific hexamerization-enhanced antibody without the P329R mutation (Overdijk et al, Mol Canc Ther 2020). Therefore, it was considered surprising that introduction of the P329R mutation in addition to the E345R mutation in IgG1-CD27-A enhances CD27 agonist activity. Furthermore, the reason why the combined effect of the E345R+P329R mutations was consistently greater for IgG1-CD27-A than for IgG1-CD27-B or IgG1-CD27-C is not known.

[0406] Example 7: Induction of human T cell proliferation by anti-CD27 antibody IgG1-CD27-A-P329R-E345R The ability of IgG1-CD27-A-P329R-E345R to increase the proliferation of TCR-stimulated human CD4 + and CD8 + T cells was analyzed in a CSFE dilution assay using human healthy donor PBMCs and compared to the prior art anti-CD27 clone IgG1-CD27-131A * , IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 *It was compared with. The T cell proliferation assay was performed as described in Example 6 with minor deviations (75,000 cells / well; concentration range 0.002 - 10 μg / mL). Samples using T cells without anti-CD3 stimulation were included to test the potential CD27 agonist activity of the antibody in the absence of T cell receptor activation (Figure 5A and Figure 5B). Such activity is undesirable as it poses a safety risk if the antibody can induce the proliferation of resting T cells.

[0407] Using FlowJo software, the percentage of proliferated T cells (Figure 5A, Figure 5B, Figure 5C, Figure 5D) was calculated as the percentage of cells with a reduction in CFSE fluorescence indicating cell division. The expansion proliferation index (Figure 5E and Figure 5F) identified the fold increase in cells in the well and was calculated using the Proliferation Modeling tool in FlowJo version 10. Manual adjustment to the peak was made if necessary to more consistently define the number of peaks present.

[0408] Neither the CD27 antibodies of the present invention nor the prior art antibodies tested here induced proliferation in unstimulated T cells, i.e., in the absence of CD3 crosslinking (Figure 5A and Figure 5B).

[0409] Most of the CD27 antibodies induced a certain degree of proliferation of activated CD4 + and CD8 + T cells at the highest antibody concentration tested (Figure 5C and Figure 5D). Based on this, the expansion proliferation index was calculated (Figure 5E and Figure 5F). The antibody IgG1-CD27-A-P329R-E345R of the present invention enhanced the proliferation of CD4 + and CD8 + T cells more significantly in vitro compared to the prior art anti-CD27 clones IgG1-CD27-131A, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215. *For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants with the F405L mutation that are not functionally relevant in the context of this experiment were used.

[0410] Example 8: Binding of C1q to Membrane-Bound CD27 Antibodies The P329R mutation has previously been described to reduce the interaction of IgG1 antibodies with C1q and FcγR (Overdijk et al, Molecular Cancer Therapeutics 2020). The effect of the P329R mutation on C1q binding of IgG1-CD27-A containing the E345R mutation was tested in a cell C1q binding assay in vitro using human healthy donor T cells. The anti-HIV gp120 antibody IgG1-b12-F405L was used as an unbound isotype control antibody (control). T cells were enriched from human healthy donor PBMCs using RosetteSep Human T cell Enrichment cocktail (Stemcell, catalog #15061) and resuspended in culture medium (RPMI 1640 [Gibco, catalog #A10491-01]) supplemented with 0.1% BSA and 1% Pen / Strep [Lonza, catalog #DE17-603E]). T cells (2×10 6 cells / well) were preincubated for 15 minutes at 37°C in a polystyrene 96-well round-bottom plate containing an antibody dilution series (eight 5-fold dilutions starting with a final assay concentration of 15 μg / mL) to bind the antibody to the T cells. The cells were then cooled on ice, supplemented with NHS as a source of human C1q (final assay concentration of 20% NHS), and incubated on ice for 45 minutes. The cells were subsequently incubated on ice for 30 minutes with an FITC-labeled rabbit anti-human C1q antibody (DAKO, catalog #F0254; 20 μg / mL) and resuspended in FACS buffer containing TO-PRO-3 (ThermoFisher, catalog #T3605; 1:5,000 dilution). C1q binding was determined by flow cytometry measuring the FITC signal in live cells.

[0411] The membrane-bound WT IgG1-CD27-A antibody did not show C1q binding (Figure 6). Introduction of the hexamerization-enhancing mutations E430G or E345R (IgG1-CD27-A-E430G and IgG1-CD27-A-E345R) resulted in C1q binding to the CD27 antibody on the T cell surface, consistent with an increase in the binding avidity of hexameric C1q protein for the hexameric antibody ring structure on the cell surface (Figure 6). Introduction of the P329R mutation in IgG1-CD27-A-E345R (IgG1-CD27-A-P329R-E345R) resulted in loss of C1q binding (Figure 6), demonstrating that IgG1-CD27-A-P329R-E345R cannot bind C1q.

[0412] These data indicate that IgG1-CD27-A-P329R-E345R cannot bind C1q upon binding to CD27 on the cell surface of T cells. This indicates that C1q binding does not contribute to the antibody-induced CD27 agonist activity of IgG1-CD27-A-P329R-E345R. This is in contrast to what has been previously described for other hexamerization-enhancing agonist antibodies. Furthermore, the lack of C1q binding indicates that IgG1-CD27-A-P329R-E345R cannot activate the classical pathway of complement activation. Therefore, it is expected that the activity of IgG1-CD27-A-P329R-E345R does not induce complement activation and CDC in unwanted T cells.

[0413] Example 9: Binding of anti-CD27 antibodies to human Fc receptors The binding of IgG1-CD27-A-P329R-E345R to human FcγR variants was analyzed using a Biacore surface plasmon resonance (SPR) system and compared to the anti-HIV gp120 antibody IgG1-b12 (control). Biacore Series S Sensor Chips CM5 (Cytiva, catalog #29104988) were covalently coated with anti-His antibody using the amine-coupling and His capture kits (Cytiva, catalog #BR100050 and catalog #29234602) according to the manufacturer's instructions. Next, 125 nM of Fcγ receptors FcγRIa, FcγRIIa (167-His[H] and 167-Arg[R]), FcγRIIb, or FcγRIIIa (176-Phe[F] and 176-Val[V]) (Sino Biological, catalog #10256-H08S-B, catalog #10374-H27H, catalog #10374-H27H1-B, catalog #10259-H27H-B, catalog #10389-H27H-B, and catalog #10389-H27H1-B) in HBS-P+ (Cytiva, catalog #BR100827) were captured onto the surface. After 3 cycles of buffer, antibody samples were injected over 36 cycles to generate binding curves using an antibody range of 0 - 3,000 nM for FcγRI and 0 - 10,000 nM for the other FcγRs. Each sample analyzed on the FcR-coated surface (active surface) was also analyzed on a parallel flow cell without FcR (reference surface) used for background correction. Dissociation from the anti-His coated surface was performed by surface regeneration using 10 mM glycine-HCl (pH 1.5) (Cytiva, catalog #BR100354). Sensorgrams were generated using Biacore Insight Evaluation software (Cytiva), and a 4-parameter logistic (4PL) fit was applied to calculate the relative binding of IgG1-CD27-A-P329R-E345R to the reference sample (control).

[0414] The binding of IgG1-CD27-A-P329R-E345R to the high-affinity receptor FcγRIa was strongly reduced compared to the control antibody, although some binding was observed at higher antibody concentrations (Figure 7A). IgG1-CD27-A-P329R-E345R did not bind to the human low-affinity receptors FcγRIIa (Figure 7B and Figure 7C), FcγRIIb (Figure 7D), or FcγRIIIa (Figure 7E and Figure 7F).

[0415] In conclusion, IgG1-CD27A-P329R-E345R shows minimal binding (FcγRIa) or no binding (FcγRIIa, FcγRIIb, and FcγRIIIa) to human IgG Fc receptors.

[0416] Example 10: Binding of anti-CD27 antibody IgG1-CD27-A-E345R-P329R to human T cells Flow cytometry was used to more precisely characterize the binding of IgG1-CD27-A-P329R-E345R to CD27 on human healthy donor T cells. The anti-HIV gp120 antibody variant IgG1-b12-P329R-E345R was used as an unbinding control antibody (control). Human PBMCs were isolated from buffy coats obtained from human healthy donors. PBMCs (1×10 5 cells / well) in FACS buffer were added to polystyrene 96-well round-bottom plates (Greiner bio-one, catalog #650101) and pelleted by centrifugation at 300×g for 3 minutes at 4°C. The cells were resuspended in 50 μL / well of serial antibody dilutions in FACS buffer (ranging from 0.0015 to 10 μg / mL in 3-fold dilution steps) and incubated at 4°C for 30 minutes. The cells were pelleted, washed twice with FACS buffer, and incubated in 50 μL / well with an FITC-conjugated secondary antibody (FITC AffiniPure F(ab') 2 fragment goat anti-human IgG, F(ab') 2The fragments were incubated with the specific substance, Jackson ImmunoResearch, Catalog #109-096-097, 1:100 dilution) for 30 minutes at 4°C in the dark. The cells were pelleted again, washed twice with FACS buffer, and incubated for 30 minutes at 4°C in the dark in a 50 μL / well staining mix for lymphocyte markers containing BV711-labeled anti-human CD19 antibody (BioLegend, Catalog #302246, 1:50), AlexaFluor700-labeled anti-human CD8a antibody (BioLegend, Catalog #301028, 1:100), APC-eFluor780-labeled anti-human CD4 antibody (Invitrogen, Catalog #47-0048-42, 1:50), PE-CF594-labeled mouse anti-human CD56 antibody (BD Biosciences, Catalog #564849, 1:100), PE-Cy7-labeled mouse anti-human CD14 antibody (BD Biosciences, Catalog #557742, 1:50), and eFluor450-labeled anti-human CD3 antibody (Invitrogen, Catalog #48-0037-42, 1:200). The cells were pelleted again, washed twice using FACS buffer, and resuspended in 80 μL of FACS buffer containing the dead cell marker 7-amino-actinomycin D (7-AAD; BD Biosciences, Catalog #51-68981E, 1:240 dilution). The samples were measured by flow cytometry on an LSRFortessa (BD) flow cytometer and analyzed using FlowJo software. The binding curves were analyzed using non-linear regression (sigmoid dose-response with variable slope) using GraphPad Prism 8 software.

[0417] The anti-CD27 antibody IgG1-CD27-A-P329R-E345R showed dose-dependent binding to healthy donor T cells, with similar binding characteristics to CD4 + and CD8 + T cells (Figure 8).

[0418] Example 11: FcγR-independent induction of CD27 cell signaling by anti-CD27 antibody IgG1-CD27-A-P329R-E345R CD27-specific monoclonal antibodies that can induce CD27 signaling independently of secondary FcγR-mediated cross-linking can be immunostimulatory in the absence of FcγR-positive cells, which is an advantage in tumors with a low frequency of FcγR-bearing cells.

[0419] The CD27 agonist activity of IgG1-CD27-A-P329R-E345R was tested in the presence or absence of FcγR-bearing cells, along with the corresponding WT antibody IgG1-CD27-A and prior art antibody IgG1-CD27-131A * , IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 * and compared. The non-binding antibody IgG1-b12-P329R-E345R was used as a negative control (control). The CD27 reporter assay was performed essentially as described in Example 2, with the exception that in this example, Thaw-and-Use GloResponse NFκB-luc2 / CD27 Jurkat cells were cultured in the presence of human FcyRIIb-expressing cells that can promote FcγR-mediated cross-linking of the membrane-bound antibody.

[0420] Thaw-and-Use effector FcγRIIb CHO-K1 cells (Promega, catalog #JA2251) were plated into 96-well flat-bottom culture plates (PerkinElmer, catalog #0815) without dilution or at three increasing dilutions (1 / 3, 1 / 9, 1 / 27) and incubated at 37°C / 5% CO 2It was incubated overnight. The supernatant of adherent FcyRIIb-expressing cells was replaced with a Thaw-and-Use NFκB-luc2 / CD27 Jurkat cell suspension containing serial dilutions of antibody (final concentration range 0.0002 - 10 μg / mL) and a fixed cell concentration in Bio-Glo Luciferase Assay Buffer (starting with a 1:1 NFκB-luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratio for non-diluted FcγRIIb CHO-K1 cells). 2 After 6 h incubation at 37°C / 5% CO 2 2, the plates were equilibrated to RT and bioluminescence was measured and presented as RLU as described in Example 2.

[0421] IgG1-CD27-A-P329R-E345R induced dose-dependent CD27 activation independently of FcγRIIb-expressing cells (Figure 9A). In contrast, the corresponding WT antibody IgG1-CD27-A, which does not have the E345R hexamerization-enhancing mutation and the P329R mutation, showed CD27 agonism only in the presence of FcγRIIb-expressing cells (Figures 9A - 9E). Similarly, CD27 activation by the prior art antibodies IgG1-CD27-131A, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 was also dependent on the presence of FcγRIIb-expressing cells and decreased gradually with decreasing NFκB-luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratio (Figures 9F - 9J).

[0422] In conclusion, these data indicate that IgG1-CD27-A-P329R-E345R can induce CD27 agonism independently of secondary FcγR-mediated cross-linking. This is in contrast to prior art anti-CD27 antibodies that are dependent on the presence of FcγR-bearing cells to induce CD27 agonism. * For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants with the F405L mutation, which is not functionally relevant in the context of this experiment, were used.

[0423] Example 12: Pharmacokinetic (PK) analysis of anti-CD27 antibody IgG1-CD27-A-P329R-E345R in the absence of target binding, studied in mice Anti-CD27 antibody IgG1-CD27-A-P329R-E345R in the absence of target binding * The pharmacokinetic characteristics were analyzed in mice, and the corresponding WT antibody IgG1-CD27-A *It was compared with. IgG1-CD27-A did not bind to mouse CD27 (Example 3, Table 2), thus, in the absence of target binding, experiments were designed to test the pharmacokinetic behavior of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R in vivo. The study was conducted by Crown Bioscience (China) by qualified staff in accordance with the approved IACUC protocol and Crown Bioscience, Inc. Standard Operating Procedures. Female SCID mice (C.B-17, 11-12 weeks old, Vital River Laboratory Animal Technology Co., Ltd. (VR, Beijing, China; 3 mice / group)) were injected intravenously with 500 μg of antibody (25 mg / kg) in a 200 μL injection volume. 40 μL blood samples were collected 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration, plasma was collected from the blood samples and stored at -80 °C until determination of total human IgG concentration by ELISA. 96-well ELISA plates (Greiner, catalog #655092) were coated overnight at 4 °C with 2 μg / mL anti-human IgG (Sanquin, The Netherlands, product #M9105, lot #8000260395), followed by blocking with PBSA (PBS supplemented with 0.2% bovine serum albumin [BSA, Roche, catalog #10735086001]) for 1 h. Next, with washing steps in between, the anti-human IgG-coated plates were incubated sequentially with plasma samples serially diluted in ELISA Buffer (PBSA supplemented with 0.05% Tween 20 [Sigma-Aldrich, catalog #P1379]) on a plate shaker at RT for 1 h, with a polyclonal peroxidase-conjugated goat anti-human IgG secondary antibody (Jackson, catalog #109-035-098) at RT for 1 h, and finally with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, catalog #11112422001). The reaction was stopped by adding 2% oxalic acid (Riedel de Haen, catalog #33506).Reference curves were generated using dilution series of each material used for injection. Absorbance was measured at 405 nm in an EL808 microtiter plate reader (BioSPX), and the total human IgG concentration (μg / mL) was plotted.

[0424] No substantial difference was found between the PK profile of IgG1-CD27-A-P329R-E345R and that of its corresponding WT antibody IgG1-CD27-A, as determined by measuring plasma IgG levels at different time points after intravenous injection in mice (Figure 10).

[0425] A steeper decline in the initial (distribution) phase was observed for IgG1-CD27-A-P329R-E345R and its WT counterpart (IgG1-CD27-A) compared to the prediction for human IgG1 in mice, but the terminal elimination of both antibodies was consistent with the predicted rate for human wild-type IgG1 based on a two-compartment model (Bleeker WK, Teeling JL, Hack CE. Blood. 2001 Nov 15;98(10):3136-42).

[0426] Taken together, this demonstrates that the introduction of the P329R and E345R mutations did not affect the pharmacokinetic properties of IgG1-CD27-A in the absence of target binding. Note: The experiments described in this example used variants of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R with an F405L mutation that are not functionally relevant in the context of this experiment.

[0427] Example 13: Induction of antibody-dependent cell phagocytosis by anti-CD27 antibody IgG1-CD27-A-P329R-E345R Antibody-dependent cell-mediated cytotoxicity (ADCC) is mainly mediated through FcγRIIIa expressed on NK cells, while antibody-dependent cell phagocytosis (ADCP) can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRI, FcγRIIa, and FcγRIII (Hayes, J.M et al 2016). To understand the effect of residual binding of anti-CD27 antibody IgG1-CD27-A-P329R-E345R to FcγRIa in the effector function of FcγRIa-expressing immune cells (Example 9), CTV-labeled CD27 + Using Burkitt lymphoma Daudi cells as target cells and human monocyte-derived macrophages (hMDM) as effector cells (E:T = 2:1), the ability of IgG1-CD27-A-P329R-E345R to induce ADCP was analyzed in vitro.

[0428] hMDM were isolated from PBMC by positive selection using CD14 microbeads (Miltenyi Biotec, catalog number 130-050-201) according to the manufacturer's instructions. PBMC were centrifuged (1,200 RPM, 5 minutes, RT) and resuspended in ice-cold monocyte isolation buffer (PBS, 0.5% BSA, 2 mM EDTA) at a density of 1.25×10 7 PBMC / mL. 20 μL of CD14 microbeads were added per 80 μL of PBMC suspension and incubated with agitation for 15 minutes at 4°C on a roller bank. 30 mL of ice-cold monocyte isolation buffer was added, and the PBMC / CD14 microbead mixture was centrifuged (300×g, 10 minutes, 4°C) and resuspended in 6 mL of ice-cold monocyte isolation buffer. The LS column (Miltenyi Biotec, catalog number 130-042-401) was rinsed with 3 mL of ice-cold monocyte isolation buffer, and 3 mL of the PBMC / CD14 microbead mixture was loaded onto each column. CD14 - cells were allowed to flow through, and the column was washed 3 times in ice-cold monocyte isolation buffer before using the plunger to elute CD14 +Monocytes were harvested into 3 mL of ice-cold monocyte isolation buffer. CD14 cells were counted using ViaStain™ Viability Dye Acridine Orange / Propidium Iodide (AOPI; Nexcelom Bioscience, catalog number CS2-0106) on a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience), and the plate was left at RT to enable cell harvesting on an UpCell™ Surface having a 100 mm + Nunc™ dish (Thermo Fisher Scientific, catalog number 174902) containing macrophage colony-stimulating factor (M-CSF; Gibco, catalog number PH9501; final concentration of 50 ng / mL) and 3 mL of monocyte suspension (i.e., 2.4×10 2 monocytes) was resuspended in Celgene® GMP DC medium (CellGenix, catalog number 20801-0500) at a density of 0.8×10 6 cells / mL. After 3 days of incubation, 2 mL of fresh medium containing 5×M-CSF was added to the plate. After 7 days of incubation (37 °C, 5% CO 6 2), the plate was left at RT for 1 - 1.5 h to detach the macrophages from the surface. The detached macrophages were pelleted by centrifugation, counted using AOPI, and resuspended in culture medium (RPMI 1640 containing 10% DBSI) at a density of 1×10 2 cells / mL. 6 Human Burkitt lymphoma Daudi cells (ATCC® CCL-213™) were labeled using the CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific, catalog number C34557) according to the manufacturer's instructions. Briefly, Cell Trace Violet (CTV) was added to 1×10

[0429] cells / mL in 1 mL of PBS to a final concentration of 0.2 μM. 6In addition to individual Daudi cells, they were incubated at 37 °C for 20 minutes in the dark (15 mL incubation volume). 10 mL of DBSI was added to inactivate unbound dye. Cells were pelleted by centrifugation (300×g, 5 minutes), washed in PBS, and counted with AOPI. CTV-labeled Daudi cells were resuspended in culture medium at a density of 0.5×10 6 cells / mL.

[0430] For the ADCP assay, hMDM (50,000 cells / well) and CTV-labeled Daudi cells (25,000 cells / well) were seeded together (E:T = 2:1) on ice in 150 μL of final volume of culture medium in a 96-well plate and incubated with anti-CD27 antibody IgG1-CD27-A-P329R-E345R or anti-CD20 antibody IgG1-CD20 (concentration range of 0.000001 - 10 μg / mL at 10-fold dilution) for 4 h (37 °C, 5% CO 2 2). After incubation, 100 μL of Human BD Fc Block™ (BD Biosciences, catalog number 564220; 1:100 in FACS buffer) was added and incubated at 4 °C for 10 minutes. Cells were pelleted by centrifugation (300×g, 5 minutes), resuspended in FACS buffer containing PE-Cy7-conjugated anti-human CD11b antibody (BioLegend, catalog number 301322; 1:80) and TO-PRO-3 (Thermo Fisher Scientific, catalog number T3605; 1:25,000), and incubated at 4 °C for 30 minutes. Cells were washed, resuspended in FACS buffer, and collected and analyzed on a FACSymphony™ A3 Cell Analyzer (BD Biosciences). Data were analyzed using FlowJo software to measure the number of viable target cells and phagocytic cells hMDM, and processed and visualized using GraphPad Prism software.

[0431] The percentage of viable Daudi cells for each condition was calculated by the following formula. TIFF2025516633000016.tif15128

[0432] The amount of phagocytic hMDM for each condition was determined as the percentage of cells of TO-PRO-3 - CD11b + CTV + and was determined as the % of cells.

[0433] IgG1-CD27-A-P329R-E345R neither increased the percentage of phagocytic hMDM nor decreased the percentage of viable Daudi cells in the phagocytosis assay using hMDM from four different human healthy donors. This demonstrates that the residual FcγRIa binding did not result in FcγRIa-mediated effector function for IgG1-CD27-A-P329R-E345R (data from a representative human healthy donor are shown in Figure 11). As demonstrated by the increase in the percentage of phagocytic hMDM and the decrease in the percentage of viable Daudi cells, the positive control antibody IgG1-CD20 efficiently induced the phagocytosis of Daudi cells expressing high levels of CD20.

[0434] In conclusion, the residual binding to FcγRIa was not sufficient to induce IgG1-CD27-A-P329R-E345R-dependent ADCP of CD27 + cells.

[0435] Example 14: Target-independent complement activation in the fluid phase by anti-CD27 antibody IgG1-CD27-A-P329R-E345R, determined by measurement of C4d deposition Fc-Fc interaction-enhanced antibodies generally exist as monomeric IgG1 molecules in solution and hexamerize on the cell surface upon target binding, forming C1q docking sites in the case of the active Fc region (Diebolder, C.A et al 2014; de Jong, R.N et al, 2016). The IgG Fc domain of the anti-CD27 antibody IgG1-CD27-A-P329R-E345R is silenced by the introduction of the P329R mutation, which results in the lack of C1q binding to membrane-bound IgG1-CD27-A-P329R-E345R (Figure 6). To confirm that IgG1-CD27-A-P329R-E345R cannot activate complement in solution in the absence of target binding, target-independent complement activation was investigated by determination of C4d deposition, considered an indicator for the activation of the classical complement pathway. Fluid-phase C4d fragment deposition by IgG1-CD27-A-P329R-E345R was analyzed by enzyme-linked immunosorbent assay (ELISA) using the MicroVue™ C4d Enzyme Immunoassay (EIA; Quidel, catalog number A008), which was performed according to the manufacturer's protocol. Heat Aggregated Gamma Globulin (HAGG; complement activator; Quidel, catalog number A114) was used as a positive control for the assay. IgG1-b12 and IgG1-b12-RGY (WO2014006217A1) were included as control antibodies. The introduction of the E345R / E430G / S440Y (RGY) Fc mutation in the IgG1 antibody has been described to induce hexamer formation in solution, resulting in fluid-phase complement activation (Diebolder, C.A et al, 2014; Wang, G., R.N et al, 2016; de Jong, R.N et al, 2016). IgG1-b12-P329R-E345R was included as an isotype control antibody.

[0436] Antibody dilutions up to a concentration of 1 mg / mL were prepared in phosphate-buffered saline (PBS), except for HAGG diluted to a concentration of 10 mg / mL. Next, the test samples were further diluted to a concentration of 100 μg / mL (monoclonal IgG) or 1,000 μg / mL (HAGG) in 90% (final concentration) normal human serum (NHS) (CompTech, lot number 42a) and incubated at 37 °C for 1 h. In parallel, "antibody-free" samples (antibody-free, 90% NHS) and "PBS alone" samples (antibody-free, NHS-free) were included as negative controls. Next, the samples were diluted 1:250 in the cooled Complement Specimen Diluent provided in the kit. On the other hand, strips coated with mouse anti-human C4d antibody were placed in a 96-well plate, and the assay wells were washed three times with 250 - 300 μL of wash buffer with a 1-minute waiting step after the first wash. The test samples were added to the wells (100 μL / well), and as a negative control, only the complement specimen diluent (blank) was used in the ELISA. In parallel, 100 μL of standards (standards A - E) and internal controls provided by the kit were added to separate wells. The plate was incubated at RT for 30 minutes. Next, the plate was washed five times with the wash buffer described above. 50 μL of C4d conjugate (peroxidase-conjugated goat anti-human C4d) was added to the wells, and the plate was incubated at RT for 30 minutes. After five wash steps with the wash buffer described above, 100 μL of C4d substrate [0.7% 2-2'-azino-di-(3-ethylbenzthiazoline sulfonic acid diammonium salt] was added, and the plate was incubated again at RT for 30 minutes. Finally, 50 μL of the stop solution provided by the kit was added, and within 1 h, the optical density was measured at 405 nm using an ELISA plate reader (EL808 BioSPX, BioTek).

[0437] IgG1-CD27-A-P329R-E345R and the control antibody IgG1-b12-P329R-E345R (having the same Fc backbone as IgG1-CD27-A-P329R-E345R) did not induce fluid-phase C4d deposition at the tested concentration of 100 μg / mL; the measured C4d levels were similar to the background levels of the control antibody (IgG1-b12) with a wild-type Fc domain and the no-antibody control (Figure 12). In contrast, the positive control antibody IgG1-b12-RGY, which is known to form hexamers in solution, induced C4d deposition up to the same level as HAGG.

[0438] These data indicate that IgG1-CD27-A-P329R-E345R did not induce target-independent, fluid-phase complement activation in vitro.

[0439] Example 15: Ability of anti-CD27 antibody IgG1-CD27-A-P329R-E345R to compete for CD70 ligand binding To determine whether the anti-CD27 antibody IgG1-CD27-A-P329R-E345R interferes with the interaction of CD27 with its natural ligand CD70, the binding of a saturating concentration of biotinylated recombinant human CD70 extracellular domain (ECD) to CD27 endogenously expressed on the human Burkitt lymphoma cell line Daudi was studied in the presence and absence of excess IgG1-CD27-A-P329R-E345R.

[0440] Daudi cells (ATCC® CCL-213™) cultured in RPMI 1640 medium (Gibco, catalog number A10491-01) supplemented with 10% donor bovine serum containing iron (DBSI; Gibco, catalog number 20731-030) were seeded into round-bottom 96-well plates (Greiner Bio One, catalog number 650261) at 50,000 cells / well. The cells were pelleted by centrifugation (300×g, 3 minutes, 4°C) and resuspended in FACS buffer (PBS, 1% BSA [Roche, catalog number 1073508600]) containing anti-CD27 or control antibody (final concentration of 50 μg / mL). Biotinylated recombinant human CD70 ECD (Abcam, catalog number ab271443) was added at a saturating concentration (6 μg / mL), and the cells were incubated at 4°C for 30 minutes.

[0441] The cells were washed twice and incubated with Brilliant Violet (BV) 421™-labeled streptavidin (BioLegend, catalog number 405225; final concentration of 0.0025 μg / mL) and R-phycoerythrin (PE)-labeled polyclonal AffiniPure F(ab') 2The fragments were resuspended in FACS buffer containing fragment goat - anti - human IgG Fc (Jackson ImmunoResearch, catalog number 109 116098; final concentration of 0.0025 μg / mL) at 4°C for 30 minutes. The cells were washed twice and resuspended in FACS buffer containing TO - PRO - 3 iodide (Thermo Fisher Scientific, catalog number T3605; 1:25,000) for analysis. Data were collected on a BD FACSymphony™ A3 flow cytometer (BD Biosciences) and analyzed using FlowJo software. For compensation, one drop of UltraComp eBeads™ Compensation Beads (Life Technologies, catalog number 01 - 2222 - 42) was added to each well. 2 μL of each antibody was added and the mix was incubated for 20 minutes. The plate was centrifuged, the beads were resuspended in FACS buffer, and measured. For viability compensation, cells were treated at 65°C for 10 minutes and mixed 1:1 with viable cells. The cells were centrifuged and resuspended in TO - PRO - 3 diluted in FACS buffer. Data were processed and visualized using GraphPad Prism.

[0442] IgG1 - CD27 - A - P329R - E345R or IgG1 - CD27 - A binds to CD27 + IgG1 - CD27 - A - P329R - E345R or IgG1 - CD27 - A did not block the binding of CD70 ECD to Daudi cells, and the CD70 binding levels were equivalent to those of Daudi cells incubated with the non - binding isotype control antibodies IgG1 - b12 - P329R - E345R or IgG1 - b12, or cells without antibody (Figure 13). Also, the prior art anti - CD27 antibodies IgG1 - CD27 - BMS986215 and IgG1 - CD27 - 131A showed a weak blocking effect on the binding of CD27 to CD70 ECD. In contrast, CD70 was unable to bind to surface CD27 on Daudi cells in the presence of the prior art anti - CD27 antibody IgG1 - CD27 - CDX1127 (Vitale et al, 2012), which was previously reported to block ligand binding (Figure 13).

[0443] As a conclusion, the binding of IgG1-CD27-A-P329R-E345R does not block the binding of CD27 by the natural ligand CD70 on Daudi cells.

[0444] Example 16: Expression of T cell activation markers upon incubation of polyclonally stimulated human PBMC with anti-CD27 antibodies The effect of IgG1-CD27-A-P329R-E345R on the expression of T cell activation markers in polyclonally activated T cells was studied using PBMC obtained from three different healthy human donors. PBMC were incubated with IgG1-CD27-A-P329R-E345R or a prior art anti-CD27 antibody for 2 days and 5 days, and then the expression of HLA-DR, CD25, CD107a, and 4-1BB was analyzed.

[0445] Freshly isolated 75,000 PBMCs per well were seeded into the cell culture medium in a 96-well U-bottom plate (Greiner Bio-One). Duplicate wells were incubated simultaneously with anti-CD3 antibody (UCHT1 clone; Stemcell; 0.1 μg / mL); and IgG1-CD27-A-P329R-E345R (0.0005 - 30 μg / mL at 3-fold dilution); or the prior art anti-CD27 antibodies IgG1-CD27-CDX1127, IgG1-CD27-131A, and IgG1-CD27-BMS986215 (30 μg / mL); or an unbound control antibody IgG1-b12-P329R-E345R (10 μg / mL). To determine the expression of each activation marker in the absence of treatment, duplicate control wells containing untreated (no anti-CD3 antibody and no anti-CD27 antibody) cells were supplemented with culture medium alone. A fluorescence minus one (FMO) control was used to set the gates for identifying activation marker-positive cells. For the FMO control, all antibodies used in the experiment except those corresponding to the activation markers in duplicate wells were added to 75,000 PBMCs per well from one donor activated with anti-CD3 antibody. Untreated cells from each donor in a single well without staining antibody were included as a negative control. To detect viable cells, untreated cells from each donor were stained in a single well with 4',6-diamidino-2-phenylindole (DAPI) alone.

[0446] After 2 or 5 days of incubation (37 °C, 5% CO 2 ) the plates were washed once with FACS buffer and antibodies for the T cell activation markers 4-1BB, CD25, CD107a, human leukocyte antigen (HLA)-DR; and CD4 + and CD8 +It was resuspended in an antibody mixture in FACS buffer containing antibodies for gating T cell subsets. After incubation at 4°C for 30 minutes, all plates were washed twice with FACS buffer and the cells were resuspended in FACS buffer. Samples were analyzed on a BD LSRFortessa Cell Analyzer using FlowJo software to determine the median fluorescence intensity (MFI) and percentage of positive cells for each T cell activation marker on CD4 + and CD8 + T cells. The change in anti-CD27 antibody-induced change in the expression level of T cell activation markers was presented as the fold change in MFI of the anti-CD27 antibody sample compared to the non-binding control antibody IgG1-b12-P329R-E345R. Samples were analyzed on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) using FlowJo software.

[0447] IgG1-CD27-A-P329R-E345R increased the expression of CD25, CD107a and 4-1BB on activated CD4 + T cells (Figure 14A). These effects were more pronounced after 2 days of incubation than after 5 days of incubation. On CD8 + T cells, incubation with IgG1-CD27-A-P329R-E345R resulted in an increase in the expression of HLA-DR, CD107a and 4-1BB both after 2 days of incubation and after 5 days of incubation (Figure 14B).

[0448] The expression of T cell activation markers with 2-day and 5-day incubations with three prior art antibodies was also evaluated. IgG1-CD27-131A and IgG1-CD27-BMS986215, CD4 + and CD8 +Equivalent increases in the expression of HLA-DR, 4-1BB, CD25, and CD107a on T cells were induced, but the effects of 2-day or 5-day incubation with IgG1-CD27-CDX1127 on T cell activation marker expression were weaker.

[0449] In conclusion, incubation of polyclonally activated PBMC with IgG1-CD27-A-P329R-E345R resulted in increased expression of the activation markers HLA-DR, CD25, CD107a, and 4-1BB on CD4 + and CD8 + T cells.

[0450] Example 17: Percentage of OVA-specific CD8 + T cells in OVA-protein immunized mice after injection of anti-CD27 antibody in a human CD27-KI mouse model The effect of IgG1-CD27-A-P329R-E345R treatment on the expansion of antigen-specific T cells in the hCD27 KI OVA model in splenocytes was analyzed by flow cytometry.

[0451] Homozygous human CD27 (hCD27)-KI mice (hCD27 KI mice) with a C57BL / 6 background were obtained from Beijing Biocytogen Co., Ltd. (strain name C57BL / 6-Cd27tm1(CD27) / Bcgen, stock number 110006). This strain was developed in collaboration with Crown Bioscience's HuGEMM™ platform and featured a humanized drug target (in this case CD27) in mice with a functional immune system. In hCD27 KI mice, exons 1-5 of the mouse CD27 gene encoding the extracellular domain were replaced with human CD27 exons 1-5. OVA-specific T cells were induced in vivo in hCD27-KI mice by subcutaneous (s.c.) injection of the immunogen ovalbumin (OVA), and the agonistic effect of IgG1-CD27-A-P329R-E345R was tested by simultaneously treating the mice intravenously (i.v.) with an antibody.

[0452] On day 0, mice were injected s.c. with 5 mg of OVA (InvivoGen, catalog number vac-pova-100, lot number EFP-42-04) and treated i.v. with IgG1-CD27-A-P329R-E345R (30 mg / kg), IgG1-CD27-CDX1127 (30 mg / kg), or IgG1-b12-P329R-E345R (30 mg / kg) via the tail vein. On days 12 and 21, the mice were boosted with OVA and treated with the same antibodies as on day 0. On days 10, 19, and 24, blood was collected into BD Microtainer® blood collection tubes containing potassium ethylenediaminetetraacetate (K2-EDTA; BD, catalog number 365974) via the cheek pouch or the saphenous vein and used immediately in further analysis. On day 28, the mice were euthanized and the spleens were excised under sterile conditions.

[0453] The excised spleen tissue in RPMI 1640 medium (Thermo Fisher Scientific, catalog number C22400500BT) was transferred to gentleMACs (trademark) C Tubes (Miltenyi Biotec, catalog number 130 - 093 - 237), and mechanically dissociated into a single cell suspension using a gentleMACS (trademark) Dissociator (Miltenyi, catalog number 130 - 093 - 235) according to the manufacturer's instructions. After dissociation, the cell suspension was filtered through a 70 μm cell strainer (Falcon, catalog number 352350). Next, the sample was washed twice by resuspension in 3 mL of wash buffer (sterile PBS [Hyclone, SH0256.01B] supplemented with 4% FBS [Gibco, catalog number 10099 141]). The cells were counted using a Cellometer Auto T4 (Nexcelom Bioscience), and the number of cells was adjusted to 2×10 6 spleen cells per tube.

[0454] 2×10 6Individual spleen cells were transferred into FACS tubes (Falcon, catalog number 352052) and resuspended in wash buffer (sterile PBS [Hyclone, SH0256.01B] supplemented with 4% FBS [Gibco, catalog number 10099 141]) supplemented with 1 μg / mL of purified rat anti-mouse CD16 / CD32 (Mouse BD Fc Block™, BD Biosciences, catalog number 553141). After a 10-minute pre-incubation in the dark at 2-8°C, 10 μL of PE-labeled OVA tetramer (MBL Life science, catalog number TS 5001 1C) was added, and the sample was gently vortexed and then further incubated in the dark at 2-8°C for 30-60 minutes. Without washing, labeled antibodies and compounds used for flow cytometry gating of T cell subsets were added. The sample was gently vortexed and incubated for an additional 30 minutes in the dark at 2-8°C. Next, the sample was washed twice by resuspension in 2 mL of wash buffer and centrifuged at 300×g for 5 minutes. Finally, the cells were resuspended in 250 μL of wash buffer and analyzed using a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences). Data were processed using Kaluza Analysis Software (Beckman Coulter).

[0455] IgG1-CD27-A-P329R-E345R increased the percentage of OVA-specific CD8 + T cells in the spleens of mice co-injected with OVA protein vaccine. The percentage of OVA-specific CD8 + T cells in mice treated with 30 mg / kg of IgG1-CD27-CDX1127 was lower than that in the IgG1-CD27-A-P329R-E345R treatment group and equivalent to that in the IgG1-b12-P329R-E345R treatment group (Figure 15). Similar observations were made in peripheral blood samples.

[0456] Example 18: OVA-specific CD8 from the spleens of OVA-immunized mice injected with anti-CD27 antibody+ IFNγ secretion by T cells The excised spleen tissue in RPMI1640 medium (see Example 17) was gently mashed with a 70-μm cell strainer (Falcon, catalog number 352350), pelleted by centrifugation (1,500 rpm, 5 minutes), and resuspended in 10 mL of Ammonium-Chloride-Potassium (ACK) Lysing Buffer (Invitrogen, catalog number A1049201). After incubation at RT for 3 - 5 minutes, the sample was washed twice with 10 - 20 mL of PBS and resuspended in 5 mL of Cellular Technology Limited (CTL) Test™ Medium (ImmunoSpot, catalog number CTLT-005) supplemented with 50 U / mL of penicillin and 50 μg / mL of streptomycin (pen / strep, Gibco, catalog number 15070-063). The collected spleen cells were filtered again through a 70-μm cell strainer and counted with a Vi-CELL™ XR Cell Viability Analyzer (Beckman Coulter), and the concentration was adjusted to 3.125×10 6 cells / mL with CTL-Test Medium containing pen / strep.

[0457] Mouse IFN-γ ELISpotPLUS kit (Mabtech, catalog number 3321-4HPW-2) was used to analyze IFNγ production by spleen cells essentially as described by the manufacturer. The pre-coated MultiScreenHTS IP Filter (MSIP) white plate (mAb AN18) was washed 4 times with 200 μL of sterile PBS per well and conditioned with 200 μL of CTL-Test Medium containing pen / strep (RT, 30 minutes). The medium was removed, and 5×10 5 spleen cells per well in duplicate were added with 2 μg / mL of OVA 257-264The peptide SIINFEKL (Invivogen, catalog number vac-sin), or the scrambled control peptide FILKSINE (SB-PEPTIDE, catalog number SB073-1MG), was incubated for 20 h at a total volume of 180 μL / well in a humidified incubator (37 °C, 5% CO 2 )). As a positive control for IFNγ production, splenocytes were incubated in parallel with a cell stimulation cocktail consisting of 500 ng / mL phorbol myristate acetate (PMA) and 10 μg / mL ionomycin (PMA + ionomycin, Dakewe Biotech, catalog number DKW ST PI). Cultures of splenocytes without peptide were included as negative controls. After incubation, the cells were removed and the plates were washed 5 times with PBS. The plates were then sequentially incubated with biotinylated detection mAb (R4-6A2; RT, 2 h), streptavidin-horseradish peroxidase (HRP; RT, 1 h), and finally 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (all provided by the kit), with 5 washing steps with PBS in between. If distinct spots appeared, the reaction was stopped by extensive washing in deionized water. Spots were counted on an AID iSpot ELISpot Reader (Autoimmun Diagnostika [AID] GMBH, ELR08IFL) using spotAID V8 software (AID). ELISpot data were analyzed using GraphPad Prism software, presented as bar graphs, and presented as the mean ± SEM of spots per well from all mice (n = 5) per treatment group.

[0458] As demonstrated by ELISpot analysis, splenocytes from all IgG1-CD27-A-P329R-E345R-treated animal groups showed increased IFNγ production in response to treatment with OVA peptide (Figure 16). Stimulation of splenocytes with scrambled control peptide either did not induce IFNγ production or induced minimal IFNγ production, suggesting that IFNγ was produced by OVA-specific T cells. In contrast, IFNγ production was not observed in splenocytes from mice treated with 30 mg / kg of IgG1-CD27-CDX1127.

[0459] Example 19: Effect of IgG1-CD27-A-P329R-E345R treatment on T cell activation in OVA-immunized mice in vivo CD8 + The effect of IgG1-CD27-A-P329R-E345R treatment on T cell activation was studied in vivo by analyzing the expression of PD-1 on CD8 + T cells derived from OVA-treated hCD27-KI mice. Mice were treated as described in Example 17. Also, the method of obtaining splenocytes and analyzing them by FACS is described in Example 17.

[0460] IgG1-CD27-A-P329R-E345R induced an increase in the percentage of CD8 + T cells expressing the activation marker PD-1 on day 28. CD8 + PD-1 + T cell percentage was low in animals treated with IgG1-CD27-CDX1127 or the control antibody IgG1-b12-P329R-E345R (Figure 17).

[0461] Example 20: Effect of IgG1-CD27-A-P329R-E345R treatment on in vivo induction of T cell subsets in OVA-immunized mice The effect of IgG1-CD27-A-P329R-E345R on the expansion and proliferation of T cell subsets was studied by analyzing the expression of CD44 and CD62L in splenocyte samples from OVA-treated hCD27-KI mice. Memory CD8 + T cells derived from the spleens of IgG1-CD27-A-P329R-E345R-treated, OVA-immunized, hCD27-KI mice were quantified by flow cytometry. Memory T cells were classified as effector memory (CD44 + CD62L - ) and pre-effector T cells (CD44 - CD62L - ; Nakajima, Y., K et al 2018). Mice were treated as described in Example 17. Also, the method of obtaining splenocytes and analyzing them by FACS is described in Example 17.

[0462] IgG1-CD27-A-P329R-E345R (30 mg / kg) induced an increase in the percentage of pre-effector T cells and effector memory CD8 + T cells in the spleen on day 28 when compared to splenocytes from mice treated with IgG1-b12-P329R-E345R (Figure 18). Within the CD45 + population, IgG1-CD27-A-P329R-E345R induced a higher percentage of pre-effector T cells and effector memory T cells than IgG1-CD27-CDX1127 (30 mg / kg), but equivalent mean percentages of these T cell populations were induced by both anti-CD27 antibodies in the CD8 + fraction of splenocytes.

[0463] Example 21: Effect of IgG1-CD27-A-P329R-E345R treatment on in vivo expansion and proliferation of T cells in OVA-immunized mice The effect of IgG1-CD27-A-P329R-E345R on the expansion and proliferation of T cells was studied by analyzing the expression of CD3 in spleen cells and blood samples from OVA-treated hCD27-KI mice. Mice were treated as described in Example 17. Also, the method of obtaining spleen cells and blood samples and analyzing them by flow cytometry is described in Example 17.

[0464] Treatment of OVA-immunized hCD27-KI mice with 30 mg / kg of IgG1-CD27-A-P329R-E345R did not increase the percentage of CD3 + T cells in the spleen compared to treatment with the non-binding control antibody IgG1-b12-P329R-E345R (Figure 19). In contrast, treatment with the benchmark antibody IgG1-CD27-CDX1127 (30 mg / kg) resulted in a decrease in CD3 + T cells in the spleen. Similar observations were made in peripheral blood samples.

[0465] Example 22: Effect of IgG1-CD27-A-P329R-E345R on T cell cytokine production in antigen-specific studies The ability of IgG1-CD27-A-P329R-E345R to increase cytokine production was studied using T cells stimulated with cognate antigen. PBMC were isolated from buffy coats obtained from healthy human donors by Ficoll-Paque density gradient separation (GE Healthcare, catalog number 17 1440 03) according to the manufacturer's instructions.

[0466] Human magnetic CD14 and CD8 microbeads (Miltenyi Biotec, catalog numbers 130 050 201 and 130 045 201, respectively) were used for positive selection of CD14 + monocytes and negative selection of CD14 - PBL from human PBMC, and CD8 from cryopreserved PBL +It was used for the positive selection of T cells. The cell suspension was centrifuged and resuspended in magnetic-activated cell sorting (MACS) buffer (Dulbecco's phosphate-buffered saline [DPBS] containing 5 mM EDTA and 1% human albumin) at 1×10 7 viable cells per 80 μL of MACS buffer. Per 1×10 7 cells, 12 μL of CD14 or CD8 microbeads were added. Subsequent MACS separation was performed using an automated magnetic cell separation device or by manual separation. Automated MACS separation was performed using an autoMACS® Pro Separator (Miltenyi Biotec) according to the manufacturer's instructions. The eluted CD14 + monocytes and CD8 + T cells were centrifuged (8 minutes, 300×g, RT), resuspended in X-VIVO 15 medium (Lonza), and counted for further use; i.e., monocyte differentiation into iDCs or electroporation of CD8 + T cells using PD-1 and / or CLDN6-specific T cell receptor (TCR) mRNA.

[0467] For the generation of monocyte-derived iDCs, up to 40×10 6 PBMC-derived CD14 + monocytes were cultured in a T175 flask in DC medium (RPMI 1640, 5% pooled human serum [PHS; One Lambda, catalog number A25761], 1× minimum essential medium non-essential amino acid solution [MEM NEAA, Life Technologies, catalog number 11140 035], 1 mM sodium pyruvate [Life Technologies, catalog number 11360 039]) supplemented with 100 ng / mL of human granulocyte / macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec, catalog number 130-093-868) and 50 ng / mL of human IL-4 (Miltenyi Biotec, catalog number 130093 924) for 5 days (37°C, 5% CO 2) After that. After 3 days of culture, half of the medium per flask was replaced. Since the medium obtained from the flask contained non-adherent monocytes, it was centrifuged (8 minutes, 300×g, RT), the supernatant was discarded, the cell pellet was resuspended in fresh DC medium, and then returned to the original flask together with 200 ng / mL of GM-CSF and 200 ng / mL of IL-4 (final concentration). After 5 days of incubation, the iDCs adhered to the culture flask were detached using 10 mL of DPBS containing 2 mM EDTA (37 °C, 10 minutes). The isolated iDCs were washed, pelleted (8 minutes, 300×g, RT), and used for electroporation with CLDN6 mRNA.

[0468] Human CD8 + T cells were electroporated with RNA encoding the alpha and beta chains of a mouse TCR specific for human CLDN6 alone or together with RNA encoding PD-1, and human monocyte-derived iDCs were electroporated with RNA encoding human CLDN6. Up to 5×10 6 individual iDCs or 15×10 6 individual CD8 + T cells were electroporated at RT in 250 μL of X-VIVO 15 medium using an ECM 830 Square Wave Electroporation System (BTX (registered trademark)). The cells were mixed with the RNA and pulsed (500 V, 3 ms for T cells or 300 V, 12 ms for iDCs) and immediately diluted with 750 μL of pre-warmed assay medium (IMDM GlutaMAX [Life technologies, catalog number 31980030] containing 5% PHS). The electroporated iDCs were transferred to 6- or 12-well plates and cultured O / N (37 °C, 5% CO 2 ) After O / N incubation, the electroporated CD8 + T cells and iDCs were evaluated by flow cytometry to determine cell purity, expression of the transfected RNA (PD-1 and CLDN6-TCR on CD8 + T cells and CLDN6 on iDCs), and CD8+ The baseline expression of CD27 and PD-1 on T cells and PD-L1 on iDCs was evaluated. Approximately 78% - 93%, 78% - 92%, and 36% - 98% of the electroporated CD8 + T cells expressed CLDN6-TCR, PD-1, and endogenous CD27, respectively. Approximately 47% - 91% and 94% - 99% of the electroporated iDCs expressed CLDN6 and endogenous PD-L1, respectively (not shown).

[0469] CD8 + T cells and iDCs were seeded in 96-well round-bottom plates at a ratio of 10:1 (7.5×10 4 T cells per well and 7.5×10 3 iDCs per well). IgG1-CD27-A-P329R-E345R was diluted in assay medium, and 25 μL of the diluted IgG1-CD27-A-P329R-E345R was added to the wells to reach a final concentration of 10 μg / mL. Similarly, the control antibodies IgG1-CD27-131A and IgG1-b12-P329R-E345R were added to reach a final concentration of 10 μg / mL. Antigen-specific T cell activity in antibody treatment was analyzed in vitro by measuring cytokines in the supernatant of T cells transduced to express CLDN6-TCR co-cultured with iDCs transduced to express and present CLDN6. The supernatant was collected after 2 days, and the concentrations of multiple pro-inflammatory cytokines and chemokines were determined by multiplex electrochemiluminescence assay (ECLIA) using a 10-spot U-PLEX ImmunoOncology Group 1 (human) kit (MSD; catalog number K151AEL 2) according to the manufacturer's instructions.

[0470] For the 10-spot U-PLEX Immuno-Oncology Group 1 kit, the biotinylated capture antibody was pre-incubated with the assigned linker having a biotin-binding domain at RT for 30 min, followed by incubation with the stop solution for 30 min. The plate was coated with a mix of capture antibodies linked by the linker by incubating at RT for 1 hr with shaking. The plate was washed 3 times with 1× MSD wash buffer. The supernatant samples or kit standards were diluted 1:2 in assay diluent, added to the wells, and incubated at RT for 2 h with constant shaking. The plate was washed 3 times with the wash buffer and incubated with the SULFO-TAG conjugate detection antibody from the kit at RT for 1 h with constant shaking. After the plate was washed 3 times with the wash buffer, read buffer B was added to catalyze the electrochemiluminescence reaction. The plate was immediately analyzed by measuring the light intensity with a MESO QuickPlex SQ 120 imager (MSD).

[0471] CD8 after 2-day incubation + The induced changes in cytokine production by IgG1-CD27-A-P329R-E345R were evaluated by multiplex ECLIA in the supernatant from T cell / iDC co-cultures (n = 4 different donors). IgG1-CD27-A-P329R-E345R uses CD8 + T cells expressing endogenous levels of PD-1 +Significant increases in the production of GM-CSF and IFNγ were induced in T cell / iDC co-cultures (Figure 20A), and increases in IL-13 and TNFα production were also observed. Considerable increases for the same cytokines were observed in cultures containing PD-1 overexpressing T cells (Figure 20B). Although cytokine levels generally decreased when T cells overexpressed PD-1, the relative increases (fold increases) in cytokine production in the presence of IgG1-CD27-A-P329R-E345R were generally high in this setting (Figure 20A and Figure 20B). In contrast, the prior art anti-CD27 antibody IgG1-CD27-131A showed minimal effect on cytokine production compared to the non-binding control antibody IgG1-b12-P329R-E345R (Figure 20A and Figure 20B).

[0472] Example 23: Expression of cytotoxicity-related molecules by antigen-specific CD8 + T cells incubated with IgG1-CD27-A-P329R-E345R Induction of T cell-mediated cytotoxicity by antibody treatment was investigated by analyzing the expression of cytotoxicity-related molecules on antigen-specific T cells by flow cytometry in co-cultures of human healthy donor T cells transduced to express CLDN6-TCR and MDA-MB-231_hCLDN6 target cells.

[0473] MDA-MB-231_hCLDN6 cells were generated by lentiviral transduction. For this purpose, 2 × 10 5 MDA-MB-231 cells were seeded per well in a 12-well tissue culture plate in 250 μL of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, catalog number 31966-047) supplemented with 10% FBS (non-heat inactivated). The cells were incubated at 37°C (7.5% CO 2) Incubated for 1 - 2 h. The supernatant containing the lentiviral vector (pL64b42E(EF1a - hClaudin6)Hygro - T2A - GFP) encoding human CLDN6 was thawed on ice and diluted with a total volume of 750 μL of DMEM / 10% FBS to obtain titers of 2×10 5 , 8×10 4 , and 3.2×10 4 TU / mL. These titers corresponded to MOIs of 1, 0.4, and 0.16, respectively. The supernatant was then added to MDA - MB - 231 cells, and the cells were incubated at 37 °C (5% CO 2 ) for 72 h without agitation. For the experiments described in this example, MDA - MB - 231 - hCLDN6 cells were cultured in DMEM / 10% FBS. The cells were passaged and harvested for experiments at 70% - 90% confluence. The cells were detached with Accutase (Thermo Fisher Scientific, catalog number A11105010) for 5 min (37 °C, 7.5% CO 2 ) and resuspended by adding culture medium. The cells were centrifuged (300×g, 4 min, RT) and counted. MDA - MB - 231_hCLDN6 cells were not cultured for more than 20 passages.

[0474] MDA - MB - 231_hCLDN6 cells were seeded at 1.2 - 1.5×10 4 cells / well in 96 - well flat - bottom plates (for flow cytometry analysis) and xCELLigence E - plates (Agilent, catalog number 05232368001; for impedance measurement) and allowed to stand at RT for 30 min. Next, the plates were incubated in an incubator and an xCELLigence real - time cell analysis (RTCA) instrument (ACEA Biosciences) for 1 day (37 °C, 5% CO 2 ).

[0475] Isolated CD8 + T cells (see Example 22) were electroporated with CLDN6 - specific TCR mRNA and incubated O / N. CD8+ After T cell isolation and electroporation, the T cell cultures contained 49% - 99% CD8 + T cells. Of these electroporated CD8 + T cells, approximately 78% - 93% expressed CLDN6 - TCR, and 59% - 98% of the CLDN6 - TCR + CD8 + cells were CD27 + . The cells were centrifuged (8 minutes, 300×g, RT), resuspended in DMEM / 10% FBS, and counted. The cells were centrifuged again and resuspended in DMEM / 10% FBS at 3×10 6 cells / mL and added to wells containing previously seeded MDA - MB - 231_hCLDN6 cells (1.5×10 5 individual CD8 + T cells / well; T cell: tumor cell ratio, effector: target ratio was 10:1). IgG1 - CD27 - A - P329R - E345R, IgG1 - CD27 - 131A, and the non - binding control antibody IgG1 - b12 - P329R - E345R were added to the co - cultures at 10 μg / mL. The expression of CD107a and GzmB was determined by flow cytometry.

[0476] After 2 - day incubation in the presence of 10 μg / mL of IgG1 - CD27 - A - P329R - E345R, the percentage of GzmB + CD107a + CD8 + T cells was significantly enhanced compared to treatment with the non - binding control antibody or the prior art anti - CD27 antibody IgG1 - CD27 - 131A (Figure 21).

[0477] In conclusion, these data indicate that IgG1 - CD27 - A - P329R - E345R was able to induce cytotoxic - related molecules on activated antigen - specific T cells.

[0478] Example 24: Ability of IgG1 - CD27 - A - P329R - E345R to Induce T Cell - Mediated Tumor Cell Lysis To evaluate T cell-mediated cytotoxicity, CLDN6-TCR electroporated CD8 + T cells were co-cultured with MDA-MB-231_hCLDN6 cells in the presence of IgG1-CD27-A-P329R-E345R, the prior art anti-CD27 antibody IgG1-CD27-131A, or the non-binding control antibody IgG1-b12-P329R-E345R for 5 days in an xCELLigence real-time cell analysis instrument (Acea Biosciences) with impedance measurements at 2-hour intervals as described in Example 23. Cell index values were derived from the impedance measurements performed at 2-hour intervals. The area under the curve (AUC) was obtained from the cell index data over the 5-day co-culture. The AUC was normalized against the co-culture treated with IgG1-b12-P329R-E345R. The scale of impedance depends on the cell number, cell morphology, and cell size as well as the strength of cell adhesion to the plate, and together these are used in this particular case as an indirect readout of tumor cell mass. A decrease in impedance in this experimental setting is considered a surrogate for tumor cell killing by CD8 + T cells. It should be noted that impedance may underestimate tumor cell killing due to T cell proliferation.

[0479] IgG1-CD27-A-P329R-E345R induced a decrease in the cell index, which is an indicator of tumor cell killing. IgG1-CD27-131A had no visible effect on the cell index and indicated a minimal ability to increase tumor cell killing (Figure 22).

[0480] Example 25: Ability of IgG1-CD27-A-P329R-E345R to induce the expansion of tumor-infiltrating lymphocytes The ability of IgG1-CD27-A-P329R-E345R to induce the expansion of tumor-infiltrating lymphocyte (TIL) subsets (CD4 + and CD8 + T cells, NK cells, and regulatory T cells [Treg]) was evaluated ex vivo using cryopreserved tumors surgically resected from NSCLC patients.

[0481] Surgically resected human NSCLC tissues were received in transport medium (HypoThermosol® FRS Preservation Solution [BioLife Solutions, catalog number 101104], 7.5 μg / mL amphotericin B [Thermo Fisher Scientific, catalog number 15290026], and 300 units / mL (U / mL) pen / strep [Thermo Fisher Scientific, catalog number 15140-122]). The samples were washed three times with wash medium (5 mL of X-VIVO 15 [Lonza], 2.5 μg / mL amphotericin B, [Thermo Fisher Scientific] and 100 U / mL pen / strep [Thermo Fisher Scientific]) and transferred to cell culture dishes. Adipose tissue and necrotic compartments were removed with a scalpel, and the tissue was cut into fragments approximately 5 mm 3 in length. Each fragment was placed into an individual cryovial, and 1 mL of freezing medium (FBS, 10% DMSO) was added to each vial. The vials were transferred to a controlled freezing chamber (Mr. Frosty freezing container) placed in an -80 °C freezer. After at least 16 h at -80 °C, the vials were transferred to liquid nitrogen for long-term storage.

[0482] Four to six cryopreserved vials containing approximately 5 mm 3 tumor fragments from one tumor specimen were thawed in a 37 °C water bath for approximately 2 minutes for each experiment, washed five times with wash medium, and transferred to cell culture dishes. The tumor fragments were further dissected with a scalpel into fragments approximately 1 mm 3 in length. Most of the fragments were used for TIL expansion and proliferation in culture with IL-2 and the treatment antibody, and the remaining fragments were used to determine the expression of unique cell surface markers at baseline without any treatment.

[0483] (On average), two tumor fragments per well were seeded into 0.1 mL of pre-warmed TIL culture medium (X-VIVO 15 [Lonza] containing 2% human serum albumin [HSA; CSL Behring, catalog number PZN-00504775], 100 U / mL pen / strep [Thermo Fisher Scientific], and 2.5 μg / mL amphotericin B [Thermo Fisher Scientific], with 45 - 50 U / mL of IL-2 (Proleukin S; Novartis Pharma, catalog number PZN-02238131)) in a 24-well plate (total volume capacity of 2 mL / well was used in the assay). IgG1-CD27-A-P329R-E345R was diluted in TIL culture medium containing 45 - 50 U / mL of IL-2, and 900 μL of this dilution was added to the wells as appropriate. The final IgG1-CD27-A-P329R-E345R concentration in the wells was 1 or 10 μg / mL. As a control, medium containing 45 - 50 U / mL of IL-2 without antibody was added to the tumor fragments in separate wells. A total of 8 - 16 wells were incubated for each experimental condition (37 °C, 5% CO 2 ) per donor.

[0484] After 3 days of culture, fresh TIL culture medium containing 45 - 50 U / mL of IL-2 and IgG1-CD27-A-P329R-E345R was added to the wells (1 mL / well, same antibody concentration as above). 5 - 14 / 17 days after the start of the assay, the cultures were monitored regularly by microscopy for the outgrowth of TILs migrating from the tissue fragments and the formation of TIL microclusters. If more than 25 TIL microclusters were observed in one well after 7 or 8 days of culture, the cells and tissue fragments from two identically treated original wells were resuspended and pooled into one well of a 6-well plate containing culture medium (total volume capacity of 5 - 6 mL / well was used in the assay), and fresh IL 2-containing TIL culture medium was added (estimated final concentration of 33 U / mL of IL-2).

[0485] Every 2 - 3 days, the cultures were replenished with fresh IL - 2 - containing TIL culture medium. The IL - 2 concentration in the medium added to the cultures was reduced to 10 U / mL, or first to 25 U / mL and then to 10 U / mL, and the wells were replenished with medium throughout the assay. On day 14 or 17, cells were harvested for flow cytometry analysis.

[0486] IgG1 - CD27 - A - P329R - E345R enhanced the expansion of TIL subtypes compared to control cultures treated with IL - 2 alone, and the greatest relative increase in cell count was in CD8 + T cells and Tregs were observed, followed by CD4 + T cells, and NK cells. For all TIL subsets, expansion was more pronounced when using 1 μg / mL of IgG1 - CD27 - A - P329R - E345R than 10 μg / mL (Table 4 and Figure 23).

[0487] (Table 4) Fold expansion of TIL treated with IgG1 - CD27 - A - P329R - E345R Tumor tissues derived from human NSCLC specimens were cultured with low - dose IL - 2 in the presence or absence of IgG1 - CD27 - A - P329R - E345R. The absolute cell counts of the indicated cell subsets were determined by flow cytometry 14 - 17 days after treatment. The fold difference in cell numbers for IgG1 - CD27 - A - P329R - E345R - treated cultures compared to IL - 2 - treated cultures is shown. The data shown are from five tumor tissues from five individual patients tested in five independent experiments. P = 0.0236, 1 μg / mL vs. 10 μg / mL IgG1 - CD27 - A - P329R - E345R (two - way ANOVA). TIFF2025516633000017.tif89166 a Patient #561 was excluded for better comparability between cell populations in the calculation of mean and SD. Abbreviations: ANOVA = analysis of variance; n.d. = not determined; NK = natural killer; NSCLC = non-small cell lung cancer; SD = standard deviation; TIL = tumor-infiltrating lymphocyte; Treg = regulatory T cell.

[0488] Example 26: BRET assay to evaluate intermolecular interactions of IgG1-CD27-A-P329R-E345R molecules on the cell surface The ability of a CD27 antibody harboring a hexamerization-enhancing mutation (E345R) that increases intermolecular Fc-Fc interactions after binding to CD27 on the cell surface was determined using a bioluminescence resonance energy transfer (BRET) assay. This molecule proximity-based assay detects protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. Energy transfer occurs only when the donor and acceptor are in close proximity (<10 nm [Wu and Brand, 1994; Dacres et al, 2012]).

[0489] First, in addition to CD27, the cell surface expression of CD20 and CD37 (as positive control molecules) was determined on the human chronic myelogenous leukemia cell line, huCD27-K562, which was genetically modified to stably express human CD27, and on Daudi cells, using an indirect immunofluorescence assay (QIFIKIT, Agilent Technologies, catalog number K0078). Cells were seeded at 100,000 cells / well and incubated with 10 μg / mL of primary antibodies (CD27: IgG1-7730-143-C102S-FEAL; CD20: IgG1-11B8-FEAR; CD37: IgG1-3009-010-FEAR). This was followed by incubation with FITC-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, catalog number 109-096-097), and in parallel, incubation with QIFIKIT beads coated with a defined number of antibody molecules. The number of antibody molecules per cell was determined by interpolating the measured MFI of the test sample in a calibration curve generated by plotting the mean fluorescence intensity (MFI) of individual bead populations against the known number of antibody molecules per bead. Samples were measured on an LSRFortessa Cell Analyzer flow cytometer (BD Biosciences) and analyzed using FlowJo software.

[0490] QiFi analysis showed moderate CD27 expression and high CD20 and CD37 expression on Daudi cells, while huCD27-K562 cells expressed high levels of CD27 but did not express CD20 and CD37 (Table 5).

[0491] (Table 5) Cell surface expression of antibody molecules per cell TIFF2025516633000018.tif28166

[0492] The BRET assay (NanoBRET (trademark) System, Promega, catalog number N1661) was essentially performed according to the manufacturer's instructions. To generate the NanoLuc (donor) and HaloTag (acceptor) tagged antibodies, variable light chain sequences (Table 1, sequences 71 - 78) having either NanoLuc or HaloTag were prepared by gene synthesis, cloned into appropriate expression vectors, and full-length antibodies were produced as described in Example 1. For analysis, 0.5×10 5 individual huCD27-K562 or Daudi cells were seeded in a 96-well round-bottom plate (Greiner Bio-One, catalog number 650101) at a total volume of 100 μL. The cells were pelleted by centrifugation (3 minutes, 300×g) and resuspended in 50 μL of assay medium (Opti-MEM I [Gibco, catalog number 11058-021] + 4% FBS [ATCC, catalog number 30-2020]) containing a mixture of NanoLuc or HaloTag tagged antibody pairs at a concentration of 5 μg / mL each. Next, 50 μL of HaloTag NanoBret 618 ligand (Promega, catalog number G980A, 1:1000 dilution in assay medium) was added. For each antibody mixture, ligand-free control samples were prepared in parallel by adding 50 μL of medium without the HaloTag NanoBret 618 ligand. The cells were incubated for 30 minutes in the dark at 37 °C, washed twice with medium, and resuspended in 100 μL of assay medium without FBS. 25 μL of NanoBRET NanoGLO substrate (Promega, catalog number N1571, 1:200 dilution in assay medium without FBS) was added to each well. The plate was shaken for 30 s and 120 μL of each sample was transferred to an OptiPlate (Perkin Elmer, catalog number 6005299). Donor luminescence was measured at 460 nm and acceptor luminescence was measured at 618 nm using an EnVision Multilabel Reader (Perkin Elmer).

[0493] BRET is in milliBRET units (mBU) = (618 nm em / 460 nm em ) was calculated at ×1000.

[0494] The results are reported as corrected BRET, which is corrected for donor contribution background or bleed-through and is calculated as the mBU ligand - mBU ligand-free control.

[0495] The proximity of NanoLuc and HaloTag-labeled IgG1-CD27-A-P329R-E345R antibodies after binding to CD27 on the cell surface was compared to the WT IgG1-CD27-A antibody with the same tags. IgG1-CD20-11B8-E430G-LNLuc and IgG1-CD37-37.3-E430G-LHalo antibodies containing the E430G mutation (WO2019243636A1) that induces hexamerization were used as positive controls for proximity-induced BRET. IgG1-CD20-11B8-E430G and IgG1-CD37-37.3-E430G have previously been shown to form heterohexamers in binding to cells expressing CD20 and CD37 using a molecular proximity assay (Oostindie, S.C. et al, Haematologica, 2019). The non-binding antibody IgG1-b12-P329R-E345R was used as a negative control.

[0496] As positive and negative controls for BRET signal induction, Daudi cells (high CD20 and CD37 expression) and huCD27-K562 cells (no CD20 and CD37 expression) were opsonized with the antibody pairs IgG1-CD20-11B8-E430G-LNLuc and IgG1-CD37-37.3-E430G-LHalo. BRET induction was detected only in Daudi cells and not in huCD27-K562 cells lacking CD20 and CD37 (Figure 24). Similarly, the non-binding control antibody pair (IgG1-b12-P329R-E345R-LNLuc + IgG1-b12-P329R-E345R-LHalo) did not induce BRET in either cell line. When huCD27-K562 cells were opsonized with a mixture of NanoLuc and HaloTag-labeled CD27 antibodies with hexamerization-enhancing mutations (IgG1-CD27-A-P329R-E345R-LNLuc + IgG1-CD27-A-P329R-E345R-LHalo), high BRET was detected, but the BRET in Daudi cells did not exceed the background level (Figure 24). The mixture of IgG1-CD27-A-LNLuc and IgG1-CD27-A-LHalo (WT) antibodies induced significantly lower BRET in huCD27-K562 cells compared to the CD27 antibodies with P329R and E345R mutations and did not induce BRET in Daudi cells. These results indicate that the BRET signal was associated with higher target expression. CD27 expression on huCD27-K562 cells was found to be approximately 26-fold higher than on Daudi cells, and the BRET level for CD27-binding IgG1-CD27-A-P329R-E345R on huCD27-K562 cells was approximately 24-fold higher than in Daudi cells.Mixtures of NanoLuc- and HaloTag-labeled non-binding and CD27-binding antibody pairs (IgG1-b12-P329R-E345R-LNLuc + IgG1-CD27-A-P329R-E345R-LHalo and IgG1-CD27-A-P329R-E345R-LNLuc + IgG1-b12-P329R-E345R-LHalo, respectively) did not induce BRET in any cell line. This supports that the observed BRET was dependent on the simultaneous interaction of donor and acceptor antibodies bound to the cell surface target.

[0497] In summary, IgG1-CD27-A-P329R-E345R induced high BRET in huCD27-K562 cells compared to its WT variant. This finding supports the enhanced proximity between membrane-bound IgG1-CD27-A-P329R-E345R molecules compared to its WT variant, consistent with the E345R-enhanced Fc-Fc interaction between cell surface-binding antibodies. Note: The experiments described in this example used a variant of IgG1-CD27-A with an F405L mutation that is not functionally relevant in the context of this experiment.

[0498] Example 27: FcγRIa + Binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages In Example 9, surface plasmon resonance (SPR) was used to evaluate the binding of IgG1-CD27-A-P329R-E345R to human FcγR variants, showing minimal binding (FcγRIa) or no binding (FcγRIIa, FcγRIIb, and FcγRIIIa) to recombinant human IgG Fc receptor molecules. This residual FcγRIa binding is CD27 +It was not sufficient to induce IgG1-CD27-A-P329R-E345R-dependent ADCP of cells (see Example 13). To further exclude the interaction of IgG1-CD27-A-P329R-E345R with FcγRIa-positive macrophages, the Fc-mediated binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages was determined.

[0499] Human CD14 as described in Example 13 + Monocytes were isolated from PBMCs from two healthy donors and differentiated into monocyte-derived macrophages by culturing the cells in medium (CellGenix, catalog number 20801-0500) supplemented with 50 ng / mL M-CSF (Gibco, catalog number PHC9501) to obtain M0 macrophages, or 50 ng / mL GM-CSF (Immunotools, catalog number 11343125) for differentiation into M1 macrophages. After 6 days of culture, the M0 and M1 phenotypes were confirmed by FACS analysis according to the expression of markers defined in Table 6. Additionally, both macrophage subtypes were confirmed to express the human Fc receptors FcγRIa, FcγRII, and FcγRIIIa (Table 6).

[0500] (Table 6) TIFF2025516633000019.tif60166

[0501] The binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages was compared to the binding of a WT IgG1 antibody (IgG1-b12) with an irrelevant antigen-binding region as a positive control for FcγRIa binding, and a variant of the same antibody (IgG1-b12-P329R-E345R) that also has the P329R mutation previously described to reduce interaction with FcγR. Since macrophages are not supposed to express CD27, the hypothesis was put forward that any observed binding occurs via FcγRIa, the only FcγR that binds monovalent IgG. Differentiated macrophages were incubated for 15 minutes with IgG1-CD27-A-P329R-E345R or control antibody (30 μg / mL in DC medium), and PE-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, catalog number 109-116-097, diluted 1:200, 30 minutes, 4°C). After incubation, the cells were washed and resuspended in 100 μL of FACS buffer containing nuclear-staining DAPI (BD Pharmingen, catalog number 564907, diluted 1:5000). Samples were measured on a FACSymphony flow cytometer (BD Biosciences) and analyzed using FlowJo software.

[0502] Binding above background (secondary antibody only) to M0 or M1 macrophages isolated from two independent donors was not observed for either IgG1-CD27-A-P329R-E345R or control IgG1-b12-P329R-E345R (Figure 25). WT IgG1-b12 containing the active Fc region consistently bound to both M0 and M1 macrophages.

[0503] In conclusion, IgG1-CD27-A-P329R-E345R and control IgG1-b12-P329R-E345R do not bind to M0 or M1 macrophages expressing FcγRIa, FcγRII, and FcγRIIIa.

[0504] Example 28: Polyclonal activation of CD8 by IgG1-CD27-A-P329R-E345R in combination with DuoBody-CD40x4-1BB + Induction of T cell proliferation Activated human CD8 + The effect of IgG1-CD27-A-P329R-E345R in combination with DuoBody-CD40x4-1BB on T cells was analyzed by flow cytometry using freshly isolated human healthy donor PBMCs that were polyclonally stimulated with a CD3 antibody.

[0505] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from human healthy donor buffy coats by density gradient centrifugation using lymphocyte separation medium (Corning, catalog number 25-072-CI) according to the manufacturer's instructions. PBS (HyClone, catalog number SH3A3830.03) supplemented with 2% donor bovine serum iron (DBSI; Gibco, catalog number 20371-030) at 10×10 6PBMCs were washed twice at a density of cells / mL and labeled with CTV using the CellTrace™ Violet Cell Proliferation Kit (Invitrogen, catalog number C34557A, diluted in PBS) according to the manufacturer's instructions. CTV-labeled PMBCs (7.5×104 cells / well) were plated in round-bottom 96-well plates (Greiner Bio-One, catalog number 650180) and mixed with anti-CD3 antibody (aCD3, clone UCHT1, final concentration 0.1 μg / mL in the assay, Stemcell, catalog number 60011) in assay medium (RPMI 1640 [Lonza, catalog number 12-115F], 10% donor bovine serum containing iron [DBSI; Gibco, catalog number 20731-030], 1% Pen / Strep [Lonza, catalog number DE17-603E]) to trigger T cell activation. Subsequently, PBMCs stimulated with aCD3 were incubated at 37 °C for 4 days in a total volume of 150 μL with IgG1-CD27-A-P329R-E345R (0.0016 - 10 μg / mL at 5-fold dilution) and DuoBody-CD40x4-1BB (0 - 0.000064 μg / mL at 5-fold dilution), either alone or in combination. The cell suspension was pelleted and incubated with FACS buffer (PBS [Lonza, catalog number BE17517Q], 0.02% sodium azide [bioWorld, catalog number 41920044 3], 0.1% BSA [Roche, catalog number 43279213], 2 mM EDTA [Sigma, catalog number BCCD3789]) containing the lymphocyte marker AF700-labeled anti-human CD8 (BD BioLegend, catalog number 301028, 1:50) at 4 °C for 30 minutes. The cells were washed three times with FACS buffer and resuspended in FACS buffer containing the viability dye TO-PRO-3 (Invitrogen, catalog number T3605). Flow cytometry data were acquired on a FACS Symphony (BD).

[0506] Viable CD8 + T cell subset (CD8 +The CTV dilution peak in 7-AAD was analyzed using the proliferation modeling tool in FlowJo software (v10.7.3), and the expansion growth index was determined according to the following formula: Number of cells at the start of culture = (G0) + (G1) / 2 + (G2) / 4 + (G3) / 8 + (G4) / 16 +...(GN / 2N) Expansion growth index = sum of total cell numbers (G0 to GN) / number of cells at the start G0 to GN are single proliferation peaks, G0 represents the fraction of non-dividing cells, and GN represents the fraction of cells that have divided N times.

[0507] CD8 + (Figure 26) A dose-dependent increase in T cell proliferation was observed across the full antibody concentration range in PBMC samples treated with IgG1-CD27-A-P329R-E345R alone. The dose-response curve of the sample treated with DuoBody-CD40x4-1BB alone showed a bell-shaped curve and reached the maximum expansion growth index at an antibody concentration of 1 μg / mL. The combination of IgG1-CD27-A-P329R-E345R with DuoBody-CD40x4-1BB increased CD8 + T cell proliferation more potently than each antibody alone, and the maximum effect was reached at the highest tested IgG1-CD27-A-P329R-E345R concentrations (2 - 10 μg / mL) combined with medium to high concentrations (0.04 - 5 μg / mL) tested for DuoBody-CD40x4-1BB.

[0508] These data indicate that the combination of IgG1-CD27-A-P329R-E345R with DuoBody-CD40x4-1BB showed a higher increase in T cell proliferation compared to each antibody alone.

[0509] Example 29: Antigen-specific stimulation assay to determine the ability of IgG1-CD27-A-P329R-E345R in combination with DuoBody-CD40x4-1BB to enhance T cell proliferation To determine the combinatorial effects of IgG1-CD27-A-P329R-E345R and DuoBody-CD40x4-1BB on T cell proliferation and cytokine production compared to single-agent activity, antigen-specific stimulation assays were performed using co-cultures of healthy human CD8+ T cells and cognate antigen-expressing immature dendritic cells (iDC).

[0510] HLA-A*02 + Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMC by magnetic-activated cell sorting (MACS) technology using anti-CD14 microbeads (Miltenyi; catalog number 130-050-201) according to the manufacturer's instructions. Peripheral blood lymphocytes (PBL, CD14-negative fraction) were cryopreserved in RPMI 1640 containing 10% DMSO (AppliChem GmbH, catalog number A3672,0050) and 10% human albumin (CSL Behring, PZN 00504775) for T cell isolation. For differentiation into iDC, 40×10 per mL 6Individual monocytes were cultured for 5 days in RPMI 1640 (Life Technologies GmbH, catalog number 61870-010) containing 5% pooled human serum (One Lambda Inc., catalog number A25761), 1 mM sodium pyruvate (Life technologies GmbH, catalog number 11360-039), 1x non-essential amino acids (Life Technologies GmbH, catalog number 11140-035), 200 ng / mL granulocyte macrophage colony-stimulating factor (GM-CSF; Miltenyi, catalog number 130-093-868) and 200 ng / mL human interleukin-4 (IL-4; Miltenyi, catalog number 130-093-924). On day 3, half of the medium was replaced with fresh medium containing supplements. On day 5, iDCs were harvested by collecting non-adherent cells and adherent cells were detached by incubation with Dulbecco's phosphate buffered saline (DPBS) containing 2 mM EDTA for 10 minutes at 37 °C. After washing with DPBS, iDCs were cryopreserved in fetal bovine serum (FBS) (Sigma-Aldrich, catalog number F7524) containing 10% dimethyl sulfoxide (DMSO) (AppliChem GmbH, catalog number A3672,0050) for future use in antigen-specific T cell assays.

[0511] Antigen-specific CD8 + One day before the start of the T cell stimulation assay, frozen PBLs and iDCs from the same donor were thawed. CD8 + T cells were isolated from PBLs by MACS technology using anti-CD8 microbeads (Miltenyi, catalog number 130-045-201) according to the manufacturer's instructions. Approximately 10×10 6 ~15×10 6 of CD8 +T cells were electroporated with 10 μg each of in vitro transcribed (IVT)-RNA encoding the alpha and beta chains of a mouse TCR (HLA-A*02 restricted) specific for human Claudin-6 (CLDN6) (described in WO 2015150327 A1) in 250 μL of X-VIVO™ 15 medium (Lonza, catalog number BE02-060Q). Cells were transferred to a 4 mm electroporation cuvette (VWR International GmbH, catalog number 732-0023) and electroporated using a BTX ECM® 830 Electroporation System (BTX; 500 V, 3 ms pulse). Immediately after electroporation, cells were transferred to fresh IMDM GlutaMAX medium (Life Technologies GmbH, catalog number 319800-030) containing 5% pooled human serum and incubated at 37°C, 5% CO 2 for at least 1 hour. T cells were labeled with 0.8 μM carboxyfluorescein succinimidyl ester (CFSE; Life Technologies GmbH, catalog number V12883) in PBS according to the manufacturer's instructions and incubated overnight in IMDM medium supplemented with 5% human serum.

[0512] Up to 5×10 6 Thawed iDCs (up to 5×10) were electroporated with 2 μg of IVT-RNA encoding full-length human CLDN6 (WO 2015150327 A1) in 250 μL of X VIVO™ 15 medium using the above electroporation system (300 V, 12 ms pulse) and incubated overnight in IMDM medium supplemented with 5% pooled human serum.

[0513] The electroporated iDCs were incubated in IMDM medium containing 5% pooled human serum in 96-well round-bottom plates at a ratio of 1:10 (DC:T cells) with electroporated, CFSE-labeled T cells in the presence of IgG1-CD27-A-P329R-E345R (0.1, 1 or 10 μg / mL), DuoBody-CD40x4-1BB (0.0022, 0.0067 or 0.2 μg / mL), or a combination of both. After 4 days of culture, the cells were stained with an APC-conjugated anti-human CD8 antibody (e.g., PE-Cy7 conjugate, BD Biosciences, catalog number 557750). CD8 + T cell proliferation was evaluated by flow cytometric analysis of CFSE dilution in CD8 + T cells. Flow cytometry data were analyzed using FlowJo software version 10.7.1. The proliferation modeling tool in FlowJo was used to evaluate the CFSE-label dilution of CD8 T cells, and the expansion growth index was calculated using the following formula: Number of cells at the start of culture = (G0) + (G1) / 2 + (G2) / 4 + (G3) / 8 + (G4) / 16 +... (GN / 2N) Expansion growth index = sum of all cells (G0 to GN) / number of cells at the start

[0514] Cytokine concentrations in cell culture supernatants were determined by multiplex electrochemiluminescence immunoassay (ECLIA) using the V-Plex Proinflammatory Panel 1 (human) assay (Meso Scale Discovery, catalog number K15049D) for the detection of human interferon (IFN)γ according to the manufacturer's protocol.

[0515] Treatment with IgG1-CD27-A-P329R-E345R or DuoBody-CD40x4-1BB alone enhanced CD8+ T cell proliferation in a dose-dependent manner compared to co-cultures without antibody treatment. The combination of IgG1-CD27-A-P329R-E345R and DuoBody-CD40x4-1BB further enhanced the single-agent activity (Figure 27). Treatment with the combination of 1 or 10 μg / mL of IgG1-CD27-A-P329R-E345R and 0.0067 or 0.2 μg / mL of DuoBody-CD40x4-1BB led to a small increase compared to treatment with the single-agent DuoBody-CD40x4-1BB but resulted in higher proliferation than treatment with each compound individually.

[0516] Treatment with IgG1-CD27-A-P329R-E345R or DuoBody-CD40x4-1BB alone enhanced the secretion of the pro-inflammatory cytokine IFNγ in a dose-dependent manner compared to co-cultures without antibody treatment (Figure 28). Treatment with the combination of 1 or 10 μg / mL of IgG1-CD27-A-P329R-E345R and 0.0067 μg / mL of DuoBody-CD40x4-1BB led to a small increase compared to treatment with the single-agent DuoBody-CD40x4-1BB but further enhanced cytokine secretion.

Brief Description of the Drawings

[0517]

Figure 1

Figure 2

Figure 3

Figure 4A-1

Figure 4A-2

Figure 4B-1

Figure 4B-2

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9-1

Figure 9-2

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14A-1

Figure 14A-2

Figure 14B-1

Figure 14B-2

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Claims

**Claim 1** (i) a first binder comprising at least one binding region that binds to CD27; and (ii) a second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 A method for reducing or preventing tumor progression or treating cancer in a subject, comprising the step of administering to the subject. **Claim 2** The method according to claim 1, wherein the first binder comprises a heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NO: 5, 6, and 7, respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NO: 9, 10, and 11, respectively. **Claim 3** The method according to claim 1 or 2, wherein the first binder comprises two binding regions capable of binding to human CD27, and the first binder comprises a heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NO: 5, 6, and 7, respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NO: 9, 10, and 11, respectively. **Claim 4** The method according to any one of the preceding claims, wherein the first binder comprises a VH region comprising the sequence shown in SEQ ID NO:

4. **Claim 5** The method according to any one of the preceding claims, wherein the first binder comprises a VL region comprising the sequence shown in SEQ ID NO:

8. **Claim 6** The method according to any one of the preceding claims, wherein the first binder comprises a VH region and a VL region comprising the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively. **Claim 7** The method according to any one of the preceding claims, wherein the first binder is an antibody, preferably a human antibody or a humanized antibody. **Claim 8** The method according to any one of the preceding claims, wherein the antibody is a full-length antibody further comprising a light chain constant region (CL) and a heavy chain constant region (CH). **Claim 9** The method according to claim 8, wherein the light chain constant region is human kappa. **Claim 10** The method according to claim 8, wherein the light chain constant region is human lambda. **Claim 11** The method according to any one of the preceding claims, wherein the first binder further comprises a heavy chain constant region, the heavy chain constant region being a human IgG isotype and optionally a modified human IgG.

12. The method according to claim 11, wherein the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4, such as human IgG1.

13. The method according to claim 11 or 12, wherein the IgG is a modified human IgG comprising one or more amino acid substitutions.

14. The method according to any one of claims 11 to 13, wherein the modified human IgG is a modified human IgG1 comprising one or more amino acid substitutions, such as two or more amino acid substitutions.

15. The method according to any one of claims 11 to 14, wherein the modified human IgG heavy chain constant region comprises up to 10 amino acid substitutions, such as up to 9, such as up to 8, such as up to 7, such as up to 6, such as up to 5, such as up to 4, such as up to 3, such as up to 2 amino acid substitutions.

16. The method according to any one of claims 11 to 15, wherein the substitution in the heavy chain constant region induces an increase in CD27 agonism compared to the same antibody except that it comprises the wild-type IgG1 antibody heavy chain constant region.

17. The method according to any one of claims 11 to 16, wherein the amino acid residue at the position corresponding to position E345 or E430 in the human IgG1 heavy chain according to Eu numbering is selected from the group comprising A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

18. The method according to any one of claims 11 to 17, wherein the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to Eu numbering is R.

19. The method according to any one of claims 11 to 18, wherein the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain according to Eu numbering is G.

20. The method according to any one of claims 11 to 19, wherein the amino acid residue at the position corresponding to position P329 in the human IgG1 heavy chain according to Eu numbering is R.

21. The method according to any one of claims 11 to 20, wherein the amino acid residues at the positions corresponding to positions E345 and P329 in the human IgG1 heavy chain according to Eu numbering are both R.

22. The method according to any one of claims 11 to 21, wherein the first binder has a pharmacokinetic profile similar to that of a parental antibody comprising a wild-type IgG1 heavy chain constant region.

23. The method according to any one of the preceding claims, wherein the first binder comprises a heavy chain constant region comprising a sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and 36.

24. The method according to any one of the preceding claims, wherein the first binder comprises a heavy chain constant region comprising the sequence shown in SEQ ID NO:

15.

25. The method according to any one of the preceding claims, wherein the first binder comprises a heavy chain constant region, and the heavy chain constant region is modified so that the first binder induces one or more Fc-mediated effector functions to a lower extent compared to the parental antibody.

26. The method according to claim 25, wherein the one or more Fc-mediated effector functions are reduced by at least 20%, such as at least 30% or at least 40%, or at least 50% or at least 60% or at least 70%, or at least 80% or at least 90%.

27. The method according to claim 25 or 26, wherein the first binder does not induce one or more Fc-mediated effector functions.

28. The one or more Fc-mediated effector functions are from the following group: complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding The method according to any one of claims 25 to 27, selected from.

29. The method according to any one of claims 25 to 28, wherein the first binder does not induce C1q binding when measured by the method of Example 8.

30. The method according to any one of the preceding claims, wherein the first binder is a monovalent antibody.

31. The method according to any one of the preceding claims, wherein the first binder is a bivalent antibody.

32. The method according to any one of the preceding claims, wherein the first binder is a monospecific antibody.

33. The method according to any one of the preceding claims, wherein the first binder is a bispecific antibody comprising a first antigen-binding region having the ability to bind to human CD27 as described in any one of the preceding claims, and a second antigen-binding region having the ability to bind to a different epitope on human CD27 or having the ability to bind to a different target.

34. The method according to any one of the preceding claims, wherein CD27 is human CD27, and in particular, the human CD27 comprises the sequence shown in SEQ ID NO:1 or the human CD27 variant shown in SEQ ID NO:

2.

35. The first binder is e. a VH region comprising the amino acid sequence shown in SEQ ID NO:4; f. a VL region comprising the amino acid sequence shown in SEQ ID NO:8; g. a CH region comprising the amino acid sequence shown in SEQ ID NO:15; and h. a CL region comprising the amino acid sequence shown in SEQ ID NO:17 The method according to any one of the preceding claims.

36. The method according to any one of the preceding claims, wherein the first binder comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:35 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

25.

37. The method according to any one of the preceding claims, wherein the first binder is in a composition or formulation comprising acetate, sorbitol, polysorbate 80 and has a pH of 5 to 6, preferably 5.

5.

38. The method according to any one of the preceding claims, wherein CD40 is human CD40, in particular human CD40 comprising the sequence shown in SEQ ID NO:62, and / or CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO:

63.

39. (a) The first binding region of the second binder comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 44, 45, and 46, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 47, YTS, and SEQ ID NO: 48, respectively; and (b) the second binding region of the second binder comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 51, 52, and 53, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 54, GAS, and SEQ ID NO: 55, respectively, the method according to any one of the preceding claims.

40. (a) The first binding region of the second binder comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 49 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO: 50; and (b) the second binding region of the second binder comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region (VL) region comprising the amino acid sequence shown in SEQ ID NO: 57, the method according to any one of the preceding claims.

41. The method according to any one of the preceding claims, wherein the second binder is a multispecific antibody, such as a bispecific antibody.

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

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

44. The second binder is (i)A first heavy chain and light chain comprising said first binding region having the ability to bind to CD40, wherein said first heavy chain comprises a first heavy chain constant region and said first light chain comprises a first light chain constant region; and (ii)A second heavy chain and light chain comprising said second binding region having the ability to bind to CD137, wherein said second heavy chain comprises a second heavy chain constant region and said second light chain comprises a second light chain constant region The method according to any one of the preceding claims, comprising:

45. (i)The amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii)The amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in said first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in said second heavy chain. The method according to claim 43 or 44.

46. The positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, in said first and second heavy chains. The method according to any one of claims 43 to 45.

47. The positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said first and second heavy chain constant regions (HC). The method according to any one of claims 43 to 46.

48. The positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU numbering of both the first and second heavy chain constant regions are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L, the method according to any one of claims 43 to 47.

49. The positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain is R, and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L, the method according to any one of claims 43 to 48.

50. The constant region of the first and / or second heavy chain, such as the second heavy chain, is (a) the sequence shown in SEQ ID NO:58 or 60 [IgG1-Fc_FEAL]; (b) a subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) a sequence having up to 6 substitutions, such as 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) comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of, the method according to any one of claims 43 to 49.

51. the constant region of the first and / or second heavy chain, such as the first heavy chain, is (a) the sequence shown in SEQ ID NO: 59 or 61 [IgG1-Fc_FEAR]; (b) a subsequence of the sequence in (a), for example, a subsequence starting from the N-terminus or C-terminus of the sequence defined in (a) and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and (c) a sequence having up to 6 substitutions, such as 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) The method according to any one of claims 43 to 50, comprising an amino acid sequence selected from the group consisting of, consisting essentially of, or consisting of said amino acid sequence.

52. The method according to any one of claims 43 to 51, wherein the second binder comprises a kappa (κ) light chain constant region.

53. The method according to any one of claims 43 to 52, wherein the second binder comprises a lambda (λ) light chain constant region.

54. The method according to any one of claims 43 to 53, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

55. The method according to any one of claims 43 to 54, wherein the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

56. The method according to any one of claims 43 to 55, wherein the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region, or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

57. The kappa (κ) light chain is (a) the sequence shown in SEQ ID NO: 16, (b) a subsequence of the sequence in (a), for example, a subsequence starting from the N-terminus or C-terminus of the sequence defined in (a) and having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids deleted; and (c) An amino acid sequence having at most 10 substitutions, such as at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution, when compared to the amino acid sequence defined in (a) or (b). The method according to any one of claims 52 to 56, comprising an amino acid sequence selected from the group consisting of **Claim 58** wherein said lambda (λ) light chain is (a) the sequence shown in SEQ ID NO: 17, (b) a subsequence of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted, starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) An amino acid sequence having at most 10 substitutions, such as at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution, when compared to the amino acid sequence defined in (a) or (b). The method according to any one of claims 53 to 57, comprising an amino acid sequence selected from the group consisting of **Claim 59** The method according to any one of the preceding claims, wherein said second binder is a binder of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. **Claim 60** The method according to any one of the preceding claims, wherein said second binder is a full-length IgG1 antibody. **Claim 61** The method according to any one of the preceding claims, wherein said second binder is an antibody of the IgG1m(f) allotype. **Claim 62** The method according to any one of the preceding claims, wherein said second binder is a bispecific antibody that binds to CD40 and CD137, and the bispecific antibody has (i) a first heavy chain comprising the amino acid sequence shown in SEQ ID NO: 64 and a first light chain comprising the amino acid sequence shown in SEQ ID NO: 65, and (ii) a second heavy chain comprising the amino acid sequence shown in SEQ ID NO: 66 and a second light chain comprising the amino acid sequence shown in SEQ ID NO:

67. **Claim 63** (a) The first binder comprises a heavy chain variable (VH) region CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NO:5, 6, and 7 respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NO:9, 10, and 11 respectively; (b) The first binding region of the second binder comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:44, 45, and 46 respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:47, YTS, and SEQ ID NO:48 respectively; and (c) The second binding region of the second binder comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:51, 52, and 53 respectively, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:54, GAS, and SEQ ID NO:55 respectively, The method according to any one of the preceding claims.

64. (a) The first binder comprises a VH region containing the amino acid sequence shown in SEQ ID NO:4 and a VL region containing the amino acid sequence shown in SEQ ID NO:8; (b) The first binding region of the second binder comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO:49 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:50; and (c) The second binding region of the second binder comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO:56 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:57, The method according to any one of the preceding claims.

65. (a) The first binder is an antibody comprising a VH region containing the amino acid sequence shown in SEQ ID NO:4, a VL region containing the amino acid sequence shown in SEQ ID NO:8, a CH region containing the amino acid sequence shown in SEQ ID NO:15, and a CL region containing the amino acid sequence shown in SEQ ID NO:17; (b) the second binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising the first binding region and the second binding arm comprising the second binding region; (c) the first binding arm of the second binder comprises a VH region comprising the amino acid sequence shown in SEQ ID NO:49, a VL region comprising the amino acid sequence shown in SEQ ID NO:50, a CH region comprising the amino acid sequence shown in SEQ ID NO:60, and a CL region comprising the amino acid sequence shown in SEQ ID NO:16; and (d) the second binding arm of the second binder comprises a VH region comprising the amino acid sequence shown in SEQ ID NO:56, a VL region comprising the amino acid sequence shown in SEQ ID NO:57, a CH region comprising the amino acid sequence shown in SEQ ID NO:61, and a CL region comprising the amino acid sequence shown in SEQ ID NO:16, The method according to any one of the preceding claims.

66. (c) the first binder comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:35 and a light chain comprising the amino acid sequence shown in SEQ ID NO:25; (d) the second binder is a bispecific antibody that binds to CD40 and CD137, the bispecific antibody having (i) a first heavy chain comprising the amino acid sequence shown in SEQ ID NO:64 and a first light chain comprising the amino acid sequence shown in SEQ ID NO:65, and (ii) a second heavy chain comprising the amino acid sequence shown in SEQ ID NO:66 and a second light chain comprising the amino acid sequence shown in SEQ ID NO:67, The method according to any one of the preceding claims.

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

68. The method according to any one of the preceding claims, wherein the tumor or cancer is a solid tumor.

69. The method according to any one of the preceding claims, wherein the tumor is a PD-L1 positive tumor.

70. The method according to any one of the preceding claims, wherein the tumor or cancer is head and neck squamous cell carcinoma (HNSCC), such as HNSCC of the oral cavity, pharynx or larynx.

71. The method according to claim 70, wherein the HNSCC is recurrent, unresectable or metastatic.

72. The method according to any one of claims 1 to 69, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

73. The method according to claim 72, wherein the NSCLC is recurrent, unresectable or metastatic.

74. The method according to claim 72 or 73, wherein the NSCLC does not have an epidermal growth factor (EGFR) - sensitive mutation and / or anaplastic lymphoma kinase (ALK) translocation and / or ROS1 rearrangement.

75. The method according to any one of claims 72 to 74, wherein the NSCLC is positive for NTRK1 / 2 / 3 (neurotrophic tyrosine kinase receptor 1 / 2 / 3) fusion and / or has a mutation in the KRAS (KRAS proto-oncogene, GTPase), BRAF (B-Raf proto-oncogene, serine / threonine kinase), or MET (MET proto-oncogene, receptor tyrosine kinase) gene and / or has a RET (ret proto-oncogene) gene rearrangement, and the subject has received previous treatment with respective targeted therapies.

76. The method according to any one of the preceding claims, wherein the subject has received previous treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, preferably at least 2 doses of a PD-1 inhibitor or a PD-L1 inhibitor.

77. The method according to any one of the preceding claims, wherein the subject has received previous treatment with a platinum-based therapy or, in case platinum is ineligible, an alternative chemotherapy, such as a gemcitabine-containing regimen.

78. The method according to any one of the preceding claims, wherein the tumor or cancer has recurred and / or progressed after treatment, such as systemic treatment with a checkpoint inhibitor.

79. The method according to any one of the preceding claims, wherein the subject has received at least 1 previous line of a systemic therapy, such as a systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

80. The method according to any one of the preceding claims, wherein the cancer or tumor has recurred and / or is refractory, or the subject has progressed after treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, administered as a monotherapy or as part of a combination therapy. Claim 81 The method according to any one of the preceding claims, wherein the previous last treatment was with a PD1 inhibitor or a PD-L1 inhibitor administered as monotherapy or as part of a combination therapy, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. Claim 82 The method according to any one of the preceding claims, wherein the time from progression in the last treatment with a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, is 6 months or less. Claim 83 The method according to any one of the preceding claims, wherein the time from the last dosing of a PD-1 inhibitor or a PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, as part of the previous last treatment is 6 months or less. Claim 84 The cancer or tumor is recurrent and / or refractory, or the subject has (i) platinum doublet chemotherapy after treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody, or (ii) treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody after platinum doublet chemotherapy The method according to any one of the preceding claims, which progresses during or after. Claim 85 (i) a first binding agent comprising at least one binding region that binds to CD27, and (ii) a second binding agent comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 A kit comprising. Claim 86 The kit according to claim 85, wherein the first binding agent is as defined in any one of claims 1 to 84, and / or the second binding agent is as defined in any one of claims 1 to 84. Claim 87 The kit according to claim 85 or 86, wherein the first binding agent, the second binding agent, and, if present, one or more additional therapeutic agents are for systemic administration, particularly for injection or infusion, such as for intravenous injection or infusion. Claim 88 The kit according to any one of claims 85 to 87, for use in a method for reducing or preventing tumor progression or treating cancer in a subject. Claim 89 The kit for use according to claim 88, wherein the tumor or cancer is as defined in any one of claims 1 to 84, and / or the subject is as defined in any one of claims 1 to 84, and / or the method is as defined in any one of claims 1 to 84.

90. (i)A first binder comprising at least one binding region that binds to CD27; (ii)A second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137; and (iii)Optionally, a pharmaceutically acceptable carrier A pharmaceutical composition comprising.

91. The pharmaceutical composition according to claim 90, wherein the first binder is as defined in any one of claims 1 to 84, and / or the second binder is as defined in any one of claims 1 to 84.

92. The pharmaceutical composition according to claim 90 or 91, for use in a method for reducing or preventing tumor progression or treating cancer in a subject.

93. The pharmaceutical composition for use according to claim 92, wherein the tumor or cancer is as defined in any one of claims 1 to 84, and / or the subject is as defined in any one of claims 1 to 84, and / or the method is as defined in any one of claims 1 to 84.

94. A first binder for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising (i)The first binder comprising at least one binding region that binds to CD27; and (ii)A second binder comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 Administering to the subject. The first binder for the use described above.

95. The first binder for use according to claim 94, wherein the method is as defined in any one of claims 1 to 84, and / or the first binder is as defined in any one of claims 1 to 84, and / or the second binder is as defined in any one of claims 1 to 84.

96. A second binding agent for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising: (i) a first binding agent comprising at least one binding region that binds to CD27; and (ii) the second binding agent comprising a first binding region that binds to CD40 and a second binding region that binds to CD137 administering to the subject, the second binding agent for said use. **Claim 97** The second binding agent for use according to claim 96, wherein the method is as defined in any one of claims 1 to 84, and / or the first binding agent is as defined in any one of claims 1 to 84, and / or the second binding agent is as defined in any one of claims 1 to 84.

Images