Binding agents capable of binding CD27 in combination therapy

Abstract

The present invention provides combination therapies for reducing or preventing tumor progression or treating cancer using a binding agent comprising at least one binding region that binds CD27 in combination with a PD1 / PD-L1 inhibitor.

Description

Technical Field

[0001] The present invention relates to combination therapies using a binding agent comprising at least one binding region that binds CD27 in combination with a PD1 / PD-L1 inhibitor to reduce tumor progression or prevent tumor progression or treat cancer. Background Art

[0002] 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 upon binding to its ligand CD70. In humans, it is expressed on the cell membranes of T, B, natural killer (NK) cells and their immediate precursors, all of which are part of the lymphoid lineage. On human T cells, CD27 is expressed on resting αβCD4 + (Treg and conventional T cells), CD8 + CD27 is expressed on 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 B cell differentiation 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 very limitedly and only transiently on activated immune cells, including T cells, B cells, NK cells, and dendritic cells (DCs).

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

[0005] To unlock their effector functions, 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. 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 antigen-specific CD4 T cell proliferation through interleukin (IL)-2-independent survival signaling. + and CD8 +CD27 also counteracts apoptosis of activated T cells during continuous division and has also been shown to inhibit the clonal expansion of T cells in mice (Carr JM et al, Proc Natl Acad Sci USA 2006 Dec 19; 130(51): 19454-9). + CD27 plays an important role in the memory differentiation of T cells. (Van de Ven K, Borst J. Immunotherapy 2015; 7(6): 655-67). Therefore, CD27 stimulation promotes the generation of effector T cells in lymphoid organs and broadens the pool of responding T cells. In human naive T cells, CD27 stimulation promotes CD4 + It promotes the differentiation of T cells into T helper type 1 cells (Th1) and supports the effector differentiation of cytotoxic T lymphocytes (Oosterwijk et al, Int Immunol. 2007 Jun; 19(6): 713-8).

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

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

[0009] In mouse tumor models, agonistic CD27 antibodies can enhance T cell function and thus enhance antitumor immunity. In a human CD27 (hCD27) transgenic lymphoma mouse model, CD27 activation using agonistic antibodies showed potent antitumor activity and induced protective immunity, which is dependent on CD4 + and CD8 +T cells (He LZ et al., J Immunol. 2013 Oct 15; 191(8): 4174-83). In addition, CD27 activation using monoclonal antibodies prevents 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), melanoma (Roberts DJ, et al., J Immunother. 2010 Oct; 33(8): 769-79), colon cancer, and thymoma (He LZ, et al., J Immunol. 2013 Oct 15; 191(8): 4174-83).

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

[0011] In WO2012 / 004367, a humanized anti-human CD27 agonist antibody (named hCD27.15) is described. It is reported that hCD27.15 does not require cross-linking of cells expressing crystallizable fragment (Fc) gamma receptors (FcγRs) to activate CD27-mediated immune response co-stimulation. However, this antibody does not bind to a common single nucleotide polymorphism (SNP) (A59T) in hCD27 and does not bind to cynomolgus monkey CD27.

[0012] WO2011 / 130434 discloses a human agonistic anti-human CD27 antibody designated 1F5, which activates CD27 upon cross-linking via cells expressing FcγRs and further blocks binding of the soluble CD70 (sCD70) ligand. 1F5 is reported to have Fc-mediated effector function activities, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) on target cells, enhances immune responses, and has anti-tumor activity in mouse models.

[0013] WO2018 / 058022 discloses the agonistic murine anti-human CD27 antibody 131A and its humanized form. 131A binds to the common hCD27 SNP A59T and cynomolgus monkey CD27. WO2018 / 058022 further discloses that antibody 131A exhibits a stronger anti-tumor response in a mouse tumor model than antibody 1F5.

[0014] WO2019 / 195452 discloses a non-ligand blocking agonist anti-human CD27 antibody named BMS-986215, which is reported to have a higher affinity for human and cynomolgus monkey CD27 than the above-mentioned 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. It is further disclosed that BMS-986215 reduces CD4 + BMS-986215 inhibits responsive T cells and binds to C1q and induces CDC, moderate ADCC, and low levels of antibody-dependent cellular phagocytosis (ADCP). It is further disclosed that BMS-986215 has only weak agonist activity in the absence of FcγR and sCD70.

[0015] Cancer cells can avoid and suppress immune responses by upregulating inhibitory immune checkpoint proteins such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) on T cells, or programmed cell death 1 ligand 1 (PD-L1) and / or programmed cell death 1 ligand 2 (PD-L2) on tumor cells, tumor stroma, or other cells within the TME. CTLA-4 and PD-1 are known to transmit signals that inhibit T cell activation. Blocking the activity of these proteins with monoclonal antibodies, thereby restoring T cell function, has delivered breakthrough therapies for cancer.

[0016] PD-1 (also known as CD279) is an immunomodulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. The protein PD-1 has two naturally occurring ligands, called PD-L1 (also known as CD274) and PD-L2 (also known as CD273). PD-L1 is expressed in a variety of cancers, including melanoma, lung cancer, kidney cancer, bladder cancer, esophageal cancer, gastric cancer, and other cancers. Therefore, when PD-L1 interacts with PD-1 in cancer, the PD-1 / PD-L1 system can inhibit the proliferation of T lymphocytes, the release of cytokines, and cytotoxicity, thereby providing cancer cells with an opportunity to avoid the T cell-mediated immune response.

[0017] Monoclonal antibodies suitable for modulating the activity of the PD-1 / PD-L1 axis are known. The PD-1 / PD-L1 interaction can be inhibited by antibodies targeting PD-1, such as pembrolizumab (also known as MK-3475, lambrolizumab, or Keytruda) and nivolumab (also known as ONO-4538, BMS-936558, or Opdivo), or by monoclonal antibodies developed to bind to PD-L1, such as atezolizumab (also known as MPDL3280A, RG7446, or Tecentriq).

[0018] Anti-CD27 antibodies must induce CD27 aggregation on the plasma membrane to induce CD27 agonism. In the case of wild-type IgG1 antibodies, CD27 aggregation can be achieved through the interaction of membrane-bound CD27 antibodies with cells bearing FcγRs (e.g., monocytes, macrophages, B cells, and other immune cells). Therefore, when the number of cells expressing FcγRs is limited, the efficiency of anti-CD27 IgG1 molecules may be low. Optimizing effector functions by modifying the Fc region of an antibody can improve the efficacy of therapeutic antibodies in treating cancer or other diseases, for example, by improving the ability of the antibody to elicit an immune response to cells expressing the antigen. Such attempts are described, for example, in WO 2013 / 004842 A2; WO 2014 / 108198 A1; WO 2018 / 146317; WO 2018 / 083126; WO 2018 / 031258 A1; Dall'Acqua, Cook et al. J Immunol 2006, 177(2):1129-1138; Moore, Chen et al. MAbs 20102(2):181-189; Desjarlais and Lazar, Exp Cell Res 2011, 317(9):1278-1285; Kaneko and Niwa, BioDrugs 2011, 25(1):1-11; Song, Myojo et al., Antiviral Res 2014, 111:60-68; Brezski and Georgiou,Curr OpinImmunol 2016,40:62-69;Sondermann and Szymkowski,Curr OpinImmunol 2016,40:78-87;Zhang,Armstrong et al.MAbs 2017,9(7):1129-1142.;Wang,Mathieu etal.Protein&Cell 2018,9(1):63-73; Diebolder FJ et al.,Science.2014Mar 14;343(6176):1260-3).

[0019] By activating the immune system, immune CPI may also cause autoimmune side effects in some patients. In addition, the engagement of the Fc domain with components of the Fc receptor or complement system may also lead to undesirable effector functions, such as activation of ADCC, ADCP and CDC, which may cause undesirable exhaustion of CD27-positive T cells. Therefore, the activation of Fc-mediated effector functions is undesirable in the context of monoclonal antibodies that block PD-1 / PD-L1 interactions. A variety of IgG antibody forms containing Fc domains that do not engage Fc receptors and / or complement systems have been developed, in which amino acid substitutions and combinations thereof (i.e., non-activating mutations) have been introduced into the constant heavy chain region of IgG1 isotype antibodies to eliminate Fc-mediated effector functions (e.g., Chiu et al., Antibodies 2019 Dec; 8(4): 55; Liu et al., Antibodies, 2020 Nov 17; 9(4): 64; 29(10): 457-66). Examples of such substitutions include introduction of the L234A-L235A-P329G inactivating mutation (Schlothauer et al., Protein Eng. Design and Selection 2016; 29(10): 457-66) or the L234F-L235E-D265A inactivating mutation (also referred to herein as FEA or FEA form, Engelberts et al., EBioMedicine 2020; 52: 102625; US10590206B2). Other inactivated formats have been developed using human IgG4 (one of the human IgG subclasses with reduced effector function) in combination with amino acid substitutions in the constant heavy chain region of the antibody to further eliminate Fc-mediated effector function (e.g., introduction of the E233P-F234V-L235A-G236del inactivating mutation described in WO2015 / 143079, or the F234A-L235A inactivating mutation described in Vafa et al. Methods 2014; 65: 114-126).

[0020] Among other things, Garber et al. discussed opportunities for combination therapies consisting of agonistic antibodies targeting T cell co-stimulatory receptors, such as 4-1BB (CD137), OX40, glucocorticoid-induced tumor necrosis factor receptor family-related receptors (GITR), and independent co-stimulators (ICOS), and monoclonal antibodies that block the PD-1 / PD-L1 axis (Garber et al. Nat Rev Drug Discov. 2020 Jan;19(1):3-5). Azpilikueta et al. (J Thorac Oncol 2016;11:524–36) published preclinical data on combination therapies containing a PD-1 blocking antibody and a 4-1BB targeting antibody in a mouse lung cancer model, demonstrating that the combination therapy was superior to monotherapy.

[0021] WO2008 / 051424A2 provides methods comprising administering agonistic antibodies targeting CD27 alone or in combination with other immunomodulators, such as antibodies targeting CD40, OX40, 4-1BB or CTLA-4.

[0022] US10668152B2 provides a method for treating cancer using a combination therapy comprising administering an anti-PD-1 antibody and an anti-CD27 antibody.

[0023] CDX-527 is a PD-L1xCD27 bispecific IgG1 antibody (Vitale et al., Cancer Immunol Immunother 2020).

[0024] WO2018 / 127916 provides a PD1-CD70 dual signal fusion protein (DSP-106) based on MIRP technology (multifunctional immune recruitment protein).

[0025] WO2015 / 016718A1 provides a method for expressing a protein that is known or expected to be expressed by stimulating CD27. + Treatment of immune cells or any condition that is alleviated by inhibition of one or more immune checkpoint proteins, for example by administering an anti-CD27 antibody in combination with an antibody that blocks the PD1 / PD-L1 interaction.

[0026] However, despite these and other attempts in the art, there remains a need for improved antibody-based immunotherapies with increased agonism and / or increased potency to engage CD27, provided as combination therapies with other immunomodulatory antibodies or antibodies that block immune checkpoints. SUMMARY OF THE INVENTION

[0028] The present invention relates to binding agents capable of binding CD27 in combination therapy.

[0029] In a first aspect, the present disclosure provides a method for reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor.

[0030] In a second aspect, the present disclosure provides a kit comprising i) a binding agent comprising at least one binding region that binds CD27 and ii) a PD1 / PD-L1 inhibitor.

[0031] In a third aspect, the present disclosure provides a kit for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the kit comprising i) a binding agent comprising at least one binding region that binds CD27 and ii) a PD1 / PD-L1 inhibitor.

[0032] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising i) a binding agent comprising at least one binding region that binds CD27; ii) a PD1 / PD-L1 inhibitor; and ii) optionally a pharmaceutically acceptable carrier.

[0033] In a fifth aspect, the present disclosure provides a pharmaceutical composition for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the pharmaceutical composition comprising i) a binding agent comprising at least one binding region that binds CD27 and ii) a PD1 / PD-L1 inhibitor.

[0034] In a sixth aspect, the present disclosure provides a binding agent for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor.

[0035] In a seventh aspect, the present disclosure provides a PD1 / PD-L1 inhibitor for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) the PD1 / PD-L1 inhibitor. Detailed Description of the Invention

[0037] definition

[0038] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof that can specifically bind to an antigen. The antibodies of the present invention comprise the Fc domain and the antigen-binding region of an immunoglobulin. Antibodies typically contain two CH2-CH3 regions and a connecting region, such as a hinge region, for example, at least an Fc domain. Therefore, the antibodies of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region or "Fc" region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. As used herein, unless otherwise contradicted by the context, the Fc region of an immunoglobulin typically contains at least the CH2 domain and the CH3 domain of the immunoglobulin CH, and may comprise a connecting region, such as a hinge region. The Fc region is generally in a dimerized form via, for example, a disulfide bond connecting two hinge regions and / or non-covalent interactions between two CH3 regions. The dimer can be a homodimer (wherein the two Fc region monomer amino acid sequences are identical) or a heterodimer (wherein the two Fc region monomer amino acid sequences are different in one or more amino acids). The Fc region fragment of a full-length antibody can be generated, for example, by digesting the full-length antibody with papain, as is well known in the art. In addition to the Fc region and the antigen-binding region, an antibody as defined herein also comprises one or both of the immunoglobulin CH1 region and the CL region. An antibody can also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody that is specific for at least two different, usually non-overlapping epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different targets, such targets can be on the same cell or different cells or cell types. As described above, unless otherwise indicated or clearly contradictory to the context, the term antibody herein includes antibody fragments that comprise at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments can be provided by any known technology, such as enzymatic cleavage, peptide synthesis and recombinant expression technology. It has been shown that the antigen binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed in the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described in WO2007059782 to Genmab); heavy chain antibodies, which consist of only two heavy chains and naturally occur in, for example, camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs, Roche, WO2011069104); chain exchange engineered domains (SEED or Seed-body), which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205);Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔADP (Regeneron, WO2010151792); asymmetric 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 diabody (Unilever; SanofiAventis, WO20100226923); di-diabody (ImClone / Eli Lilly); knob-in-hole antibody formats (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545); DuetMab (MedImmune, US2014 / 0348839); electrostatically steered antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545); CrossMAb (Roche, WO2011117329); LUZ-Y (Genentech); biclonal (Merus, WO2013157953); dual-targeting domain antibody (GSK / Domantis); two-in-one antibody or dual-action Fab recognizing two targets (Genentech, NovImmune, Adimab); cross-linking Mab (Karmanos Cancer Center); covalent fusion mAb (AIMM); CovX-body (CovX / Pfizer); FynomAb (Covagen / Janssen ilag); DutaMab (Dutalys / Roche); iMab (MedImmune); IgG-like bispecific (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);Dual affinity retargeting molecules (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538); BEAT (Glenmark); Zybody (Zyngenia); methods with a common light chain (Crucell / Merus, US7262028) or a common heavy chain (κλBody of NovImmune, WO2012023053), and fusions comprising a polypeptide sequence fused to an antibody fragment containing an Fc region, such as scFv-fusions, such as BsAb of ZymoGenetics / BMS, HERCULES of 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 fusion (Genentech / Roche); scFv fusion (Novartis); scFv fusion (Immunomedics); scFv fusion (Changzhou Adam Biotech Inc, CN 102250246); TvAb (Roche, WO 2012025525, WO 2012025530); mAb2 (f-Star, WO2008 / 003116); and dual scFv-fusions. It should be understood that, unless otherwise indicated, the term antibody includes 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; for example, antibody mixtures (recombinant polyclonal) generated by technologies developed by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, and fusion proteins as described in WO2014 / 031646. Although these different antibody fragments and forms are generally included within the meaning of antibodies, they are collectively and independently unique features of the present invention, exhibiting different biological properties and utilities.

[0039] "Agonistic antibodies" to natural receptors are compounds that bind to the receptor to form a receptor-antibody complex and activate the receptor, thereby initiating pathway signaling and further biological processes.

[0040] The terms "agonism" and "agonism" are used interchangeably herein and refer to or describe an antibody that is capable of substantially inducing, promoting or enhancing CD27 biological activity or activation, directly or indirectly. Optionally, an "agonistic CD27 antibody" is an antibody that is capable of activating the CD27 receptor by a mechanism similar to the CD27 ligand known as CD70 (tumor necrosis factor superfamily member 7, TNFSF7; CD27 ligand, CD27L), which results in activation of one or more intracellular signaling pathways, which may include activation of the NF-KB and MAPK8 / JNK pathways. "Agonism" as defined herein can be determined according to Example 2 herein.

[0041] As used herein, a "CD27 antibody" or "anti-CD27 antibody" is an antibody that specifically binds to the protein CD27, particularly human CD27.

[0042] As used herein, "variant" refers to a protein or polypeptide sequence that is different from a parent or reference sequence in one or more amino acid residues. A variant can, for example, have at least 80%, such as 90%, or 95%, or 97%, or 98%, or 99% sequence identity with a parent or reference sequence. In addition, or alternatively, a variant can differ from a parent or reference sequence by 12 or less, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations, such as substitutions, insertions, or deletions of amino acid residues. Thus, "variant antibodies" or "antibody variants" used interchangeably herein refer to antibodies that differ from a parent or reference antibody in one or more amino acid residues, such as in the antigen binding region, the Fc region, or both. Likewise, a "variant Fc region" or "Fc region variant" refers to an Fc region that is different from a parent or reference Fc region in one or more amino acid residues, optionally differing from the parent or reference Fc region amino acid sequence by 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, such as substitutions, insertions or deletions of amino acid residues. The parent or reference Fc region is typically the Fc region of a human wild-type antibody, which, depending on the context, may be a specific isotype. The variant Fc region in a dimerized form may be a homodimer or a heterodimer, for example, wherein one of the amino acid sequences of the dimerized Fc region comprises a mutation, while the other is identical to the parent or reference wild-type amino acid sequence. Examples of wild-type (typically a parent or reference sequence) IgG CH and variant IgG constant region amino acid sequences comprising Fc region amino acid sequences are listed in Table 3.

[0043] As used herein, the term "immunoglobulin heavy chain" or "heavy chain of immunoglobulin" is intended to refer to one of the heavy chains of an immunoglobulin. A heavy chain generally comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the immunoglobulin isotype. The heavy chain constant region generally comprises three domains, CH1, CH2, and CH3. As used herein, the term "immunoglobulin" is intended to refer to a class of structurally related glycoproteins that consists of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four chains possibly interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, for example, Fundamental Immunology Ch. 7 Paul, W., 2nd ed. Raven Press, NY 1989). Within the structure of an immunoglobulin, two heavy chains are interconnected via disulfide bonds in the so-called "hinge region". Like the heavy chain, each light chain generally comprises several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically comprises one domain, CL. In addition, the VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions in the form of loops determined in sequence and / or structure), also known as complementarity determining regions (CDRs), interspersed in more conserved regions called framework regions (FRs). Each VH and VL typically comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Unless otherwise indicated or inconsistent with the context, 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)).

[0044] As used herein, the terms "half molecule", "Fab-arm" and "arm" refer to a heavy chain-light chain pair. 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 is generated by recombining the half molecules from each of the first and second antibodies into the resulting bispecific antibody by any known method. In this context, "recombinant" is not intended to be limited to any particular recombinant method and thus includes all methods described below for generating bispecific antibodies, including, for example, by "half molecule exchange" and "Fab arm exchange" also described in the art. The method allows recombination, as well as recombination at the nucleic acid level and / or by co-expression of the two half molecules in the same cell.

[0045] As used herein, the term "antigen binding region" or "binding region" or antigen binding domain refers to the region of an antibody that is capable of binding to an antigen. This binding region is generally defined by the VH and VL domains of the antibody, which can be further subdivided into hypervariable regions (or hypervariable regions that can be hypervariable in the form of loops determined in sequence and / or structure), also known as complementarity determining regions (CDRs), interspersed in more conserved regions called framework regions (FRs). An antigen can be any molecule, such as a polypeptide, such as a polypeptide present on a cell, bacterium, or virion. Unless otherwise indicated by the context, the terms "antigen binding region" and "antigen binding site" and "antigen binding domain" are used interchangeably in the context of the present invention.

[0046] Unless contradicted by the context, the terms "antigen" and "target" are used interchangeably in the context of the present invention.

[0047] As used herein, the term "binding" when determined by biolayer interferometry using an antibody as a ligand and an antigen as an analyte refers to the binding of an antibody to a predetermined antigen or target, typically with a specific affinity corresponding to 1E 6 M or smaller, such as 5E 7 M or smaller, 1E 7 M or smaller, such as 5E 8 M or smaller, such as 1E 8 M or smaller, such as 5E 9 M or smaller, or for example 1E 9 M or smaller K D The binding affinity of the antigen is at least 10 times, such as at least 100 times, such as at least 1000 times, such as at least 10,000 times, such as at least 100,000 times, than the affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). D The affinity of the antigen is sufficient to bind to the target antigen.

[0048] As used herein, the term "K D “(M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction and is expressed by k d Divide by k a get.

[0049] As used herein, the term "K d “(sec -1 ) refers to the dissociation rate constant for a specific antibody-antigen interaction. This value is also called k off value or dissociation rate.

[0050] As used herein, the term "k a ”(M -1 x sec-1 ) refers to the association rate constant for a specific antibody-antigen interaction. This value is also called k on value or binding rate.

[0051] As used herein, the term "CD27" refers to a human protein named 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 a signal peptide, and amino acid residues 20-240 are a mature polypeptide. Unless otherwise indicated by the context, CD27 may also refer to variants, isoforms, and orthologs of CD27. A naturally occurring variant of human CD27 comprising the A59T mutation is shown in SEQ ID NO: 2.

[0052] 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, no signal peptide is defined.

[0053] The term "antibody binding region" refers to the region of antigen, which comprises the epi-position of antibody binding. The antibody binding region can be determined by using the epi-position of biolayer interferometry, by alanine scanning or by shuffling assay (using the antigen construct exchanged between the region of the antigen and the region of another species and determining whether the antibody is still in conjunction with the antigen). The amino acid that participates in the antibody binding region and interacts with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and by the crystallography of the antibody bound to its antigen.

[0054] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. An epitope is typically composed of surface groups of a molecule such as amino acids, sugar side chains, or a combination thereof, and typically has specific three-dimensional structural characteristics and specific charge characteristics. The difference between conformational and non-conformational epitopes is that, in the presence of a denaturing solvent, the binding to the former, but not the latter, is lost. An epitope can 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 the antibody when the antibody binds to the antigen (in other words, the amino acid residue is within or immediately adjacent to the footprint of a specific antibody).

[0055] As used herein, the terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb" and the like refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice or rats) having a genome comprising a human heavy chain transgene and a light chain transgene fused to immortalized cells. Monoclonal antibodies can also be produced by recombinantly modified host cells or systems using cell extracts that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

[0056] As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region gene, or any allotype thereof, such as IgG1m(za) and IgG1m(f). In addition, each heavy chain isotype can be combined with a kappa or lambda light chain.

[0057] When used in this article, the term "full-length antibody" means that this antibody is not a fragment, but contains all domains of the specific isotype found in nature usually for this isotype, for example VH, CH1, CH2, CH3, hinge, VL and CL domains of IgG1 antibody. In full-length variant antibodies, when compared with full-length parent or wild-type antibody, heavy chain and light chain constant domains and variable domains can especially contain amino acid substitutions that improve antibody functional properties. Full-length antibodies according to the present invention can be produced by a method comprising the following steps: (i) CDR sequences are cloned into a suitable vector comprising complete heavy chain sequence and complete light chain sequence, and (ii) complete heavy chain and light chain sequence are expressed in a suitable expression system. For technical personnel, it is well known to produce full-length antibodies from CDR sequences or complete variable region sequences. Therefore, technical personnel will know how to generate full-length antibodies according to the present invention.

[0058] As used herein, the term "human antibody" is intended to include antibodies comprising variable regions and framework regions and human immunoglobulin constant domains derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies wherein CDR sequences derived from the germline of another non-human species (e.g., mouse) have been transplanted onto human framework sequences.

[0059] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody containing human antibody constant domains and modified to contain non-human variable domains with a high level of sequence homology to human variable domains. This can be achieved by transplanting six non-human antibody complementary determining regions (CDRs) that together form an antigen binding site onto homologous human receptor framework regions (FRs) (see WO92 / 22653 and EP0629240). In order to fully rebuild the binding affinity and specificity of parental antibodies, it may be necessary to replace the framework residues of parental antibodies (i.e., non-human antibodies) into human framework regions (back mutations). Structural homology modeling can help identify the amino acid residues important to the binding properties of antibodies in the framework regions. Therefore, humanized antibodies can include non-human CDR sequences, primarily one or more amino acid back mutations in which human framework regions optionally include non-human amino acid sequences, and fully human constant regions. Optionally, other amino acid modifications that are not necessarily back mutations can be applied to obtain humanized antibodies with preferred features such as affinity and biochemical properties.

[0060] As used herein, the terms "Fc region" or "Fc domain" are used interchangeably and refer to the region of the heavy chain constant region, which includes at least the hinge region, the CH2 region, and the CH3 region in the direction from the N-terminus to the C-terminus of the antibody. The Fc region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0061] Unless otherwise indicated or clearly contradicted by the context, the term "parent polypeptide" or "parent antibody" is to be understood as a polypeptide or antibody that is identical to a polypeptide or antibody according to the present invention, but wherein the parent polypeptide or parent antibody has not been mutated. For example, the antibody IgG1-CD27-A of the present invention is the parent antibody of IgG1-CD27-A-P329R-E345R.

[0062] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the Eu numbering (Eu-index) described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition-US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991). However, the hinge region may also be any other subtype as described herein.

[0063] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering as described in Kabat (ibid). However, the CH1 region may also be of any other subtype as described herein.

[0064] 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 as described in Kabat (ibid). However, the CH2 region may also be of any other subtype as described herein.

[0065] 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 as described in Kabat (ibid). However, the CH3 region may also be of any other subtype as described herein.

[0066] As used herein, the terms "Fc-mediated effector function" or "Fc effector function" are used interchangeably and are intended to refer to functions that result from binding of a polypeptide or antibody to its target or antigen on a cell membrane, wherein 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 cellular cytotoxicity (ADCC), (v) Fc-gamma receptor (FcγR) binding, (vi) antibody-dependent, FcγR-mediated antigen cross-linking, (vii) antibody-dependent cellular phagocytosis (ADCP), (viii) complement-dependent cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x) complement receptor binding of opsonized antibodies mediated by antibodies, (xi) opsonization, and (xii) a combination of any one of (i) to (xi).

[0067] As used herein, the terms "reduced Fc effector function" or "reduced Fc-mediated effector function" are used interchangeably and are intended to refer to a reduction in the Fc effector function of an antibody when directly compared to the Fc effector function of a parent polypeptide or antibody in the same assay.

[0068] As used herein, the terms "inert," "inert," or "non-activating" refer to an Fc region that is at least unable to bind to any FcγR, unable to induce Fc-mediated cross-linking of FcγRs, or unable to induce FcγR-mediated cross-linking of a target antigen via two Fc regions of a single antibody, or unable to bind to C1q. Thus, in certain embodiments of the present invention, the Fc region is inert. Thus, in certain embodiments, some or all of the Fc-mediated effector functions are diminished or completely absent.

[0069] As used herein, the term "oligomerization" is intended to refer to the process of converting monomers to a limited degree of polymerization. The antibodies according to the present invention can form oligomers, such as hexamers, via non-covalent association of the Fc region after target binding, for example at the cell surface. Oligomerization of anti-CD27 antibodies after cell surface binding via Fc:Fc interactions can increase CD27 aggregation, leading to activation of CD27 intracellular signaling. The ability of antibodies comprising E345R or E430G mutations to form oligomers (e.g., hexamers) after cell surface binding can be assessed as described in de Jong RN et al, PLoS Biol. 2016 Jan 6; 14(1): e1002344. Fc-Fc-mediated oligomerization of antibodies occurs through intermolecular association of the Fc regions between adjacent antibodies after target binding to the (cell) surface, and this oligomerization can be enhanced by introducing E345R or E430G mutations (numbered according to the Eu-index).

[0070] As used herein, the term "aggregation" refers to the oligomerization of antibodies through non-covalent interactions.

[0071] As used herein, the term "Fc-Fc enhancement" is intended to refer to increasing the binding strength between the Fc regions of two antibodies containing Fc regions or stabilizing the interaction between them so that the antibodies form oligomers, such as hexamers, on the cell surface. This enhancement can be obtained by certain amino acid mutations in the Fc region of the antibody, such as E345R or E430G. In the context of the present invention, the term "monovalent antibody" refers to an antibody molecule that can interact with a specific epitope on an antigen and has only one antigen binding domain (e.g., one Fab arm). In the context of bispecific antibodies, "monovalent antibody binding" refers to the binding of a bispecific antibody to a specific epitope on an antigen and has only one antigen binding domain (e.g., one Fab arm).

[0072] In the context of the present invention, the term "monospecific antibody" refers to an antibody that has binding specificity for only one epitope. The antibody can be a monospecific monovalent antibody (i.e., carrying only one antigen-binding region) or a monospecific bivalent antibody (i.e., an antibody having two identical antigen-binding regions).

[0073] The term "bispecific antibody" refers to an antibody comprising two different antigen binding domains, such as two different Fab arms or two Fab arms with different CDR regions. In the context of the present invention, a bispecific antibody is specific for at least two different epitopes. Such epitopes can be on the same or different antigens or targets. If the epitopes are on different antigens, such antigens can be on the same cell or different cells, cell types or structures, such as extracellular matrix or vesicles and soluble proteins. Thus, a bispecific antibody can be able to cross-link multiple antigens, such as two different cells. Specific bispecific antibodies of the present invention can bind to CD27 and a second target.

[0074] The term "bivalent antibody" refers to an antibody having two antigen-binding regions that bind to one or two epitopes on a target or antigen or to one or two epitopes on the same antigen. Thus, a bivalent antibody can be a monospecific bivalent antibody or a bispecific bivalent antibody.

[0075] The terms "amino acid" and "amino acid residue" are used interchangeably herein and should not be construed as limiting. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, as well as side chains (R groups) specific to each amino acid. In the context of the present invention, amino acids can be classified based on structural and chemical properties. Thus, the class of amino acids can be reflected in one or both of the following tables:

[0076] Table 20. Major classifications based on the structure and general chemical characteristics of the R group

[0077] category amino acids acidic residues D and E basic residues K, R, and H Hydrophilic uncharged residues S, T, N, and Q Aliphatic uncharged residues G, A, V, L, and I Nonpolar uncharged residues C, M and P Aromatic residues F, Y and W

[0078] Table 21. Alternative physical and functional classifications of amino acid residues

[0079]

[0080]

[0081] The substitution of one amino acid to another can be classified as conservative or non-conservative substitution. In the context of the present invention, a "conservative substitution" is one in which an amino acid is substituted by another amino acid having similar structure and / or chemical characteristics, wherein an amino acid residue is substituted by another amino acid residue of the same class as defined in either of the two tables above: for example, leucine can be substituted by isoleucine because they are all aliphatic, branched-chain hydrophobic. Similarly, aspartic acid can be substituted by glutamic acid because they are all small negatively charged residues.

[0082] In the context of the present invention, substitutions in antibodies are represented by:

[0083] Original amino acid - position - substituted amino acid;

[0084] With reference to generally accepted amino acid nomenclature, three-letter codes or one-letter codes, including the codes "Xaa" or "X," are used to represent any amino acid residue. Thus, Xaa or X can generally represent any of the 20 naturally occurring amino acids. As used herein, the term "naturally occurring" refers to any 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 comprises a lysine to arginine substitution at amino acid position 409.

[0085] The substitution of an amino acid at a given position to any other amino acid is called:

[0086] Original amino acid position; or e.g. "K409"

[0087] For modifications where the original amino acid and / or substituted amino acids may include more than one but not all amino acids, the more than one amino acid may be separated by ", " or " / ". For example, substitution of lysine at position 409 with arginine, alanine or phenylalanine is:

[0088] “Lys409Arg, Ala, Phe” or “Lys409Arg / Ala / Phe” or “K409R, A, F” or “K409R / A / F” or “K409 to R, A or F”.

[0089] These names are used interchangeably in the context of the present invention but have the same meaning and purpose.

[0090] In addition, the term "substituted" includes substitutions with any of the 19 naturally occurring amino acids or other amino acids, or with other amino acids such as non-natural amino acids. For example, substitution of amino acid K at position 409 includes each of the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. Incidentally, this is equivalent to the designation 409X, where X represents 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. This also applies by analogy to each and every position mentioned herein, specifically including any such substitution herein.

[0091] Antibodies according to the present invention may also comprise deletions of amino acid residues. Such deletions may be indicated as "del" and include, for example, K409del. Thus, in such embodiments, the lysine in position 409 has been deleted from the amino acid sequence.

[0092] 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 terms are intended to refer not only to a specific subject cell, but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent 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, NSO cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli and other eukaryotic hosts such as plant cells and fungi.

[0093] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express an antibody or target antigen, such as CHO cells, PER.C6 cells, NSO cells, HEK-293 cells, Expi293F cells, plant cells, or fungi, including yeast cells.

[0094] For purposes of the present invention, use Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) to determine the sequence identity between two amino acid sequences, as implemented in the Needle program of EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), preferably 5.0.0 version or higher version.The parameter used is: gap opening penalty is 10, gap extension penalty is 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using-nobrief option to obtain) of the Needle labeled " longest identity " is used as identity percentage, and is calculated as follows:

[0095] (Identical residues x 100) / (length of alignment - total number of gaps in the alignment).

[0096] The retention of similar residues can also or alternatively be measured by a similarity score, as determined by using a BLAST program (e.g., BLAST 2.2.8 obtained through NCBI, using standard settings BLOSUM62, open gap = 11 and extension gap = 1). Suitable variants typically exhibit at least about 45%, e.g., 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 more (e.g., about 99%) similarity to the parent sequence.

[0097] As used herein, the term "internalized" or "internalization" refers to a biological process in which a molecule, such as an antibody according to the present invention, is engulfed by a cell membrane and taken into the interior of the cell. Internalization may also be referred to as "endocytosis."

[0098] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells (e.g., neutrophils, granulocytes, mast cells, and basophils). Some effector cells express Fc receptors (FcgR) or complement receptors and perform specific immune functions. In some embodiments, effector cells such as natural killer cells can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells expressing FcgR participate in the specific killing of target cells and / or present antigens to other components of the immune system, or combine with cells presenting antigens. In some embodiments, ADCC can be further enhanced by the classical complement activation driven by antibodies, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CR) such as CR3 expressed on myeloid cells. Recognition of complement fragments by CR on effector cells can promote enhanced 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 can phagocytose target antigens, target particles, or target cells, which can be mediated by antibody binding and FcγR expressed by effector cells. The expression of specific FcR or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, it has been found that the expression of FcγRI is upregulated by interferon γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of cells carrying FcγRI 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 direct phagocytosis by effector cells or indirectly by enhancing antibody-mediated phagocytosis.In certain embodiments herein wherein the antibody has an inert Fc region, the antibody does not induce Fc-mediated effector function.

[0099] As used herein, "effector T cells" or "Teff" refers to T lymphocytes that perform immune response functions (e.g., killing tumor cells and / or activating anti-tumor immune responses), which can lead to the elimination of tumor cells from the body. Examples of Teff phenotypes include CD3 + CD4 + and CD3 + CD8 + Teffs may secrete, contain or express markers such as IFNγ, granzyme B and ICOS. It should be understood that Teffs may not be completely limited to these phenotypes.

[0100] As used herein, "memory T cells" refer to T lymphocytes that remain in the body for a long time after the infection is removed. Examples of memory T cells include central memory T cells (CD45RA-CCR7+) and effector memory T cells (CD45RA-CCR7-). It should be understood that memory T cells may not be completely limited to these phenotypes.

[0101] As used herein, "regulatory T cells" or "Tregs" refer to T lymphocytes that regulate the activity of other T cells and / or other immune cells, typically by suppressing their activity. Examples of Treg phenotypes are CD3 + CD4 + CD25 + CD127dim. Tregs may further express Foxp3. It should be understood that Tregs may not be completely limited to this phenotype.

[0102] As used herein, the term "complement activation" refers to the activation of the classical complement pathway, which is triggered by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on the surface. C1 is a complex composed of a heterotetramer of 6 recognition proteins, C1q, and 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 that begin with the cleavage of C4 into C4a and C4b and the cleavage of C2 into C2a and C2b. C4b deposits and forms an enzymatically active convertase called C3 convertase together with C2a, which cleaves the complement component C3 into C3b and C3a, which forms the C5 convertase. The C5 convertase cleaves C5 into C5a and C5b, and the last component is deposited on the membrane, thereby triggering the late events of complement activation, in which the terminal complement components C5b, C6, C7, C8, and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores in cell membranes, which leads to cell lysis, also known as complement dependent cytotoxicity (CDC).In certain embodiments herein wherein the antibody has an inert Fc region, the antibody does not induce complement activation.

[0103] Complement activation can be assessed by using C1q binding efficacy, CDC kinetics CDC assays (as described in WO2013 / 004842, WO2014 / 108198) or by the cellular deposition of C3b and C4b as described in Beurskens et al., J Immunol April 1, 2012 vol. 188 no. 7, 3532-3541.

[0104] As used herein, the term "Clq binding" refers to the binding of Clq in the context of Clq binding to an antibody bound to its antigen. Antibody binding to its antigen is understood to occur both in vivo and in vitro in the context described herein. Clq binding can be assessed, for example, by using antibodies immobilized on an artificial surface or by using antibodies that bind to a predetermined antigen on the surface of a cell or virion, as described in Example 8 herein. Binding of Clq to an antibody oligomer is understood herein to be a multivalent interaction resulting in high-affinity binding. For example, a reduction in Clq binding resulting from the introduction of a mutation into an antibody of the invention can be measured by comparing Clq binding of the mutant antibody with Clq binding of its parent antibody (an antibody of the invention without the mutation in the same assay).

[0105] The term "treating" refers to administering an effective amount of a therapeutically active antibody of the present invention for the purpose of alleviating, improving, preventing or eradicating (curing) symptoms or disease states.

[0106] The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the necessary dosage and for the necessary period of time. A therapeutically effective amount of an antibody can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody variant are outweighed by the therapeutically beneficial effects.

[0107] The term "pharmacokinetic profile" as used herein can be determined from plasma IgG levels over time as described in Example 12 herein.

[0108] 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 considered to be involved in T cell activation. Other synonyms of CD137 include, but are not limited to, 4-1BB ligand receptor, CD137, T cell antigen 4-1BB homologue and T cell antigen ILA. In one embodiment, CD137 (4-1BB) is human CD137 (4-1BB) with UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO: 130. Amino acids 1-23 of SEQ ID NO: 130 correspond to the signal peptide of human CD137; amino acids 24-186 of SEQ ID NO: 130 correspond to the extracellular domain of human CD137; and the remainder of the protein, amino acids 187-213 and 214-255 from SEQ ID NO: 130, are the transmembrane domain and the cytoplasmic domain, respectively.

[0109] The "programmed death-1 (PD-1)" receptor refers to an immunoinhibitory receptor belonging to the CD28 family.

[0110] As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, such as macaque (cynomolgus monkey), African elephant, wild boar, and mouse PD-L1 (cf., e.g., Genbank accession numbers NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), as well as analogs that share at least one common epitope with hPD-L1. The sequence of human PD-L1 is also shown in SEQ ID NO: 98 (mature sequence) and SEQ ID NO: 129, wherein amino acids 1-18 are predicted to be a signal peptide. As used herein, the term "PD-L2" includes human PD-L2 (hPD-L2), variants, isoforms and species homologs of hPD-L2, and analogs having at least one common epitope with hPD-L2. The ligands of PD-1 (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells (such as dendritic cells or macrophages) and other immune cells. The binding of PD-1 to PD-L1 or PD-L2 leads to downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 are able to shut down T cells expressing PD-1, which leads to inhibition of anti-cancer immune responses. The interaction between PD-1 and its ligands leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune escape of cancer cells. Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is cumulative when the interaction of PD-1 with PD-L2 is also blocked.

[0111] The term "PD-1" refers to programmed cell death-1 and includes any variants, conformations, isoforms and species homologs of PD-1 that are naturally expressed by cells or expressed by cells transfected with the PD-1 gene. Preferably, "PD-1" refers to human PD-1, in particular to a protein having an amino acid sequence as shown in SEQ ID NO: 58 of the sequence listing (NCBI Reference Sequence: NP_005009.2), or preferably a protein encoded by a nucleic acid sequence as shown in SEQ ID NO: 60 of the sequence listing (NCBI Reference Sequence: NM_005018.2). Alternative names for "PD-1" include CD279 and SLEB2.

[0112] The term "PD-1" includes post-translationally modified variants, isoforms and species homologs of human PD-1 that are naturally expressed by cells or expressed in / on cells transfected with the PD-1 gene.

[0113] The term "PD-1 variant" shall encompass (i) PD-1 splice variants, (ii) PD-1 post-translational modified variants, particularly including variants with different N-glycosylation states, and (iii) PD-1 conformational variants. Such variants may include soluble forms of PD-1.

[0114] PD-1 is a type I membrane protein belonging to the immunoglobulin superfamily (The EMBO Journal (1992), vol. 11, issue 11, p. 3887-3895). The human PD-1 protein comprises an extracellular domain containing amino acids at positions 24 to 170 of the sequence shown in SEQ ID NO: 58 of the sequence listing, a transmembrane domain (amino acids at positions 171 to 191 of the sequence shown in SEQ ID NO: 58) and a cytoplasmic domain (amino acids at positions 192 to 288 of the sequence shown in SEQ ID NO: 58). As used herein, the term "PD-1 fragment" should encompass any fragment of the PD-1 protein, preferably an immunogenic fragment. The term also encompasses, for example, the above-mentioned domains of the full-length protein or any fragment of these domains, particularly immunogenic fragments. The amino acid sequence of the preferred extracellular domain of the human PD-1 protein is listed in SEQ ID NO: 59 of the sequence listing.

[0115] The Fc region can have a lysine at its C-terminus. The source of the lysine is a naturally occurring sequence found in humans, and these Fc regions are derived from this sequence. During the cell culture production process of recombinant antibodies, the terminal lysine can be cut off by proteolysis of endogenous carboxypeptidases, thereby producing a constant region with the same sequence but lacking a C-terminal lysine. For the purpose of preparing antibodies, the DNA encoding the terminal lysine can be omitted from the sequence so that antibodies are produced in the absence of lysine. The antibodies produced by the nucleic acid sequence encoding or not encoding the terminal lysine are substantially the same in sequence and function, because when, for example, antibodies produced in a CHO-based production system are used, the degree of processing of the terminal lysine is generally very high (Dick, LW et al. Biotechnol. Bioeng. 2008; 100: 1132–1143). Therefore, it should be understood that proteins according to the present invention, such as antibodies, can be generated in the case of encoding or not encoding or having a terminal lysine. According to the present invention, it should also be understood that a sequence with a terminal lysine, such as a constant region sequence with a terminal lysine, can be understood as a corresponding sequence without a terminal lysine, and a sequence without a terminal lysine can also be understood as a corresponding sequence with a terminal lysine.

[0116] Aspects and embodiments of the present disclosure

[0117] In a first aspect, the present disclosure provides a method for reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor.

[0118] Binding agents that bind to CD27

[0119] In one embodiment of the invention, the binding agent comprises at least one antigen binding region capable of binding to human CD27, wherein the binding agent comprises heavy chain variable (VH) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, and light chain variable (VL) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

[0120] In a further embodiment of the invention, the binding agent comprises two such antigen-binding regions, comprising VH region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and VL region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively. Provided herein are anti-CD27 antibodies that are capable of binding to human CD27 and further bind to human CD27 variants comprising the A59T mutation.

[0121] In one embodiment of the present invention, the binding agent binds, for example, CD27 on T cells and exhibits agonistic activity when bound to its target. Provided herein are binding agents that stimulate T cell activation and proliferation. The binding agent can further stimulate memory formation and survival of T cells. Such binding agents can be used, for example, to treat cancer. The binding agent can further bind to cynomolgus monkey CD27, which is useful for toxicological studies of the binding agent.

[0122] In one embodiment, the binding agent is an isolated antibody.

[0123] In one embodiment, the binding agent is an antibody. In another embodiment, the binding agent is a human antibody. In another embodiment, the binding agent is a humanized antibody. In another embodiment, the binding agent is a chimeric antibody.

[0124] In a preferred embodiment, the binding agent is a full-length antibody. Therefore, the binding agent of the present invention may further comprise a light chain constant region (CL) and a heavy chain constant region (CH). CH preferably comprises a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0125] It is well known in the art that mutations can be made in the VH and VL of an antibody to, for example, increase the affinity of the antibody for its target antigen, reduce its potential immunogenicity, and / or increase the yield of the antibody expressed by the host cell. Thus, in some embodiments, binding agents comprising variants of the CDR, VH, and / or VL sequences of the binding agents according to the present invention are also contemplated, in particular functional variants of the VH and / or VL regions as shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively. Functional variants can differ in one or more amino acids, for example in one or more CDRs, compared to the parent VH and / or VL sequence, but still allow the antigen-binding region to retain at least a substantial portion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) or even all of the affinity and / or specificity of the parent antibody. Typically, such functional variants retain significant sequence identity to the parent sequence. Exemplary variants include those that differ from their respective parent VH or VL regions by 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations, such as substitutions, insertions, or deletions of amino acid residues. Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of the parent sequence primarily by conservative amino acid substitutions; for example, 12, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in the variant may be conservative. In further embodiments of the invention, the binding agent may comprise up to 1, 2, or 3 mutations in the VH CDR region and / or VL CDR region, respectively. Such mutations may be substitutions. Preferably, such substitutions do not significantly alter the binding affinity and / or binding specificity of the binding agent of the invention. Thus, the present invention encompasses variants of the binding agents of the invention having the same functional characteristics as the binding agents 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.

[0126] In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 80% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 85% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 90% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 95% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 96% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 97% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 98% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence that is at least 99% identical to the VH region set forth in SEQ ID NO: 4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence set forth in SEQ ID NO: 4.

[0127] In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 80% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 85% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 90% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 95% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 96% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 97% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, a binding agent comprises a VH region comprising a sequence that is at least 98% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence that is at least 99% identical to the VH region set forth in SEQ ID NO: 8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence set forth in SEQ ID NO:8.

[0128] In another embodiment of the invention, the binding agent comprises VH and VL regions comprising the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

[0129] The binding agent used in the method according to the invention may comprise a light chain constant region which is a human kappa light chain. In another embodiment, it may comprise a human lambda light chain constant region.

[0130] The binding agent may preferably further comprise a heavy chain constant region, which is a human IgG isotype. It may optionally comprise a modified human IgG constant region. Such human IgG comprises an Fc region, which comprises a CH2 and CH3 region. By modifying the IgG constant region in the Fc region, for example, the Fc effector function of the antibody can be modulated or Fc-Fc interactions can be increased, thereby increasing the tendency of the antibody to form clusters such as hexamers. In one embodiment of the invention, the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4. In one embodiment, it is IgG1. In another embodiment, it is IgG2. In yet another embodiment, it is IgG3. In a further embodiment, it is IgG4. In a specific embodiment, the IgG is a modified human IgG comprising one or more amino acid substitutions in the Fc region. In one embodiment, it can be a human IgG1 comprising one or more amino acid substitutions in the Fc region. In a further embodiment of the invention, IgG1 comprises two or more amino acid substitutions in the Fc region. In one embodiment, the IgG1 Fc region has two amino acid substitutions.

[0131] In a further embodiment of the invention, the modified human IgG heavy chain constant region comprises up to 10 amino acid substitutions in the Fc region. In another embodiment, it comprises up to 9 amino acid substitutions. In another embodiment, it comprises up to 8 amino acid substitutions. In another embodiment, it comprises up to 7 amino acid substitutions. In another embodiment, it comprises up to 6 amino acid substitutions. In another embodiment, it comprises up to 5 amino acid substitutions. In another embodiment, it comprises up to 4 amino acid substitutions. In another embodiment, it comprises up to 3 amino acid substitutions. In another embodiment, it comprises up to 2 amino acid substitutions in the Fc region.

[0132] Mutations in the amino acid residues at positions corresponding to E430, E345, and S440 in the human IgG1 heavy chain, where the amino acid residues are numbered according to the EU index, can improve the ability of the antibody to induce CDC. Without being bound by theory, it is believed that by substituting one or more amino acids at these positions, oligomerization of the antibody can be stimulated, thereby modulating Fc-mediated effector functions, such as increasing C1q binding, complement activation, CDC, ADCP, internalization, or other related functions that may provide in vivo efficacy.

[0133] In a further embodiment of the invention, the binding agent is a variant antibody comprising an antigen binding region and a variant Fc region.

[0134] In certain embodiments, an antibody variant that binds human CD27 comprises:

[0135] (a) a heavy chain comprising a VH region and a human IgG1 CH region comprising a mutation in one or more of E430, E345, and S440, the VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO: 5, a VH CDR2 comprising the sequence set forth in SEQ ID NO: 6, and a VH CDR3 comprising the sequence set forth in SEQ ID NO: 7, wherein amino acid residues are numbered according to the EU index;

[0136] (b) a light chain comprising a VL region comprising a VL CDR1 comprising the sequence shown in SEQ ID NO:9, a VL CDR2 comprising the sequence shown in SEQ ID NO:10, and a VL CDR3 comprising the sequence shown in SEQ ID NO:11.

[0137] In certain other embodiments, the antibody variant that binds human CD27 comprises:

[0138] (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, amino acid residues being numbered according to the EU index, and

[0139] (b) a light chain comprising a VL region comprising SEQ ID NO: 8.

[0140] Variant antibodies of the invention that bind to human CD27 comprise a variant Fc region or a variant human IgG1 CH region comprising a mutation in one or more of P329, E430, and E345. Hereinafter, references to mutations in the Fc region may similarly apply to mutations in the human IgG1 CH region, and vice versa.

[0141] As described herein, when numbering according to the Eu index, the position of the amino acid to be mutated in the Fc region can be given relative to (i.e., "corresponding to") its position in the naturally occurring (wild-type) human IgG1 heavy chain. Thus, if the parent Fc region already contains one or more mutations and / or if the parent Fc region is, for example, an IgG2, IgG3, or IgG4 Fc region, the position of the amino acid corresponding to the amino acid residue numbered according to the Eu index (e.g., E430) in the human IgG1 heavy chain can be determined by comparison. Specifically, the parent Fc region is aligned with the wild-type human IgG1 heavy chain sequence to identify the residue corresponding to the position of E430 in the human IgG1 heavy chain sequence. Any wild-type human IgG1 constant region amino acid sequence can be used for this purpose, including any of the different human IgG1 allotypes listed in Table 3.

[0142] In one embodiment of the invention, modifications in the IgG Fc region induce increased CD27 agonism compared to a wild-type IgG Fc region of the same antibody but comprising the same isotype, such as IgG1. This can be achieved, for example, by introducing an amino acid other than E at an amino acid position corresponding to position E345 and / or E430 in a human IgG1 heavy chain numbered according to Eu. In one embodiment of the invention, the amino acid residue at the position corresponding to position E345 in a human IgG1 heavy chain numbered according to Eu 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 embodiment of the invention, the amino acid residue at the position corresponding to position E430 in a human IgG1 heavy chain numbered according to Eu 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.

[0143] In a preferred embodiment, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain numbered according to Eu is R. Thus, a binding agent of the invention may comprise an E345R substitution in the Fc region. In another embodiment of the invention, the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain numbered according to Eu is G. Thus, a binding agent of the invention may comprise an E430G substitution in the Fc region. In another embodiment, the binding agent comprises an amino acid substitution selected from the group consisting of: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, and E345Y.

[0144] Thus, provided is a binding agent in the form of an antibody or antibodies having enhanced Fc-Fc interactions that upon antibody binding lead to antibody-dependent clustering of CD27 on the cell surface, thereby increasing the agonistic effect of the binding agent of the invention.

[0145] In another embodiment of the binding agent for use according to the 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 binding agent for use according to the invention may further comprise a mutation in position 329.

[0146] In a further embodiment of the invention, the binding agent comprises an amino acid residue R at a position corresponding to position P329 in the heavy chain of human IgG1 numbered according to Eu. Thus, the binding agent of the invention may comprise a P329R substitution in the Fc region. Without being bound by theory, it is believed that a binding agent comprising an E345R mutation in the Fc region (e.g., as set forth in SEQ ID NO: 13) has increased serum clearance. The inventors have found that introducing a further mutation at position 329, such as P329R (e.g., as set forth in SEQ ID NO: 15), restores the clearance of the binding agent to the level of a binding agent comprising, for example, a wt IgG1 as set forth in SEQ ID NO: 12.

[0147] In another preferred embodiment, 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. This provides a binding agent that has increased CD27 receptor agonism and comparable pharmacokinetic properties, such as, for example, serum clearance, when compared to a binding agent comprising the same VH and VL regions and comprising the same IgG1 heavy chain constant region, except comprising the wild-type amino acid P at position 329 and the wild-type amino acid E at position 345.

[0148] Thus, in embodiments, the binding agent has increased receptor agonism upon binding to CD27 and further has comparable pharmacokinetic properties, e.g., similar or even identical pharmacokinetic properties, when compared to the pharmacokinetic properties of a binding agent comprising the same VH and VL regions but comprising a wild-type IgG1 heavy chain constant region (e.g., as set forth in SEQ ID NO: 12). In other words, the binding agent may have a pharmacokinetic property that is not significantly different from the pharmacokinetic property of the same binding agent (except for comprising a wild-type IgG1 heavy chain constant region).

[0149] In other embodiments of the present invention, the binding agent comprises a variant Fc region according to any one of the aforementioned 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 corresponding to one or more amino acid residues at E430, E345, and P329 is produced in a parent Fc region of a human IgG Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. Preferably, the parent Fc region is a naturally occurring (wild type) human IgG Fc region, such as a human wild type IgG1, IgG2, IgG3, or IgG4 Fc region, or a mixed isotype thereof. Therefore, in addition to the mutation (in one or more amino acid residues selected from E430, E345, and P329), the variant Fc region can be a human IgG1, IgG2, IgG3, or IgG4 isotype, or a mixed isotype thereof.

[0150] In one embodiment, the parent Fc region and / or human IgG1 CH region is a wild-type human IgG1 isotype.

[0151] Thus, except for the mutations (in E430 or E345 or P329), the variant Fc region may be a human IgG1 Fc region.

[0152] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(f) isotype.

[0153] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(z) isotype.

[0154] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(a) isotype.

[0155] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(x) isotype.

[0156] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is a human wild-type IgG1 of mixed isotypes, eg, IgG1m(za), IgG1m(zax), IgG1m(fa), and the like.

[0157] Thus, in addition to the mutations (in E430 or E345 or P329), the variant Fc region and / or human IgG1 CH region may be of human IgG1m(f), IgG1m(a), IgG1m(x), IgG1m(z) allotype or a mixed allotype of any two or more thereof.

[0158] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(za) isotype.

[0159] In a specific embodiment, the parent Fc region is a human wild-type IgG2 isotype.

[0160] In a specific embodiment, the parent Fc region is a human wild-type IgG3 isotype.

[0161] In a specific embodiment, the parent Fc region is a human wild-type IgG4 isotype.

[0162] The CH region amino acid sequences of specific examples of wild-type human IgG isotypes and IgG1 allotypes are listed in Table 3.

[0163] In another embodiment, the binding agent comprises a heavy chain constant region comprising an amino acid 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. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 12. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 13. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 14. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 15. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 18. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 19. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 20. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 21. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 22. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 23. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 27. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 28. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 29. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 30. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 31. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 32. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 33. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 34. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 36.

[0164] In embodiments, the binding agent comprises:

[0165] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0166] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0167] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 15 and

[0168] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0169] In another embodiment, the binding agent comprises:

[0170] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0171] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0172] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 12 and

[0173] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0174] In another embodiment, the first combination comprises:

[0175] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0176] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0177] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 13 and

[0178] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0179] In another embodiment, the binding agent comprises:

[0180] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0181] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0182] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 14 and

[0183] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0184] In another embodiment, the binding agent comprises:

[0185] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0186] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0187] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 18 and

[0188] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0189] In another embodiment, the binding agent comprises:

[0190] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0191] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0192] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 19 and

[0193] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0194] In another embodiment, the binding agent comprises:

[0195] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0196] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0197] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 20 and

[0198] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0199] In another embodiment, the binding agent comprises:

[0200] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0201] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0202] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 21 and

[0203] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0204] In another embodiment, the binding agent comprises:

[0205] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0206] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0207] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 22 and

[0208] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0209] In another embodiment, the binding agent comprises:

[0210] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0211] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0212] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 23 and

[0213] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0214] In another embodiment, the binding agent comprises:

[0215] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0216] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0217] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 27 and

[0218] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0219] In another embodiment, the binding agent comprises:

[0220] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0221] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0222] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 28 and

[0223] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0224] In another embodiment, the binding agent comprises:

[0225] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0226] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0227] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 29 and

[0228] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0229] In another embodiment, the binding agent according to the invention comprises:

[0230] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0231] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0232] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 30 and

[0233] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0234] In another embodiment, the binding agent comprises:

[0235] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0236] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0237] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 31 and

[0238] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0239] In another embodiment, the binding agent comprises:

[0240] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0241] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0242] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 32 and

[0243] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0244] In another embodiment, the binding agent comprises:

[0245] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0246] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0247] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 33 and

[0248] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0249] In another embodiment, the binding agent comprises:

[0250] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0251] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0252] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 34 and

[0253] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0254] In another embodiment, the binding agent comprises:

[0255] a. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0256] b. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0257] c. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 36 and

[0258] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO:16.

[0259] In an alternative embodiment, the CL region may be the amino acid sequence shown in SEQ ID NO:17.

[0260] In embodiments, the binding agent comprises:

[0261] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0262] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0263] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 15 and

[0264] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0265] In another embodiment, the binding agent comprises:

[0266] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0267] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0268] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 12 and

[0269] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0270] In another embodiment, the binding agent comprises:

[0271] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0272] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0273] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 13 and

[0274] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0275] In another embodiment, the binding agent comprises:

[0276] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0277] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0278] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 14 and

[0279] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0280] In another embodiment, the binding agent comprises:

[0281] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0282] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0283] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 18 and

[0284] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0285] In another embodiment, the binding agent comprises:

[0286] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0287] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0288] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 19 and

[0289] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0290] In another embodiment, the binding agent comprises:

[0291] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0292] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0293] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 20 and

[0294] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0295] In another embodiment, the binding agent comprises:

[0296] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0297] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0298] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 21 and

[0299] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0300] In another embodiment, the binding agent comprises:

[0301] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0302] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0303] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 22 and

[0304] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0305] In another embodiment, the binding agent comprises:

[0306] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0307] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0308] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 23 and

[0309] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0310] In another embodiment, the binding agent comprises:

[0311] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0312] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0313] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 27 and

[0314] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0315] In another embodiment, the binding agent comprises:

[0316] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0317] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0318] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 28 and

[0319] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0320] In another embodiment, the binding agent comprises:

[0321] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0322] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0323] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 29 and

[0324] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0325] In another embodiment, the binding agent comprises:

[0326] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0327] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0328] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 30 and

[0329] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0330] In another embodiment, the binding agent comprises:

[0331] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0332] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0333] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 31 and

[0334] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0335] In another embodiment, the binding agent comprises:

[0336] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0337] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0338] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 32 and

[0339] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0340] In another embodiment, the binding agent comprises:

[0341] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0342] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0343] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 33 and

[0344] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0345] In another embodiment, the binding agent comprises:

[0346] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0347] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0348] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 34 and

[0349] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0350] In another embodiment, the binding agent comprises:

[0351] e. VH region, which comprises the amino acid sequence shown in SEQ ID NO: 4

[0352] f. VL region, which comprises the amino acid sequence shown in SEQ ID NO: 8

[0353] g. CH region, which comprises the amino acid sequence shown in SEQ ID NO: 36 and

[0354] h. CL region comprising the amino acid sequence shown in SEQ ID NO: 17;

[0355] In another embodiment, the binding agent 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.

[0356] In another embodiment, the binding agent 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.

[0357] In another embodiment, the binding agent comprises a modified heavy chain constant region so that the binding agent induces Fc-mediated effector functions to a lesser extent relative to the same binding agent except for the modification. Examples herein are CD27 binding antibodies of the present invention comprising P329R and E345R substitutions. Compared to antibodies comprising the same sequence but not comprising P329R substitutions, and also compared to the same antibodies comprising the same sequence but not comprising P329R and E345R substitutions (e.g., wild-type IgG1 heavy chains), such antibodies induce one or more Fc-mediated effector functions to a lesser extent. In one embodiment, the Fc-mediated effector function is reduced by at least 20%. In another embodiment, the Fc-mediated effector function is reduced by at least 30%. In another embodiment, the Fc-mediated effector function is reduced by at least 40%. In another embodiment, the Fc-mediated effector function is reduced by at least 50%. In another embodiment, the Fc-mediated effector function is reduced by at least 60%. In another embodiment, the Fc-mediated effector function is reduced by at least 70%. In another embodiment, the effector function of Fc mediation is reduced by at least 80%. In another embodiment, the effector function of Fc mediation is reduced by at least 90%. In another embodiment, the binding agent does not induce one or more Fc-mediated effector functions. One or more Fc effector functions that are reduced or not induced at all can be selected from the group consisting of 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. Therefore, in one embodiment, the binding agent induces CDC by reducing at least 20%, for example, at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or reducing by at least 90% relative to the same binding agent but with a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce CDC.

[0358] In another embodiment, the binding agent induces CDCC to an extent that is at least 20% less, e.g., 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% less, relative to the same binding agent but with a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce CDCC.

[0359] In another embodiment, the binding agent induces ADCC to an extent that is at least 20% less, e.g., 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% less, relative to an identical binding agent but having a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce ADCC.

[0360] In another embodiment, the binding agent induces ADCP to a degree that is at least 20% lower, e.g., 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% lower, relative to the same binding agent but with a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce ADCP.

[0361] In another embodiment, the binding agent induces C1q binding to an extent that is at least 20% less, 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% less, relative to an identical binding agent but having a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce C1q binding. Preferably, C1q binding is determined as described in Example 8.

[0362] In another embodiment, the binding agent induces FcγR binding to a degree that is at least 20% less, 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% less, relative to an identical binding agent but having a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce FcγR binding. Preferably, FcγR binding is determined as described in Example 9.

[0363] In one embodiment, the binding agent has reduced CIq binding and reduced FcγR binding compared to a binding agent comprising the same amino acid sequence but not comprising the P329R substitution.

[0364] In one embodiment, the binding agent used in any aspect or embodiment herein is a human antibody, except for the recited mutations.

[0365] In an embodiment of the invention, the binding agent is a monovalent antibody.

[0366] In another embodiment, the binding agent is a bivalent antibody.

[0367] Furthermore, the binding agents of the invention may be monospecific antibodies.

[0368] In one embodiment, the binding agent used in any aspect or embodiment herein is a monoclonal antibody, eg, a human monoclonal antibody, eg, a human bivalent monoclonal antibody, eg, a human bivalent full-length monoclonal antibody.

[0369] In a preferred embodiment, the binding agent used in any aspect or embodiment herein is an IgG1 antibody, e.g., a full-length IgG1 antibody, e.g., a human full-length IgG1 antibody, optionally a human monoclonal full-length bivalent IgG1, κ antibody, e.g., a human monoclonal full-length bivalent IgG1m(f), κ antibody, in addition to the optional enumerated mutations in the Fc region.

[0370] The binding agents used in connection with the present invention are advantageously present in a bivalent monospecific format, comprising two antigen-binding regions that bind to the same epitope. However, bispecific formats in which one antigen-binding region binds to a different epitope are also contemplated. Thus, unless contradicted by the context, the binding agents used in accordance with any aspect or embodiment herein may be monospecific antibodies or bispecific antibodies.

[0371] Thus, in another embodiment, the binding agent is a bispecific antibody comprising a first antigen-binding region that is capable of binding to human CD27 as described herein and a second antigen-binding region that is capable of binding to a different epitope on human CD27. In another embodiment, the binding agent is a bispecific antibody comprising a first antigen-binding region that is capable of binding to human CD27 as described herein and a second antigen-binding region that is capable of binding to a different target. Such targets can be on different cells or on the same cell as CD27.

[0372] In an embodiment of the present invention, the binding agent is capable of binding to human CD27 having the sequence set forth in SEQ ID NO: 1. However, human CD27 may be expressed as a variant in some individuals. Therefore, in another embodiment, the binding agent is capable of further binding to a human CD27 variant, such as the human CD27 variant set forth in SEQ ID NO: 2. In another embodiment, the binding agent is capable of further binding to cynomolgus monkey CD27, such as the one set forth in SEQ ID NO: 3.

[0373] In a further embodiment of the invention, the binding agent is capable of binding to human T cells expressing CD27.

[0374] In another embodiment of the invention, the binding agent is capable of binding to cynomolgus monkey T cells expressing CD27.

[0375] In one embodiment of the present invention, the full-length IgG1 antibody has the C-terminal lysine of the HC cleaved off. Such antibodies are also considered "full-length antibodies."

[0376] In another embodiment of the invention, the binding agent is capable of inducing human T cells such as CD4 + and CD8 + Proliferation of T cells, such as T helper cells and cytotoxic T cells. Such activity can be determined as described in Example 6 or 7 herein.

[0377] In another embodiment of the invention, the binding agent is capable of inducing activation of Jurkat reporter T cells expressing human CD27, such as described in Example 2 herein.

[0378] In another embodiment of the invention, the binding agent is capable of inducing activation of Jurkat reporter T cells expressing human CD27 in the absence of cross-linking of Fcγ receptor IIb, such as described in Example 11 herein.

[0379] In another embodiment of the invention, the binding agent is capable of inducing CD4 T cells with a central memory T cell phenotype. + and CD8 + Proliferation of T cells.

[0380] In another embodiment of the invention, the binding agent is capable of inducing IFNy production.

[0381] In another embodiment of the invention, the binding agent is in a composition or formulation comprising acetate, sorbitol, polysorbate 80, and having a pH of 5 to 6, preferably a pH of 5.5.

[0382] PD1 / PD-L1 inhibitors

[0383] In one embodiment, a PD1 / PD-L1 inhibitor blocks inhibitory signals associated with PD-1. In one embodiment, a PD1 / PD-L1 inhibitor is an antibody or fragment thereof that disrupts or inhibits inhibitory signaling associated with PD-1. In one embodiment, a PD1 / PD-L1 inhibitor is a small molecule inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, a PD1 / PD-L1 inhibitor is a peptide-based inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, a PD1 / PD-L1 inhibitor is an inhibitory nucleic acid molecule that disrupts or inhibits inhibitory signaling.

[0384] Inhibition or blocking of PD-1 signaling as described herein results in prevention or reversal of immunosuppression and establishment or enhancement of T cell immunity against cancer cells. In one embodiment, inhibition of PD-1 signaling as described herein reduces or suppresses immune system dysfunction. In one embodiment, inhibition of PD-1 signaling as described herein results in less dysfunction of dysfunctional immune cells. In one embodiment, inhibition of PD-1 signaling as described herein results in less dysfunction of dysfunctional T cells.

[0385] In one embodiment, PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence shown in SEQ ID NO: 98.

[0386] In one embodiment, PD1 is human PD1. Preferably, PD1 has or comprises the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 59, or the amino acid sequence of PD1 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 59, or is an immunogenic fragment thereof.

[0387] In one embodiment, a PD1 / PD-L1 inhibitor prevents the interaction between PD-1 and PD-L1.

[0388] PD1 / PD-L1 inhibitors can be antibodies, antigen-binding fragments thereof, or constructs comprising antibody portions of antigen-binding fragments thereof having the desired specificity. Antibodies or antigen-binding fragments thereof are as described herein. Antibodies or antigen-binding fragments thereof that are PD1 / PD-L1 inhibitors particularly include antibodies or antigen-binding fragments thereof that bind to PD-1, and antibodies or antigen-binding fragments thereof that bind to PD-L1. As described herein, antibodies or antigen-binding fragments may also be conjugated to other moieties. In particular, the antibodies or antigen-binding fragments thereof are chimeric, humanized, or human antibodies.

[0389] In one embodiment, the antibody that acts as a PD1 / PD-L1 inhibitor is an isolated antibody.

[0390] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody, fragment, or construct thereof that prevents the interaction between PD-1 and PD-L1.

[0391] PD1 / PD-L1 inhibitors can be inhibitory nucleic acid molecules, such as oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules and aptamers (e.g., DNA or RNA aptamers), particularly antisense oligonucleotides. In one embodiment, the PD1 / PD-L1 inhibitor as siRNA interferes with mRNA, thereby blocking translation, such as translation of PD-1 protein.

[0392] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody, an antigen binding portion thereof, or a construct thereof that disrupts or inhibits the interaction between the PD-1 receptor and one or more of its ligands, PD-L1 and / or PD-L2. Antibodies that bind to PD-1 or PD-L1 and disrupt or inhibit the interaction between PD-1 and one or more of its ligands are known in the art. In certain embodiments, the antibody, its antigen binding portion, or its construct specifically binds to PD-1. In certain embodiments, the antibody, its antigen binding portion, or its construct specifically binds to PD-L1.

[0393] In certain preferred embodiments, the PD1 / PD-L1 inhibitor is an antibody that binds to PD-1, such as a PD-1 blocking antibody. In certain preferred embodiments, the PD1 / PD-L1 inhibitor is an antibody that binds to PD-L1, such as a PD-L1 blocking antibody.

[0394] Exemplary PD1 / PD-L1 inhibitors include, but are not limited to, anti-PD-1 antibodies, such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606, and US 2015 / 0079109), lamblizumab (e.g., disclosed as hPD109A and its humanized derivatives h409A1, h409A16, and h409A17 in WO 2008 / 156712), AB137132 (Abcam), EH12.2H7, and RMP1-14 (#BE0146; BioXcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix Bioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; see U.S. Pat. No. 8,008,449; WO 2015 / 35606, and US 2015 / 0079109), 2013 / 173223; WO 2006 / 121168), pembrolizumab (KEYTRUDA; MK-3475; Merck; see WO 2008 / 156712), pidilizumab (CT-011; CureTech; see Hardy et al., 1994, Cancer Res., 54(22):5793-6 and WO 2009 / 101611), PDR001 (Novartis; see WO 2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see WO 2012 / 145493), TSR-042 (see WO 2014 / 179664), cemiplimab (REGN-2810; Regeneron; H4H7798N; cf. US2015 / 0203579 and WO 2015 / 112800), JS001 (TAIZHOU JUNSHIPHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70:136), AMP-224 (GSK-2661380; cf. Li et al., 2016, Int J Mol Sci 17(7):1151 and WO 2010 / 027827 and WO 2011 / 066342), PF-06801591 (Pfizer), tislelizumab (BGB-A317; BeiGene; see WO 2015 / 35606, U.S. Patent No. 9,834,606 and US2015 / 0079109), BI 754091, SHR-1210 (see WO 2015 / 085847 and WO Antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in 2006 / 121168, INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang et al., 2017, J. Hematol. Oncol. 70:136), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540), cetrelimab (JNJ-63723283; JNJ-3283; see Calvo et al., J. Clin. Oncol. 36, no. 5_suppl (2018) 58), genolimzumab (CBT-501; see Patel et al., J. ImmunoTher. Cancer, 2017, 5(Suppl 2): ​​P242), sasanlimab (PF-06801591; see Youssefet et al., J. Clin. Oncol. 36, no. 5_suppl (2018) 58), al.,Proc.Am.Assoc.Cancer Res.Ann.Meeting2017;Cancer Res 2017;77(13Suppl):Abstract)、toripalimab (JS-001;see US2016 / 0272708)、camrelizumab (SHR-1210;INCSHR-1210;see US2016 / 376367;Huang et al.,Clin.Cancer Res.2018;24(6):1296-1304)、spartalizumab (PDR001;see WO 2017 / 106656;Naing et al.,J.Clin.Oncol.34,no.15_suppl(2016)3060-3060)、BCD-100 (JSCBIOCAD, Russia;see WO 2018 / 103017), balstilimab (AGEN2034; see WO 2017 / 040790), sintilimab (IBI-308; see WO 2017 / 024465 and WO 2017 / 133540), ezabenlimab (BI-754091; see US 2017 / 334995; Johnson et al., J. Clin. Oncol. 36, no.5_suppl(2018)212-212), zimberelimab (GLS-010; see WO 2017 / 025051), LZM-009 (see US2017 / 210806), AK-103 (see WO 2017 / 071625, WO2017 / 166804 and WO 2018 / 036472), retifanlimab (MGA-012; see WO 2017 / 019846), Sym-021 (see WO 2017 / 055547), CS1003 (see CN107840887), anti-PD-1 antibodies, as described, for example, in US 7,488,802, US 8,008,449, US 8,168,757, WO 03 / 042402, WO 2010 / 089411 (further disclosing anti-PD-L1 antibodies), WO 2010 / 036959, WO 2011 / 159877 (further disclosing antibodies against TIM-3), WO 2011 / 082400, WO 2011 / 161699, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2012 / 145493 (further disclosing antibodies against PD-L1), WO 2015 / 035606, WO 2014 / 055648 (further disclosing anti-KIR antibodies), US2018 / 0185482 (further disclosing anti-PD-L1 and anti-TIGIT antibodies), US8,008,449, US8,779,105, US6,808,710, US8,168,757, US2016 / 0272708 and US8,354,509, such as small molecule antagonists of the PD-1 signaling pathway disclosed in, for example, Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumarand Ramachandra, 2018, BioDrugs, 32(5):481-497, such as siRNA against PD-1 disclosed in, for example, WO 2019 / 000146 and WO 2018 / 103501, such as WO Soluble PD-1 protein as disclosed in WO 2018 / 222711 and oncolytic viruses comprising a soluble form of PD-1 as disclosed, for example, in WO 2018 / 022831.

[0395] In certain embodiments, the PD1 / PD-L1 inhibitor is nivolumab (OPDIVO; BMS-936558) or a biosimilar thereof, pembrolizumab (KEYTRUDA; MK-3475) or a biosimilar thereof, pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514) or a biosimilar thereof, TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI 754091, or SHR-1210.

[0396] In certain embodiments, the PD1 / PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody or antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of one of the anti-PD1 or anti-PD-L1 antibodies or antigen-binding fragments described herein, e.g., the CDRs of an anti-PD1 or anti-PD-L1 antibody or antigen-binding fragment selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplizumab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0397] In certain embodiments, the PD1 / PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody or antigen-binding fragment thereof, which comprises the heavy chain variable region and the light chain variable region of one of the above-mentioned anti-PD1 or anti-PD-L1 antibodies or antigen-binding fragments, such as the heavy chain variable region and the light chain variable region of an anti-PD1 or anti-PD-L1 antibody or antigen-binding fragment selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplizumab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0398] In certain embodiments, the PD1 / PD-L1 inhibitor is an anti-PD-1 or anti-PD-L1 antibody or antigen-binding fragment thereof selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplizumab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0399] In certain preferred embodiments, the PD1 / PD-L1 inhibitor is an antibody that binds to PD-L1 or PD-L1. In certain preferred embodiments, the PD1 / PD-L1 inhibitor is an antibody that is an antagonist of the PD1 / PD-L1 interaction. In certain preferred embodiments, the PD1 / PD-L1 inhibitor is a PD1 blocking antibody or a PD-L1 blocking antibody.

[0400] In certain embodiments, the PD1 / PD-L1 inhibitor is an antibody of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, such as an antibody of the IgG1 isotype. In one embodiment, the PD1 / PD-L1 inhibitor is an antibody of the IgG1 isotype. In one embodiment, the PD1 / PD-L1 inhibitor is an antibody of the IgG2 isotype. In one embodiment, the PD1 / PD-L1 inhibitor is an antibody of the IgG3 isotype. In one embodiment, the PD1 / PD-L1 inhibitor is an antibody of the IgG4 isotype.

[0401] In certain embodiments, the PD1 / PD-L1 inhibitor is a full-length antibody or an antibody fragment, such as a full-length IgG1 antibody.

[0402] In certain embodiments, the PD1 / PD-L1 inhibitor is a monospecific antibody.

[0403] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody that binds to PD1 and comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 99, 100, and 101, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 102, LAS, and SEQ ID NO: 103, respectively.

[0404] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody that binds to PD1 and comprises a VH region comprising the amino acid sequence of SEQ ID NO: 104 and a VL region comprising the amino acid sequence of SEQ ID NO: 105.

[0405] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody that binds to PD1 and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 106 and a light chain comprising the amino acid sequence of SEQ ID NO: 107.

[0406] In a preferred embodiment, the PD1 / PD-L1 inhibitor is pembrolizumab or a biosimilar thereof.

[0407] In a preferred embodiment, the PD1 / PD-L1 inhibitor is nivolumab or a biosimilar thereof.

[0408] In a preferred embodiment, the PD1 / PD-L1 inhibitor is atezolizumab or a biosimilar thereof.

[0409] In some embodiments, the PD1 / PD-L1 inhibitor is a PD1 inhibitor, such as a PD1 blocking antibody. In some embodiments, the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor, such as a PD-L1 blocking antibody.

[0410] In certain embodiments, the PD1 / PD-L1 inhibitor is a PD1 inhibitor selected from the group consisting of pembrolizumab, nivolumab, cemiplizumab, dotalimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-224, AMP-514, or biosimilars thereof.

[0411] In certain embodiments, the PD1 inhibitor is selected from pembrolizumab, nivolumab, cemiplizumab, dotalimumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-514, or biosimilars thereof.

[0412] In certain embodiments, the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor selected from the following: Atezolizumab, Avelumab, Durvalumab, KN035, CK-301, Acasunlimab, AUNP12, CA-170, BMS-986189, or their respective biosimilars.

[0413] In certain embodiments, the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, acalabrutinib, or biosimilars thereof.

[0414] In a further preferred embodiment, the PD1 / PD-L1 inhibitor is an antibody that binds to PD-1. The antibody that binds to PD-1 may comprise a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise or have the sequences shown in SEQ ID NO: 49, SEQ ID NO: 46, and SEQ ID NO: 45, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise or have the sequences shown in SEQ ID NO: 52, QAS, and SEQ ID NO: 50, respectively. A specific but non-limiting example of such an antibody is MAB-19-0202.

[0415] The terms "heavy chain variable region" (also referred to as "VH") and "light chain variable region" (also referred to as "VL") are used herein in their most general sense and encompass any sequence that can contain complementarity determining regions (CDRs) interspersed among other regions, also referred to as framework regions (FRs). The framework regions, among other things, separate the CDRs so that they can form an antigen binding site, particularly after VH and VL fold and pair. Preferably, each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. That is, the terms "heavy chain variable region" and "light chain variable region" should not be interpreted as being limited to sequences that can be found in native antibodies or in the VH and VL sequences exemplified herein (SEQ ID NOs: 54 to 57 of the Sequence Listing). These terms include any sequence that can contain and appropriately position the CDR, such as sequences derived from the VL and VH regions of a natural antibody or sequences derived from sequences shown in SEQ ID NOs: 54 to 57 of the sequence listing. It will be understood by those skilled in the art that, in particular, the sequences of the framework regions can be modified (including variants with respect to amino acid substitutions and variants with respect to sequence length, i.e., insertion or deletion variants) without losing the characteristics of VH and VL, respectively. In a preferred embodiment, any modification is limited to the framework regions. However, those skilled in the art will also be fully aware that CDRs, hypervariable regions, and variable regions can also be modified without losing the ability to bind to PD-1. For example, the CDR regions will be identical or highly homologous to the regions specified herein. By "highly homologous," it is expected that 1 to 5, preferably 1 to 4, such as 1 to 3 or 1 or 2 substitutions can be produced in the CDRs. In addition, the hypervariable and variable regions can be modified so that they show substantial homology to the regions specifically disclosed herein.

[0416] In antibodies that bind to PD-1, the CDRs, as specified herein, have been identified using two different CDR identification methods. The first numbering scheme used herein is based on Kabat (Wu and Kabat, 1970; Kabat et al., 1991), and the second is the IMGT numbering (Lefranc, 1997; Lefranc et al., 2005). In the third method, the intersection of the two identification schemes was used.

[0417] Antibodies that bind to PD-1 may comprise one or more CDRs, CDR groups, or combinations of CDR groups as described herein, comprising the CDRs and their intermediate framework regions (also referred to herein as framework regions or FRs) or portions of the framework regions. Preferably, the portion will include at least about 50% of one or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of antibodies prepared by recombinant DNA techniques may result in the introduction of N-terminal or C-terminal residues into the variable region encoded by the introduced linker to facilitate cloning or other manipulation steps, including the introduction of a linker to connect the variable region of the present disclosure to another protein sequence, including an immunoglobulin heavy chain, other variable domains (e.g., in the production of diabodies), or a protein tag.

[0418] The antibody that binds to PD-1 may comprise a heavy chain variable region (VH) comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of the VH sequence as set forth in any one of SEQ ID NO: 56. In one embodiment, the antibody comprises a heavy chain variable region (VH), wherein the VH comprises a sequence as set forth in any one of SEQ ID NO: 56. In one embodiment, the antibody comprises a light chain variable region (VL), wherein the VH comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of the VL sequence as set forth in any one of SEQ ID NO: 57. In one embodiment, the antibody comprises a light chain variable region (VL), wherein the VL comprises a sequence as set forth in any one of SEQ ID NO: 57.

[0419] The antibody that binds to PD-1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or has the sequence shown in SEQ ID NO: 56, and the VL comprises or has the sequence shown in SEQ ID NO: 57, or a variant of each of these sequences. Another example of an antibody that binds to PD-1 can comprise a VH comprising or having the sequence shown in SEQ ID NO: 56 or a variant thereof, and a VL comprising or having the sequence shown in SEQ ID NO: 57 or a variant thereof. A specific but non-limiting example of such an antibody is MAB-19-0618. Antibody MAB-19-0618 is derived from MAB-19-0202. The present disclosure also encompasses variants of the heavy chain variable region (VH) and the light chain variable region (VL), as well as combinations of these variant VH and VL.

[0420] The antibody that binds to PD-1 may comprise a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region comprising or having the sequence shown in SEQ ID NO: 38 or 128 and a heavy chain variable region (VH) comprising or having the sequence shown in SEQ ID NO: 56, and the light chain comprises a light chain constant region comprising or having the sequence shown in SEQ ID NO: 42 and a light chain variable region (VL) comprising or having the sequence shown in SEQ ID NO: 57.

[0421] The antibody that binds to PD-1 may comprise a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region comprising or having the sequence shown in SEQ ID NO: 38 or 128 and a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 56, and the light chain comprises a light chain constant region comprising or having the sequence shown in SEQ ID NO: 42 and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 57. For example, the CDR1, CDR2, and CDR3 sequences are as specified herein.

[0422] The antibody that binds to PD-1 can be a monoclonal, chimeric, or monoclonal, humanized antibody or a fragment of such an antibody. The antibody can be a whole antibody or an antigen-binding fragment thereof, including, for example, a bispecific antibody.

[0423] In an antibody that binds to PD-1, one or more, preferably two, heavy chain constant regions may be modified such that binding of C1q to the antibody is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, compared to a wild-type antibody. In one embodiment, C1q binding can be determined by ELISA.

[0424] As used herein, "wild type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.

[0425] In an antibody that binds to PD-1, one or more, preferably two, heavy chain constant regions may be modified such that binding of one or more IgG Fc-γ receptors to the antibody is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, compared to a wild-type antibody. In one embodiment, the one or more IgG Fc-γ receptors are selected from at least one of Fc-γRI, Fc-γRII, and Fc-γRIII. In one embodiment, the IgG Fc-γ receptor is Fc-γRI.

[0426] In one embodiment, the antibody that binds to PD-1 is unable to induce Fc-γRI-mediated effector function, or wherein the induced Fc-γRI-mediated effector function is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0427] In one embodiment, the antibody that binds to PD-1 is unable to induce at least one of complement dependent cytotoxicity (CDC)-mediated lysis, antibody dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis, or wherein at least one of complement dependent cytotoxicity (CDC)-mediated lysis, antibody dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis is induced to a reduced extent, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0428] Antibody-dependent cell-mediated cytotoxicity is also referred to herein as “ADCC.” ADCC describes the cell-killing ability of effector cells, particularly lymphocytes, as described herein, which preferably requires that the target cells be labeled with antibodies.

[0429] ADCC preferably occurs when the antibody binds to the antigen on the tumor cell and the antibody Fc domain engages the Fc receptor (FcR) on the surface of the immune effector cell. Several Fc receptor families have been identified, and specific cell populations characteristically express determined Fc receptors. ADCC can be considered as a mechanism for directly inducing varying degrees of immediate tumor destruction, which results in antigen presentation and induces T cell responses against the tumor. Preferably, the induction of ADCC in vivo will result in T cell responses against the tumor and host-derived antibody responses.

[0430] Complement dependent cytotoxicity is also referred to herein as "CDC". CDC is another method of cell killing that can be directed by antibodies. IgM is the most efficient isotype for complement activation. IgG1 and IgG3 are also very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex results in the activation of multiple C1q binding sites on the C1q of participating antibody molecules (e.g., IgG molecules). HThe C1q domain is exposed near the C1q domain (C1q is one of the three subcomponents of complement C1). Preferably, these unexposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into one of high-affinity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of the effector cell chemoattractants / activators C3a and C5a. Preferably, the complement cascade terminates with the formation of a membrane attack complex, which creates pores in the cell membrane, facilitating the free flow of water and solutes into and out of the cell and can lead to apoptosis.

[0431] In one embodiment, the antibody that binds to PD-1 has reduced or depleted effector function. In one embodiment, the antibody does not mediate ADCC or CDC or both.

[0432] In one embodiment, the heavy chain constant regions of one or more, preferably two, antibodies that bind to PD-1 have been modified such that neonatal Fc receptor (FcRn) binding to the antibody is not affected compared to wild-type antibodies.

[0433] In one embodiment, the PD-1 to which the antibody is capable of binding is human PD-1. In one embodiment, PD-1 has or comprises the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 59, or the amino acid sequence of PD-1 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 59, or is an immunogenic fragment thereof. In one embodiment, the antibody has the ability to bind to a native epitope of PD-1 present on the surface of a living cell.

[0434] In one embodiment, an antibody that binds to PD-1 comprises a heavy chain constant region, wherein the heavy chain constant region comprises an aromatic or non-polar amino acid at the position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering and an amino acid other than glycine at the position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering.

[0435] As used herein, the term "amino acid corresponding to position ..." and similar expressions refer to the amino acid position numbering in the human IgG1 heavy chain. By alignment with human IgG1, corresponding amino acid positions in other immunoglobulins can be found. Thus, an amino acid or fragment in one sequence "corresponds to" an amino acid or fragment in another sequence is an amino acid or fragment that is aligned with the other amino acid or fragment using a standard sequence alignment program (e.g., ALIGN, ClustalW or a similar program, typically at default settings) and has at least 50%, at least 80%, at least 90% or at least 95% identity with a human IgG1 heavy chain. How to align sequences or fragments in a sequence and thereby determine the position in the sequence that corresponds to an amino acid position according to the present disclosure is well known in the art.

[0436] For example, for the amino acid sequence of SEQ ID NO: 38 in the sequence listing according to the present disclosure, the amino acid positions corresponding to positions 234 to 236 in the human IgG1 heavy chain according to EU numbering are amino acid positions 117 to 119 of SEQ ID NO: 38, wherein F is at position 117 (corresponding to position 234 in the human IgG1 heavy chain according to EU numbering), E is at position 118 (corresponding to position 235 in the human IgG1 heavy chain according to EU numbering), and R is at position 119 (corresponding to position 236 in the human IgG1 heavy chain according to EU numbering). In the sequence shown below, the FER amino acid sequence is underlined and shown in bold letters.

[0437]

[0438] Unless otherwise indicated herein or clearly contradicted by context, all references to amino acid positions in the antibody heavy chain constant region throughout this disclosure refer to positions corresponding to the corresponding positions in the human IgG1 heavy chain according to the EU numbering as set forth in Kabat (described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition—US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991)).

[0439] In one embodiment, the antibody that binds to PD-1 comprises a heavy chain constant region that has reduced or depleted Fc-mediated effector function, or that induces Fc-mediated effector function to a lesser extent than another antibody comprising the same antigen binding region and a heavy chain constant region (CH) comprising the hinge, CH2, and CH3 regions of human IgG1.

[0440] In a specific embodiment, the heavy chain constant region (CH) of the antibody that binds to PD-1 is modified such that the antibody induces Fc-mediated effector function to a lesser extent than the same antibody except that it comprises an unmodified heavy chain constant region (CH).

[0441] As used herein, the term "Fc-mediated effector function" refers to such function particularly selected from the group consisting of IgG Fc receptor (FcgammaR, FcγR) binding, C1q binding, ADCC, CDC, and any combination thereof.

[0442] In the context of the present disclosure, the term "having reduced or depleted Fc-mediated effector function" as used in relation to an antibody (including a multispecific antibody) means that the antibody causes an overall reduction in Fc-mediated effector function, particularly selected from the group consisting of IgG Fc receptor (FcgammaR, FcγR) binding, C1q binding, ADCC or CDC, preferably an overall reduction of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more compared to a human IgG1 antibody comprising (i) the same CDR sequences as said antibody, particularly comprising the same first and second antigen-binding regions as said antibody, and (ii) two heavy chains comprising the hinge, CH2 and CH3 regions of human IgG1. "Depleted Fc-mediated effector function" or similar phrases include complete or substantially complete inhibition, i.e., reduction to zero or substantially to zero.

[0443] In the context of the present disclosure, the term "induces Fc-mediated effector functions to a lesser extent" as used in relation to an antibody (including a multispecific antibody) means that the antibody induces Fc-mediated effector functions to a lesser extent compared to a human IgG1 antibody comprising (i) the same CDR sequences as said antibody, in particular comprising the same first and second antigen-binding regions as said antibody, and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions, such functions in particular being selected from the group consisting of IgGFc receptor (FcgammaR, FcγR) binding, C1q binding, ADCC or CDC.

[0444] Fc-mediated effector function can be determined by measuring binding of the binding agent to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. In particular, Fc-mediated effector function can be determined by measuring binding of the binding agent to C1q and / or IgG Fc-γRI.

[0445] In one embodiment involving the use of an antibody that binds to PD-1, the amino acid at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is a basic amino acid.

[0446] The terms "amino acid" and "amino acid residue" are used interchangeably herein and should not be construed as limiting. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, as well as side chains (R groups) specific to each amino acid. In the context of the present disclosure, amino acids can be classified based on structural and chemical properties.

[0447] In the present disclosure, amino acid residues are represented by using the following abbreviations. In addition, unless otherwise expressly stated, the amino acid sequences of peptides and proteins are identified from N-terminus to C-terminus (left to right end), with the N-terminus being identified as the first residue. Amino acids are represented by their 3-letter abbreviations, 1-letter abbreviations, or full names, as shown below. Ala: A: Alanine; Asp: D: Aspartic acid; Glu: E: Glutamic acid; Phe: F: Phenylalanine; Gly: G: Glycine; His: H: Histidine; Ile: I: Isoleucine; Lys: K: Lysine; Leu: L: Leucine; Met: M: Methionine; Asn: N: Asparagine; Pro: P: Proline; Gln: Q: Glutamine; Arg: R: Arginine; Ser: S: Serine; Thr: T: Threonine; Val: V: Valine; Trp: W: Tryptophan; Tyr: Y: Tyrosine; Cys: C: Cysteine.

[0448] Naturally occurring amino acids can also be generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

[0449] In one embodiment of the use of an antibody incorporating PD-1, the basic amino acid at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is selected from the group consisting of lysine, arginine and histidine. In one embodiment, the basic amino acid at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is arginine (G236R). Such amino acid substitutions are also referred to herein as G236R. The term "G236R" means that at position 236 in the human IgG1 heavy chain according to EU numbering, the amino acid glycine (G) is replaced by arginine (R). In the present disclosure, similar terms are used for other amino acid positions and amino acids. Unless otherwise indicated, the amino acid positions mentioned in these terms are amino acid positions in the human IgG1 heavy chain according to EU numbering.

[0450] In one embodiment of the use of an antibody that binds to PD-1, the amino acid at the position corresponding to position 234 in the human IgG1 heavy chain according to EU numbering is an aromatic amino acid. In one embodiment, the aromatic amino acid at this position is selected from the group consisting of phenylalanine, tryptophan, and tyrosine.

[0451] In one embodiment of the use of an antibody that binds to PD-1, the amino acid at the position corresponding to position 234 in the human IgG1 heavy chain according to EU numbering is a non-polar amino acid. In one embodiment, the non-polar amino acid at this position is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. In one embodiment, the non-polar amino acid at this position is selected from the group consisting of isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0452] In one embodiment involving the use of an antibody that binds to PD-1, the amino acid at the position corresponding to position 234 in the human IgG1 heavy chain according to EU numbering is phenylalanine (L234F).

[0453] Exemplary combinations of possible amino acids at positions corresponding to positions 234 and 236 in a human IgG1 heavy chain according to EU numbering are listed in the table below:

[0454] Table 22:

[0455]

[0456]

[0457] For example, at the positions corresponding to positions 234 and 236 in a human IgG1 heavy chain according to EU numbering, in particular the following amino acids may be present in the heavy chain constant region of an antibody that binds to PD-1: 234F / 236R, 234W / 236R, 234Y / 236R, 234A / 236R, 234L / 236R, 234F / 236K, 234W / 236K, 234Y / 236K, 234A / 236K, 234L / 236K, 234F / 236H, 234W / 236H, 234Y / 236H, 234A / 236H or 234L / 236H.

[0458] The aforementioned amino acids or amino acid substitutions at positions 234 and 236 may be present in only one heavy chain of the antibody that binds to PD-1 or in both heavy chains of the antibody that binds to PD-1. The corresponding amino acids present in the first and second heavy chains of the antibody may be selected independently of each other.

[0459] For example, at least one heavy chain of an antibody that binds to PD-1 may comprise the following sequence (SEQ ID NO: 38):

[0460]

[0461] In one embodiment of an antibody that binds to PD-1, the heavy chain, wherein the amino acids at positions corresponding to positions 234 and 236 in a human IgG1 heavy chain according to EU numbering are as specified above, and further, the amino acid at the position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering is an acidic amino acid. In one embodiment, the acidic amino acid at this position is selected from aspartic acid or glutamic acid. In one embodiment, the amino acid at the position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering is glutamic acid (L235E).

[0462] In one embodiment relating to an antibody that binds to PD-1, in the heavy chain constant region, the amino acids at positions corresponding to positions 234, 235, and 236 in a human IgG1 heavy chain according to EU numbering are a nonpolar or aromatic amino acid at position 234, an acidic amino acid at position 235, and a basic amino acid at position 236.

[0463] Exemplary combinations of possible amino acids at positions corresponding to positions 234, 235 and 236 in a human IgG1 heavy chain according to EU numbering are listed in the table below:

[0464] Table 23:

[0465]

[0466]

[0467] For example, at the positions corresponding to positions 234, 235 and 236 in a human IgG1 heavy chain according to EU numbering, in particular the following amino acids may be present in the heavy chain constant region of an antibody that binds to PD-1: 234F / 235E / 236R, 234W / 235E / 236R, 234Y / 235E / 236R, 234A / 235E / 236R, 234L / 235E / 236R, 234F / 235D / 236R, 234W / 235D / 236R, 234Y / 235D / 236R, 234A / 235D / 236R, 234L / 235E / 236R, 234F / 235L / 236R, 234W / 235D / 236R. 5L / 236R, 234Y / 235L / 236R, 234A / 235L / 236R, 234L / 235L / 236R, 234F / 23 5A / 236R, 234W / 235A / 236R, 234Y / 235A / 236R, 234A / 235A / 236R, 234L / 235 A / 236R, 234F / 235E / 236K, 234W / 235E / 236K, 234Y / 235E / 236K, 234A / 235E / 236K、234L / 235E / 236K、234F / 235D / 236K、234W / 235D / 236K、234Y / 235D / 236K, 234A / 235D / 236K, 234L / 235D / 236K, 234F / 235L / 236K, 234W / 235L / 236K, 234Y / 235L / 236K, 234A / 235L / 236K, 234L / 235L / 236K, 234F / 235A / 2 36K, 234W / 235A / 236K, 234Y / 235A / 236K, 234A / 235A / 236K, 234L / 235A / 2 36K, 234F / 235E / 236H, 234W / 235E / 236H, 234Y / 235E / 236H, 234A / 235E / 23 6H, 234L / 235E / 236H, 234F / 235D / 236H, 234W / 235D / 236H, 234Y / 235D / 23 6H, 234A / 235D / 236H, 234L / 235D / 236H, 234F / 235L / 236H, 234W / 235L / 236 H, 234Y / 235L / 236H, 234A / 235L / 236H, 234L / 235L / 236H, 234F / 235A / 236H , 234W / 235A / 236H, 234Y / 235A / 236H, 234A / 235A / 236H or 234L / 235A / 236H.

[0468] The aforementioned amino acids or amino acid substitutions at positions 234, 235 and 236 may be present in only one heavy chain of the antibody or in both heavy chains of the antibody. The corresponding amino acids present in the first and second heavy chains of the antibody may be selected independently of each other.

[0469] For example, at least one heavy chain of an antibody that binds to PD-1 may comprise the following sequence (SEQ ID NO: 128 or 38):

[0470]

[0471]

[0472] Unless the context indicates otherwise, any permutation and combination of all described amino acid substitutions at positions 234, 236 and 235 in this application, for example as shown in Tables 22 and 23, if applicable, should be considered to be disclosed by the description of the present application. For example, in one embodiment of the antibody, the first heavy chain comprises the amino acids FER at positions corresponding to positions 234 to 236 in a human IgG1 heavy chain according to EU numbering, or the first heavy chain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 38, and the second heavy chain of the antibody comprises further amino acids, for example, comprises, consists essentially of, or consists of the amino acid AAG or LLG at positions corresponding to positions 234 to 236 in a human IgG1 heavy chain according to EU numbering, or the second heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 37 or 43. In another embodiment of the antibody, the first and second heavy chains comprise the same amino acids at positions corresponding to positions 234 to 236 in a human IgG1 heavy chain according to EU numbering, i.e., the same aromatic or non-polar amino acid, e.g., F, at the position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering and the same amino acid other than glycine, e.g., R, at the position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering, e.g., a specific combination of FER or FLR.

[0473] In one embodiment, the antibody that binds to PD-1 comprises at least one or two heavy chain constant regions in which the amino acid corresponding to position 234 is phenylalanine, the amino acid corresponding to position 235 is glutamic acid, and the amino acid corresponding to position 236 is arginine (L234F / L235E / G236R=FER).

[0474] In one embodiment, the antibody that binds to PD-1 comprises one or more heavy chain constant regions (CH) comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of the heavy chain constant region sequence shown in SEQ ID NO: 38.

[0475] In one embodiment, the antibody that binds to PD-1 comprises one or more, for example, two, heavy chain constant regions (CH), wherein the heavy chain constant region comprises the sequence shown in SEQ ID NO: 38.

[0476] In one embodiment, the antibody that binds to PD-1 comprises a heavy chain having the sequence shown in SEQ ID NO: 139 and a light chain having the sequence shown in SEQ ID NO: 140.

[0477] The antibody is preferably of the IgG1 isotype.

[0478] As used herein, the term "isotype" refers to the class of immunoglobulins encoded by the heavy chain constant region genes. When referring to the IgG1 isotype herein, the term is not limited to a specific isotype sequence, such as a specific IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, such as IgG1, than to another isotype. Thus, for example, the IgG1 antibodies disclosed herein may be sequence variants of naturally occurring IgG1 antibodies, including variations in the constant region.

[0479] IgG1 antibodies can exist in a variety of polymorphic variants known as allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some embodiments herein. Common allotype variants in the human population are those designated by the letters a, f, n, z, or a combination thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In further embodiments, the human IgG Fc region comprises human IgG1.

[0480] There are two types of light chains in mammals, namely, lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is largely conserved among different isotypes of immunoglobulins, while the variable portion is highly diverse and is responsible for antigen recognition.

[0481] For example or in an embodiment, the antibody, preferably the monoclonal antibody used according to the present invention is an IgG1, κ isotype or a λ isotype, which preferably comprises a human IgG1 / κ or human IgG1 / λ constant part, or the antibody, preferably the monoclonal antibody, is derived from an IgG1, λ (lambda) or IgG1, κ (kappa) antibody, preferably derived from a human IgG1, λ (lambda) or human IgG1, κ (kappa) antibody.

[0482] In one embodiment, the antibody that binds to PD-1 comprises a light chain having a light chain constant region (LC) comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the amino acid sequence of the LC sequence as shown in SEQ ID NO: 42. In one embodiment, the antibody comprises a light chain having a light chain constant region (LC) comprising the sequence as shown in SEQ ID NO: 42.

[0483] In one embodiment of the invention, the antibody that binds to PD-1 is a full-length IgG1 antibody, such as, for example, IgG1, κ. In one embodiment of the invention, the binding agent is a full-length IgG1 antibody, such as, for example, IgG1, κ.

[0484] In one embodiment, an antibody that binds to PD-1 can be derivatized, linked, or co-expressed with other binding specificities. In another embodiment, the antibody can be derivatized with, linked to, or co-expressed with another functional molecule, such as another peptide or protein (e.g., a Fab' fragment). For example, it can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities (e.g., another antibody) (e.g., to produce a bispecific or multispecific antibody).

[0485] The antibody that binds to PD-1 can be a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies that bind to PD-1 may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0486] The present disclosure includes the use of bispecific and multispecific molecules comprising at least one first binding specificity for PD-1 and a second binding specificity (or additional binding specificities) for a second target epitope (or for additional target epitopes).

[0487] In one embodiment, the first antigen-binding region of the multispecific antibody that binds to PD-1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as described herein.

[0488] In one embodiment of the use of a multispecific antibody that binds to PD-1, the antibody comprises a first and a second binding arm derived from a full-length antibody, such as a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody as described above. In one embodiment, the first and second binding arms are derived from a monoclonal antibody. For example, or in a preferred embodiment, the first and / or second binding arms are derived from an IgG1, κ isotype or a λ isotype, preferably comprising a human IgG1 / κ or human IgG1 / λ constant portion.

[0489] The first antigen-binding region of the multispecific or bispecific antibody used according to the present invention that binds to PD-1 may comprise the heavy and light chain variable regions of an antibody that competes with PD-L1 and / or PD-L2 for binding to PD-1. In one embodiment involving the use of a multispecific or bispecific antibody, the first antigen-binding region that binds to PD-1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as described herein.

[0490] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response, rather than the cognitive and activation phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, for example, lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

[0491] "Target cell" refers to any undesirable cell in a subject (eg, a human or animal) that can be targeted by an antibody. In a preferred embodiment, the target cell is a tumor cell.

[0492] In another embodiment, the PD1 / PD-L1 inhibitor is a multispecific antibody, such as a bispecific antibody.

[0493] In a preferred embodiment, the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor comprising a first antigen-binding region that binds CD137 and a second antigen-binding region that binds PD-L1.

[0494] In one embodiment, PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence shown in SEQ ID NO: 98. In one embodiment, CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO: 97.

[0495] In one embodiment, a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 80, 81, and 82, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 84, GAS, and SEQ ID NO: 85, respectively; and b) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 87, 88, and 89, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 91, DDN, and SEQ ID NO: 92, respectively.

[0496] In one embodiment, a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 79, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 83; and b) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 86, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 90.

[0497] In one embodiment, the PD-L1 inhibitor is a multispecific antibody, such as a bispecific antibody.

[0498] In one embodiment, the PD-L1 inhibitor is in the form of a full-length antibody or an antibody fragment.

[0499] In one embodiment, the PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises

[0500] i) a polypeptide comprising said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and

[0501] ii) a polypeptide comprising said first light chain variable region (VL) and a first light chain constant region (CL);

[0502] and the second binding arm comprises

[0503] iii) a polypeptide comprising said second heavy chain variable region (VH) and a second heavy chain constant region (CH), and

[0504] iv) a polypeptide comprising said second light chain variable region (VL) and a second light chain constant region (CL).

[0505] In one embodiment, the PD-L1 inhibitor comprises

[0506] i) a first heavy chain and a first light chain comprising said antigen binding region capable of binding to CD137, the first heavy chain comprising a first heavy chain constant region, and the first light chain comprising a first light chain constant region; and

[0507] ii) a second heavy chain and a second light chain, which comprise the antigen-binding region capable of binding to PD-L1, the second heavy chain comprises a second heavy chain constant region, and the second light chain comprises a second light chain constant region.

[0508] In one embodiment, (i) in the first heavy chain constant region (CH), the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L, and in the second heavy chain constant region (CH), the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, or (ii) in the first heavy chain, the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, and in the second heavy chain, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L.

[0509] In one embodiment, in said first and second heavy chains the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering are F and E, respectively.

[0510] In one embodiment, in said first and second heavy chain constant regions (HC), the positions corresponding to positions L234, L235 and D265 in a human IgGl heavy chain according to EU numbering are F, E and A, respectively.

[0511] In one embodiment, in the PD-L1 inhibitor, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering of both the first and the second heavy chain constant region are F and E, respectively, and (i) the position of the first heavy chain constant region corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L, and the position of the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, or (ii) the position of the first heavy chain constant region corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L.

[0512] In one embodiment, in the PD-L1 inhibitor, the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering of both the first and the second heavy chain constant region are F, E and A, respectively, and wherein (i) the position of the first heavy chain constant region corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L, and the position of the second heavy chain constant region corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, or (ii) the position of the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L.

[0513] In one embodiment, the constant region of the first and / or second heavy chain (e.g., the second heavy chain) in the PD-L1 inhibitor comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0514] a) the sequence represented by SEQ ID NO: 94 or 96 [IgG1-Fc_FEAL];

[0515] 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

[0516] c) sequences 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).

[0517] In one embodiment, in the PD-L1 inhibitor, the constant region of the first and / or second heavy chain (e.g., the first heavy chain) comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

[0518] a) the sequence shown in SEQ ID NO: 93 or 95 [IgG1-Fc_FEAR];

[0519] 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

[0520] c) sequences 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).

[0521] In one embodiment, the PD-L1 inhibitor comprises a kappa (κ) light chain constant region.

[0522] In one embodiment, the PD-L1 inhibitor comprises a lambda (λ) light chain constant region.

[0523] In one embodiment, the first light chain constant region of the PD-L1 inhibitor is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0524] In one embodiment, the second light chain constant region of the PD-L1 inhibitor is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0525] In one embodiment, the first light chain constant region of the PD-L1 inhibitor 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.

[0526] In one embodiment, the kappa (κ) light chain of the PD-L1 inhibitor comprises an amino acid sequence selected from the group consisting of:

[0527] a) the sequence shown in SEQ ID NO: 16,

[0528] 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

[0529] c) sequences having up to 10 substitutions, such as up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in a) or b).

[0530] In one embodiment, the lambda (λ) light chain of the PD-L1 inhibitor comprises an amino acid sequence selected from the group consisting of:

[0531] a) the sequence shown in SEQ ID NO: 17,

[0532] 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

[0533] c) sequences having up to 10 substitutions, such as up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in a) or b).

[0534] In one embodiment, the PD-L1 inhibitor is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0535] In one embodiment, the PD-L1 inhibitor is a full-length IgG1 antibody.

[0536] In one embodiment, the PD-L1 inhibitor is an antibody of the IgG1m(f) allotype.

[0537] In one embodiment, the PD-L1 inhibitor is a bispecific antibody that binds to CD137 and PD-L1, the bispecific antibody having i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 75 and a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 76, and ii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 77 and a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 78.

[0538] In one embodiment, the PD-L1 inhibitor is acalabrutinib or a biosimilar thereof.

[0539] Subject and tumor or cancer to be treated

[0540] The subject treated according to the present disclosure is preferably a human subject.

[0541] In one embodiment, the tumor or cancer is a solid tumor.

[0542] In one embodiment, the tumor is a PD-L1 positive tumor.

[0543] In one embodiment, the tumor or cancer is head and neck squamous cell carcinoma (HNSCC), such as HNSCC of the oral cavity, pharynx, or larynx.

[0544] In one embodiment, the HNSCC is recurrent, unresectable, or metastatic.

[0545] In one embodiment, the tumor or cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

[0546] In one embodiment, the NSCLC is recurrent, unresectable, or metastatic.

[0547] In one embodiment, the NSCLC does not have an epidermal growth factor (EGFR) sensitizing mutation and / or anaplastic lymphoma kinase (ALK) translocation and / or ROS1 rearrangement.

[0548] In one embodiment, the NSCLC is NTRK1 / 2 / 3 (neurotrophic receptor tyrosine kinase 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 previously received treatment with the corresponding targeted therapy.

[0549] In one embodiment, the subject has previously received treatment with a PD-1 inhibitor or a PD-L1 inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody), preferably at least two doses of the PD-1 inhibitor or the PD-L1 inhibitor.

[0550] In one embodiment, the subject has previously received treatment with a platinum-based therapy or an alternative chemotherapy if platinum is not adequate, such as a gemcitabine-containing regimen.

[0551] In one embodiment, the tumor or cancer relapses and / or progresses following treatment (e.g., systemic treatment with a checkpoint inhibitor).

[0552] In one embodiment, the subject has received at least one prior line of systemic therapy, e.g., systemic therapy comprising a PD-1 inhibitor or a PD-L1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0553] In one embodiment, the cancer or tumor has relapsed and / or is refractory, or the subject has progressed following treatment with a PD-1 inhibitor or PD-L1 inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody), administered as monotherapy or as part of a combination therapy.

[0554] In one embodiment, the last prior treatment was with a PD1 inhibitor or a PD-L1 inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

[0555] In one embodiment, the time to progression from the last treatment with a PD-1 inhibitor or PD-L1 inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) is 6 months or less.

[0556] In one embodiment, the time from the last dose of a PD-1 inhibitor or PD-L1 inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) as part of the last prior treatment is 6 months or less.

[0557] In one embodiment, the cancer or tumor has relapsed and / or is refractory, or the subject has progressed during or after i) treatment with an anti-PD-1 antibody or anti-PD-L1 antibody followed by platinum doublet chemotherapy, or ii) platinum doublet chemotherapy followed by treatment with an anti-PD-1 antibody or anti-PD-L1 antibody.

[0558] In a second aspect, the present disclosure provides a kit comprising i) a binding agent comprising at least one binding region that binds CD27 and ii) a PD1 / PD-L1 inhibitor.

[0559] In one embodiment of the kit according to the second aspect, the binding agent is as defined in any aspect or embodiment of the present disclosure.

[0560] In one embodiment of the kit according to the second aspect, the PD1 / PD-L1 inhibitor is as defined in any aspect or embodiment of the present disclosure.

[0561] In one embodiment of the kit according to the second aspect, the binding agent, the PD1 / PD-L1 inhibitor and, if present, the one or more additional therapeutic agents are for systemic administration, in particular for injection or infusion, such as intravenous injection or infusion.

[0562] In a third aspect, the present disclosure provides a kit for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the kit comprising i) a binding agent comprising at least one binding region that binds CD27 and ii) a PD1 / PD-L1 inhibitor.

[0563] In one embodiment of the kit for use according to the third aspect, the kit is as defined in any aspect or embodiment of the disclosure.

[0564] In one embodiment of the kit for use according to the third aspect, the tumor or cancer is as defined in any aspect or embodiment of the disclosure.

[0565] In one embodiment of the kit for use according to the third aspect, the subject is as defined in any aspect or embodiment of the disclosure.

[0566] In one embodiment of the kit for use according to the third aspect, the method is as defined in any aspect or embodiment of the disclosure.

[0567] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising i) a binding agent comprising at least one binding region that binds CD27; ii) a PD1 / PD-L1 inhibitor; and iii) optionally a pharmaceutically acceptable carrier.

[0568] In one embodiment of the pharmaceutical composition according to the fourth aspect, the binding agent is as defined in any aspect or embodiment of the present disclosure.

[0569] In one embodiment of the pharmaceutical composition according to the fourth aspect, the PD1 / PD-L1 inhibitor is as defined in any aspect or embodiment of the present disclosure.

[0570] In a fifth aspect, the present disclosure provides a pharmaceutical composition for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the pharmaceutical composition comprising i) a binding agent comprising at least one binding region that binds CD27 and ii) a PD1 / PD-L1 inhibitor.

[0571] 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.

[0572] 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.

[0573] 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 disclosure.

[0574] 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 disclosure.

[0575] In a sixth aspect, the present disclosure provides a binding agent for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor.

[0576] In one embodiment of the binding agent for use according to the sixth aspect, the method is as defined in any aspect or embodiment of the present disclosure.

[0577] In one embodiment of the binding agent for use according to the sixth aspect, the binding agent is as defined in any aspect or embodiment of the present disclosure.

[0578] In one embodiment of the binding agent for use according to the sixth aspect, the PD1 / PD-L1 inhibitor is as defined in any aspect or embodiment of the present disclosure.

[0579] In a seventh aspect, the present disclosure provides a PD1 / PD-L1 inhibitor for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) the PD1 / PD-L1 inhibitor.

[0580] In one embodiment of the PD1 / PD-L1 inhibitor for use according to the seventh aspect, the method is as defined in any aspect or embodiment of the present disclosure.

[0581] In one embodiment of the PD1 / PD-L1 inhibitor for use according to the seventh aspect, the binding agent is as defined in any aspect or embodiment of the disclosure.

[0582] In one embodiment of the PD1 / PD-L1 inhibitor for use according to the seventh aspect, the PD1 / PD-L1 inhibitor is as defined in any aspect or embodiment of the present disclosure.

[0583] The citation of the documents and studies referred to herein is not intended to admit that any of the foregoing is relevant prior art. All statements regarding the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the contents of these documents.

[0584] This specification (including the following examples) is presented to enable those of ordinary skill in the art to prepare and use various embodiments. The description of specific equipment, technology and application is provided only as an example. Various modifications to the example described herein will be apparent to those of ordinary skill in the art, and the general principles defined herein can be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the example described and shown herein, but meet the scope consistent with the claims.

[0585] This public project

[0586] 1. A method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor.

[0587] 2. The method of claim 1 , wherein the binder comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0588] 3. The method of claim 1 or 2, wherein the binding agent comprises two binding regions capable of binding to human CD27, wherein the antibody comprises heavy chain variable (VH) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, and light chain variable (VL) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

[0589] 4. The method of any of the preceding items, wherein the binding agent comprises a VH region comprising the sequence shown in SEQ ID NO: 4.

[0590] 5. The method of any of the preceding items, wherein the binding agent comprises a VL region comprising the sequence shown in SEQ ID NO: 8.

[0591] 6. The method of any of the preceding items, wherein the binding agent comprises VH and VL regions comprising the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

[0592] 7. The method of any of the preceding items, wherein the binding agent is an antibody, preferably a human antibody or a humanized antibody.

[0593] 8. The method of any one 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).

[0594] 9. The method of claim 8, wherein the light chain constant region is human κ.

[0595] 10. The method of claim 8, wherein the light chain constant region is human λ.

[0596] 11. The method of any of the preceding items, wherein the binding agent further comprises a heavy chain constant region that is of the human IgG isotype, optionally a modified human IgG.

[0597] 12. The method of claim 11, wherein the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4, such as human IgG1.

[0598] 13. The method of claim 11 or 12, wherein the IgG is a modified human IgG comprising one or more amino acid substitutions.

[0599] 14. The method of 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.

[0600] 15. The method of any one of items 11 to 14, wherein the modified human IgG heavy chain constant region comprises at most 10 amino acid substitutions, for example at most 9, for example at most 8, for example at most 7, for example at most 6, for example at most 5, for example at most 4, for example at most 3, for example at most 2 amino acid substitutions.

[0601] 16. The method of any one of items 11 to 15, wherein the substitution in the heavy chain constant region induces increased CD27 agonism compared to the same antibody except that it comprises a wild-type IgG1 antibody heavy chain constant region.

[0602] 17. The method of 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 numbered according to Eu is selected from the group consisting of: A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

[0603] 18. The method of 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.

[0604] 19. The method of 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.

[0605] 20. The method of 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.

[0606] 21. The method of 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.

[0607] 22. The method of any one of items 11 to 21, wherein the binding agent has a pharmacokinetic profile as a parent antibody comprising a wild-type IgG1 heavy chain constant region.

[0608] 23. The method of any of the preceding items, wherein the binding agent comprises a heavy chain constant region comprising a 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.

[0609] 24. The method of any of the preceding items, wherein the binding agent comprises a heavy chain constant region comprising the sequence shown in SEQ ID NO: 15.

[0610] 25. The method of any of the preceding items, wherein the binding agent comprises a heavy chain constant region that is modified such that the binding agent induces one or more Fc-mediated effector functions to a lesser extent relative to a parent antibody.

[0611] 26. The method of 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%.

[0612] 27. The method of claim 25 or 26, wherein the binding agent does not induce one or more Fc-mediated effector functions.

[0613] 28. The method of any one of items 25 to 27, wherein the one or more Fc-mediated effector functions are selected from the group consisting of 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.

[0614] 29. The method of any one of items 25 to 28, wherein the binding agent does not induce C1q binding when measured by the method of Example 8.

[0615] 30. The method of any of the preceding items, wherein the binding agent is a monovalent antibody.

[0616] 31. The method of any of the preceding items, wherein the binding agent is a bivalent antibody.

[0617] 32. The method of any of the preceding items, wherein the binding agent is a monospecific antibody.

[0618] 33. The method of any of the preceding items, wherein the binding agent is a bispecific antibody comprising a first antigen binding region capable of binding to human CD27 according to any of the preceding items and a second antigen binding region capable of binding to a different epitope on human CD27 or capable of binding to a different target.

[0619] 34. The method of any of the preceding items, wherein CD27 is human CD27, in particular said human CD27 comprises the sequence shown in SEQ ID NO: 1 or the human CD27 variant shown in SEQ ID NO: 2.

[0620] 35. The method of any of the preceding items, wherein the binding agent comprises:

[0621] a. a VH region comprising the amino acid sequence shown in SEQ ID NO: 4;

[0622] b. a VL region comprising the amino acid sequence shown in SEQ ID NO: 8;

[0623] c. a CH region comprising the amino acid sequence shown in SEQ ID NO: 15; and

[0624] d. CL region, which comprises the amino acid sequence shown in SEQ ID NO: 17.

[0625] 36. The method of any of the preceding items, wherein the binding agent 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.

[0626] 37. The method according to any of the preceding items, wherein PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence shown in SEQ ID NO: 98.

[0627] 38. The method of any of the preceding items, wherein PD1 is human PD1 or an immunogenic fragment thereof, preferably, the PD1 has or comprises the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 59, or the amino acid sequence of PD1 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 59.

[0628] 39. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1 or PD-L1, preferably an antibody that is an antagonist of the PD1 / PD-L1 interaction and / or a PD1 or PD-L1 blocking antibody.

[0629] 40. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is an antibody of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, such as an antibody of the IgG1 isotype.

[0630] 41. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is a full-length antibody or an antibody fragment, such as a full-length IgG1 antibody.

[0631] 42. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is a monospecific antibody.

[0632] 43. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 99, 100, and 101, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 102, LAS, and SEQ ID NO: 103, respectively.

[0633] 44. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, the antibody comprising a VH region comprising the amino acid sequence shown in SEQ ID NO: 104 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 105.

[0634] 45. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, the antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 106 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 107.

[0635] 46. ​​The method of any one of the preceding items, wherein

[0636] a) The binding agent is an antibody comprising 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.

[0637] b) The PD1 / PD-L1 inhibitor is pembrolizumab or a biosimilar thereof.

[0638] 47. The method of any one of items 1 to 42, wherein

[0639] a) The binding agent is an antibody comprising 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.

[0640] b) The PD1 / PD-L1 inhibitor is nivolumab or its biosimilar.

[0641] 48. The method of any one of items 1 to 42, wherein

[0642] a) The binding agent is an antibody comprising 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.

[0643] b) The PD1 / PD-L1 inhibitor is atezolizumab or a biosimilar thereof.

[0644] 49. The method of any one of items 1-42, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, or an antigen-binding fragment thereof, wherein the antibody that binds to PD1 comprises VH region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NO:49, 46 and 45, respectively, and VL region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NO:52, QAS and SEQ ID NO:50, respectively.

[0645] 50. The method of claim 49, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of the VH sequence shown in SEQ ID NO:56.

[0646] 51. The method of claim 50, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH), wherein the VH comprises the sequence shown in SEQ ID NO: 56.

[0647] 52. The method of any one of items 49-51, wherein the antibody that binds to PD1 comprises a light chain variable region (VL), which comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of the VL sequence shown in SEQ ID NO:57.

[0648] 53. The method of claim 52, wherein the antibody that binds to PD1 comprises a light chain variable region (VL), wherein the VL comprises the sequence shown in SEQ ID NO: 57.

[0649] 54. The method of any one of items 49-53, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or has a sequence as shown in SEQ ID NO:56, and the VL comprises or has a sequence as shown in SEQ ID NO:57.

[0650] 55. The method of any one of items 49-54, wherein the antibody that binds to PD1 comprises a heavy chain constant region, wherein the heavy chain constant region comprises an aromatic or non-polar amino acid at a position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering, and comprises an amino acid other than glycine at a position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering.

[0651] 56. The method of claim 55, wherein the amino acid at the position corresponding to position 236 is a basic amino acid.

[0652] 57. The method of claim 56, wherein the basic amino acid is selected from the group consisting of lysine, arginine and histidine.

[0653] 58. The method of claim 56 or 57, wherein the basic amino acid is arginine (G236R).

[0654] 59. The method of any one of items 55-58, wherein the amino acid at the position corresponding to position 234 is an aromatic amino acid.

[0655] 60. The method of claim 59, wherein the aromatic amino acid is selected from the group consisting of phenylalanine, tryptophan, and tyrosine.

[0656] 61. The method of any one of items 55-58, wherein the amino acid at the position corresponding to position 234 is a non-polar amino acid.

[0657] 62. The method of claim 61, wherein the non-polar amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0658] 63. The method of claim 61 or 62, wherein the non-polar amino acid is selected from the group consisting of isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0659] 64. The method of any one of items 55-63, wherein the amino acid at position corresponding to position 234 is phenylalanine (L234F).

[0660] 65. The method of any one of items 55-64, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acid at the position corresponding to position 235 in the human IgG1 heavy chain according to EU numbering is an acidic amino acid.

[0661] 66. The method of claim 65, wherein the acidic amino acid is aspartic acid or glutamic acid.

[0662] 67. The method of any one of items 55-66, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acid at the position corresponding to position 235 in the human IgG1 heavy chain according to EU numbering is glutamic acid (L235E).

[0663] 68. The method of any one of items 55-67, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acids at positions corresponding to positions 234, 235, and 236 are a non-polar or aromatic amino acid at position 234, an acidic amino acid at position 235, and a basic amino acid at position 236.

[0664] 69. The method of any one of items 55-68, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acid corresponding to position 234 is phenylalanine, the amino acid corresponding to position 235 is glutamic acid, and the amino acid corresponding to position 236 is arginine (L234F / L235E / G236R).

[0665] 70. The method of any one of items 49-69, wherein the heavy chain constant region of the antibody that binds to PD1 comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of the HC sequence shown in SEQ ID NO:38.

[0666] 71. The method of any one of items 49-70, wherein the heavy chain constant region of the antibody that binds to PD1 comprises the sequence shown in SEQ ID NO:38.

[0667] 72. The method of any one of items 49-71, wherein the isotype of the heavy chain constant region of the antibody that binds to PD1 is IgG1.

[0668] 73. The method of any one of items 49-72, wherein the antibody that binds to PD1 comprises a heavy chain having a sequence as shown in SEQ ID NO: 139 and a light chain having a sequence as shown in SEQ ID NO: 140.

[0669] 74. The method of any one of items 49-73, wherein the antibody that binds to PD1 is a monoclonal, chimeric or humanized antibody or a fragment of such an antibody.

[0670] 75. The method of any one of items 49-74, wherein the antibody that binds to PD1 has reduced or depleted Fc-mediated effector function.

[0671] 76. The method of any one of items 49-75, wherein the binding of complement protein C1q to the constant region of the antibody that binds to PD1 is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0672] 77. The method of any one of items 49-76, wherein binding of one or more IgG Fc-γ receptors to the PD1 binding antibody is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0673] 78. The method of claim 77, wherein the one or more IgG Fc-γ receptors are selected from at least one of Fc-γRI, Fc-γRII, and Fc-γRIII.

[0674] 79. The method of claim 77 or 78, wherein the IgG Fc-γ receptor is Fc-γRI.

[0675] 80. The method of any one of items 49-79, wherein the antibody that binds to PD1 is unable to induce Fc-γRI-mediated effector function, or wherein the induced Fc-γRI-mediated effector function is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0676] 81. The method of any one of items 49-80, wherein the antibody that binds to PD1 is unable to induce at least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis, or wherein at least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis is induced to a reduced extent, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0677] 82. The method of any one of items 49-81, wherein binding of the neonatal Fc receptor (FcRn) to the PD1 binding antibody is unaffected compared to the wild-type antibody.

[0678] 83. The method of any one of items 49-82, wherein the antibody that binds to PD1 binds to a native epitope of PD1 present on the surface of living cells.

[0679] 84. The method of any one of items 49-83, wherein the antibody that binds to PD1 is a multispecific antibody comprising a first antigen binding region that binds to PD1 and at least one additional antigen binding region that binds to another antigen.

[0680] 85. The method of claim 84, wherein the antibody that binds to PD1 is a bispecific antibody comprising a first antigen-binding region that binds to PD1 and a second antigen-binding region that binds to another antigen.

[0681] 86. The method of item 84 or 85, wherein the first antigen binding region that binds to PD1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown in any one of items 50 to 54.

[0682] 87. The method of any one of items 49-86, wherein

[0683] a) the binding agent comprises a VH region and a VL region, the VH region comprises the amino acid sequence shown in SEQ ID NO: 4, and the VL region comprises the amino acid sequence shown in SEQ ID NO: 8;

[0684] b) The PD1-binding antibody comprises a VH region and a VL region, wherein the VH comprises or has the sequence shown in SEQ ID NO: 56, and the VL comprises or has the sequence shown in SEQ ID NO: 57.

[0685] 88. The method of any one of items 49-87, wherein

[0686] a) the binding agent is an antibody comprising a VH region, a VL region, a CH region, and a CL region, wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 4, the VL region comprises the amino acid sequence set forth in SEQ ID NO: 8, the CH region comprises the amino acid sequence set forth in SEQ ID NO: 15, and the CL region comprises the amino acid sequence set forth in SEQ ID NO: 17;

[0687] b) The PD1-binding antibody comprises a VH region, a VL region, a CH region, and a CL region, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 56, the VL region comprises the amino acid sequence shown in SEQ ID NO: 57, the CH region comprises the amino acid sequence shown in SEQ ID NO: 38, and the CL region comprises the amino acid sequence shown in SEQ ID NO: 42.

[0688] 89. The method of any one of items 1-41, wherein the PD1 / PD-L1 inhibitor is a multispecific antibody, such as a bispecific antibody.

[0689] 90. The method of claim 89, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor comprising a first antigen binding region that binds to CD137 and a second antigen binding region that binds to PD-L1.

[0690] 91. The method of claim 90, wherein CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO: 97.

[0691] 92. The method of claim 90 or 91, wherein

[0692] a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 79, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 83;

[0693] and

[0694] b) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 86, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 90.

[0695] 93. The method of any one of items 90-92, wherein a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 80, 81, and 82, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 84, GAS, and SEQ ID NO: 85, respectively; and b) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 87, 88, and 89, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 91, DDN, and SEQ ID NO: 92, respectively.

[0696] 94. The method of any one of items 90-93, wherein

[0697] a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence set forth in SEQ ID NO: 79, and the light chain variable region (VL) comprises the amino acid sequence set forth in SEQ ID NO: 83;

[0698] and

[0699] b) The second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 90.

[0700] 95. The method of any one of items 90-94, wherein the PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises

[0701] i) a polypeptide comprising said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and

[0702] ii) a polypeptide comprising said first light chain variable region (VL) and a first light chain constant region (CL);

[0703] And the second binding arm comprises

[0704] iii) a polypeptide comprising said second heavy chain variable region (VH) and a second heavy chain constant region (CH), and

[0705] iv) a polypeptide comprising said second light chain variable region (VL) and a second light chain constant region (CL).

[0706] 96. The method of any one of items 90-95, wherein the PD-L1 inhibitor comprises

[0707] i) a first heavy chain and a first light chain comprising said antigen binding region capable of binding to CD137, said first heavy chain comprising a first heavy chain constant region, and said first light chain comprising a first light chain constant region; and

[0708] ii) a second heavy chain and a second light chain comprising the antigen-binding region capable of binding to PD-L1, the second heavy chain comprising a second heavy chain constant region, and the second light chain comprising a second light chain constant region.

[0709] 97. The method of claim 95 or 96, wherein (i) in the first heavy chain constant region (CH), the amino acid in the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L, and in the second heavy chain constant region (CH), the amino acid in the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, or (ii) in the first heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, and in the second heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L.

[0710] 98. The method of any one of items 95-97, wherein in the first heavy chain and the second heavy chain, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively.

[0711] 99. The method of any one of items 95-98, wherein in the first and second heavy chain constant regions (HC), 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.

[0712] 100. The method of any one of items 95-99, wherein the positions of both the first and second heavy chain constant regions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively, and wherein (i) the position of the first heavy chain constant region corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L, and the position of the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, or (ii) the position of the first heavy chain constant region corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L.

[0713] 101. The method of any one of items 95-100, wherein the positions of both the first and second heavy chain constant regions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering are F, E and A, respectively, and wherein (i) the position of the first heavy chain constant region corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L, and the position of the second heavy chain constant region corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, or (ii) the position of the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L.

[0714] 102. The method of any one of items 95 to 101, wherein the constant region of the first and / or second heavy chain (e.g., the second heavy chain) comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0715] a) the sequence represented by SEQ ID NO: 94 or 96 [IgG1-Fc_FEAL];

[0716] 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

[0717] c) sequences 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).

[0718] 103. The method of any one of items 95 to 102, wherein the constant region of the first and / or second heavy chain (e.g., the first heavy chain) comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0719] a) the sequence shown in SEQ ID NO: 93 or 95 [IgG1-Fc_FEAR];

[0720] 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

[0721] c) sequences 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).

[0722] 104. The method of any one of items 95-103, wherein the PD-L1 inhibitor comprises a kappa (κ) light chain constant region.

[0723] 105. The method of any one of items 95-104, wherein the PD-L1 inhibitor comprises a lambda (λ) light chain constant region.

[0724] 106. The method of any one of items 95-105, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0725] 107. The method of any one of items 95-106, wherein the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0726] 108. The method of any one of items 95-107, 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.

[0727] 109. The method of any one of items 104-108, wherein the kappa (κ) light chain comprises an amino acid sequence selected from the group consisting of:

[0728] a) the sequence shown in SEQ ID NO: 16,

[0729] 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

[0730] c) sequences having up to 10 substitutions, such as up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in a) or b).

[0731] 110. The method of any one of items 105-109, wherein the lambda (λ) light chain comprises an amino acid sequence selected from the group consisting of:

[0732] a) the sequence shown in SEQ ID NO: 17,

[0733] 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

[0734] c) sequences having up to 10 substitutions, such as up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in a) or b).

[0735] 111. The method of any one of items 90-110, wherein the PD-L1 inhibitor is an antibody of the IgG1m(f) allotype.

[0736] 112. The method of any one of items 90-111, wherein the PD-L1 inhibitor is a bispecific antibody that binds to CD137 and PD-L1, the bispecific antibody having i) a first heavy chain comprising the amino acid sequence shown in SEQ ID NO: 75 and a first light chain comprising the amino acid sequence shown in SEQ ID NO: 76, and ii) a second heavy chain comprising the amino acid sequence shown in SEQ ID NO: 77 and a second light chain comprising the amino acid sequence shown in SEQ ID NO: 78.

[0737] 113. The method of any one of items 90-112, wherein the PD-L1 inhibitor is acalabrutinib or a biosimilar thereof.

[0738] 114. The method of any one of items 90-113, wherein

[0739] a) the binding agent comprises heavy chain variable (VH) region CDR1, CDR2 and CDR3 and light chain variable (VL) region CDR1, CDR2 and CDR3, wherein the heavy chain variable (VH) region CDR1, CDR2 and CDR3 comprise the sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, and the light chain variable (VL) region CDR1, CDR2 and CDR3 comprise the sequences shown in SEQ ID NOs: 9, 10 and 11, respectively;

[0740] b) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 80, 81 and 82, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 84, GAS and SEQ ID NO: 85, respectively; and

[0741] c) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 91, DDN and SEQ ID NO: 92, respectively.

[0742] 115. The method of any one of items 90-114, wherein

[0743] a) the binding agent comprises a VH region and a VL region, the VH region comprises the amino acid sequence shown in SEQ ID NO: 4, and the VL region comprises the amino acid sequence shown in SEQ ID NO: 8;

[0744] b) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 79, and the light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 83; and

[0745] c) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 90.

[0746] 116. The method of any one of items 90-115, wherein

[0747] a) the binding agent is an antibody comprising a VH region, a VL region, a CH region, and a CL region, wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 4, the VL region comprises the amino acid sequence set forth in SEQ ID NO: 8, the CH region comprises the amino acid sequence set forth in SEQ ID NO: 15, and the CL region comprises the amino acid sequence set forth in SEQ ID NO: 17;

[0748] b) the PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first binding region, and the second binding arm comprising a second binding region;

[0749] c) the first binding arm of the PD-L1 inhibitor comprises a VH region, a VL region, a CH region, and a CL region, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 79, the VL region comprises the amino acid sequence of SEQ ID NO: 83, the CH region comprises the amino acid sequence of SEQ ID NO: 95, and the CL region comprises the amino acid sequence of SEQ ID NO: 16; and

[0750] d) The second binding arm of the PD-L1 inhibitor comprises a VH region, a VL region, a CH region, and a CL region, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 86, the VL region comprises the amino acid sequence shown in SEQ ID NO: 90, the CH region comprises the amino acid sequence shown in SEQ ID NO: 96, and the CL region comprises the amino acid sequence shown in SEQ ID NO: 17.

[0751] 117. The method of any one of items 90-116, wherein

[0752] a) the binding agent comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 25;

[0753] b) The PD-L1 inhibitor is a bispecific antibody that binds to CD137 and PD-L1, the bispecific antibody having i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a first light chain comprising the amino acid sequence of SEQ ID NO: 76, and ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a second light chain comprising the amino acid sequence of SEQ ID NO: 78.

[0754] 118. The method of any one of items 90-117, wherein

[0755] a) the binding agent comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 25;

[0756] b) The PD-L1 inhibitor is acalabrutinib or a biosimilar thereof.

[0757] 119. The method of any one of items 1-38, wherein the PD1 / PD-L1 inhibitor is a PD1 inhibitor selected from the following: pembrolizumab, nivolumab, cemiprilimab, dotalimab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-224, AMP-514 or their respective biosimilars.

[0758] 120. The method of any one of items 1-38, wherein the PD1 inhibitor is selected from pembrolizumab, nivolumab, cemiplizumab, dotalimumab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tilelizumab, toripalimab, INCMGA00012 (MGA012), AMP-514 or their respective biosimilars.

[0759] 121. The method of any one of items 1-38, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor selected from the following: atezolizumab, avelumab, durvalumab, KN035, CK-301, acalabrutinib, AUNP12, CA-170, BMS-986189 or their respective biosimilars.

[0760] 122. The method of any one of items 1-38, wherein the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, acalabrutinib, or their respective biosimilars.

[0761] 123. The method of any of the preceding items, wherein the subject is a human subject.

[0762] 124. The method of any of the preceding items, wherein the tumor or cancer is a solid tumor.

[0763] 125. The method according to any of the preceding items, wherein the tumor is a PD-L1 positive tumor.

[0764] 126. The method of 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.

[0765] 127. The method of claim 126, wherein the HNSCC is recurrent, unresectable, or metastatic.

[0766] 128. The method of any one of items 1-125, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), e.g., squamous or non-squamous NSCLC.

[0767] 129. The method of claim 128, wherein the NSCLC is recurrent, unresectable, or metastatic.

[0768] 130. The method of claim 128 or 129, wherein the NSCLC does not have an epidermal growth factor (EGFR) sensitizing mutation and / or anaplastic lymphoma (ALK) translocation and / or ROS1 rearrangement.

[0769] 131. The method of any one of items 128-130, wherein the NSCLC is NTRK1 / 2 / 3 (neurotrophic receptor tyrosine kinase 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 previously received treatment with the corresponding targeted therapy.

[0770] 132. The method of any of the preceding items, wherein the subject has previously received treatment with a PD1 inhibitor or a PD-L1 inhibitor (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody), preferably at least two doses of the PD1 inhibitor or the PD-L1 inhibitor.

[0771] 133. The method of any of the preceding items, wherein the subject has previously been treated with a platinum-based therapy or an alternative chemotherapy if platinum is not adequate, such as a gemcitabine-containing regimen.

[0772] 134. The method of any of the preceding items, wherein the tumor or cancer recurs and / or progresses after treatment (e.g., systemic treatment with a checkpoint inhibitor).

[0773] 135. The method of any of the preceding items, wherein the subject has received at least one prior line of systemic therapy, e.g., systemic therapy comprising a PD1 inhibitor or a PD-L1 inhibitor, e.g., an anti-PD1 antibody or an anti-PD-L1 antibody.

[0774] 136. The method of any of the preceding items, wherein the cancer or tumor has relapsed and / or is refractory, or the subject has progressed following treatment with a PD1 inhibitor or PD-L1 inhibitor (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody), administered as monotherapy or as part of a combination therapy.

[0775] 137. The method of any of the preceding items, wherein the last prior treatment was with a PD1 inhibitor or a PD-L1 inhibitor, e.g., an anti-PD1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

[0776] 138. The method of any of the preceding items, wherein the time to progression from the last treatment with a PD1 inhibitor or PD-L1 inhibitor (eg, an anti-PD1 antibody or an anti-PD-L1 antibody) is 6 months or less.

[0777] 139. The method of any of the preceding items, wherein the time from the last administration of a PD1 inhibitor or PD-L1 inhibitor (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody) as part of the last treatment is 6 months or less.

[0778] 140. The method of any of the preceding items, wherein the cancer or tumor has relapsed and / or is refractory, or the subject has progressed during or after:

[0779] i) treatment with anti-PD1 antibody or anti-PD-L1 antibody followed by platinum doublet chemotherapy, or

[0780] ii) Platinum doublet chemotherapy followed by treatment with anti-PD1 antibody or anti-PD-L1 antibody.

[0781] 141. A kit comprising

[0782] i) a binding agent comprising at least one binding region that binds CD27, and

[0783] ii) PD1 / PD-L1 inhibitors.

[0784] 142. The kit of claim 141 , wherein the binding agent is as defined in any one of claims 1 to 140 and / or the PD1 / PD-L1 inhibitor is as defined in any one of claims 1 to 140.

[0785] 143. The kit according to item 141 or 142, wherein the binding agent, the PD1 / PD-L1 inhibitor and (if present) one or more additional therapeutic agents are for systemic administration, in particular for injection or infusion, such as intravenous injection or infusion.

[0786] 144. A kit according to any one of items 141 to 143, for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject.

[0787] 145. A kit for use according to item 144, wherein the tumor or cancer is as defined in any one of items 1-140, and / or the subject is as defined in any one of items 1-140, and / or the method is as defined in any one of items 1-140.

[0788] 146. A pharmaceutical composition comprising

[0789] i) a binding agent comprising at least one binding region that binds CD27;

[0790] ii) PD1 / PD-L1 inhibitors; and

[0791] iii) optionally a pharmaceutically acceptable carrier.

[0792] 147. A pharmaceutical composition according to claim 146, wherein the binding agent is as defined in any one of claims 1 to 140 and / or the PD1 / PD-L1 inhibitor is as defined in any one of claims 1 to 140.

[0793] 148. A pharmaceutical composition according to item 146 or 147, for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject.

[0794] 149. A pharmaceutical composition for use according to claim 148, wherein the tumor or cancer is as defined in any one of claims 1 to 140, and / or the subject is as defined in any one of claims 1 to 140, and / or the method is as defined in any one of claims 1 to 140.

[0795] 150. A binding agent for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) the binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor

[0796] 151. A binding agent for use according to item 150, wherein the method is as defined in any one of items 1-140, and / or the binding agent is as defined in any one of items 1-140, and / or the PD1 / PD-L1 inhibitor is as defined in any one of items 1-140.

[0797] 152. A PD1 / PD-L1 inhibitor for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) the PD1 / PD-L1 inhibitor.

[0798] 153. A PD1 / PD-L1 inhibitor for use according to claim 152, wherein the method is as defined in any one of claims 1 to 140, and / or the binding agent is as defined in any one of claims 1 to 140, and / or the PD1 / PD-L1 inhibitor is as defined in any one of claims 1 to 140.

[0799] Further aspects of the disclosure are disclosed herein.

[0800] Example 1: Generation of DuoBody-PD-L1x4-1BB and anti-human CD27 antibodies and their Fc variants

[0801] Anti-human CD27 antibodies were generated by immunization and hybridoma generation at Aldevron GmbH (Freiburg, Germany). The cDNA encoding human CD27 (full length and ECD) was cloned into an Aldevron proprietary expression plasmid. Anti-CD27 antibodies were generated by immunizing OmniRat animals (transgenic rats expressing a diverse antibody repertoire with fully human idiotypes; Ligand Pharmaceuticals Inc.) with gold particles coated with human CD27 cDNA using a handheld particle bombardment device ("gene gun") intradermally. Serum samples were collected after a series of immunizations and tested for full-length human CD27 expression by flow cytometry on HEK cells transiently transfected with the above-mentioned expression plasmid. 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.

[0802] Based on binding to primary T cells and diversity in in vitro CD27 binding competition assays, six antibodies were selected from a panel of 71 CD27 antibodies for further characterization. These six antibodies are designated herein as IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E, and IgG1-CD27-F.

[0803] The variable regions of the heavy and light chains of interest, which in some cases have single point mutations to remove amino acid residues considered unfavorable for production (e.g., free cysteines or glycosylation sites), are gene synthesized and cloned into expression vectors containing the framework sequences of human antibody light chains and human IgG1 heavy chains.

[0804] Six different antibody Fc variants were generated by introducing one or more of the following amino acid mutations, according to Eu numbering: E345R, E430G, P329R, G237A, K326A, E333A, as shown in Tables 1 and 3 below. After in vitro functional characterization as described below, CD27-specific IgG1-CD27-A (VH SEQ ID NO: 4; VL SEQ ID NO: 8) was considered to have the best biological properties. The sequences of the prior art CD27-targeting antibodies used as benchmarks herein were obtained as follows: IgG1-CD27-15 (WO2012004367; SEQ ID NOs: 3 and 4), IgG1-CD27-131A (WO2018 / 058022; SEQ ID NOs: 10 and 15), IgG1-CD27-CDX1127 (WO2016145085; SEQ ID NOs: 1 and 2), and IgG1-CD27-BMS986215 (WO2019195452A1; SEQ ID NOs: 8 and 9). The VH and VL sequences of type I anti-human CD20 antibodies have been previously described in WO2019 / 145455A1 (SEQ ID NOs: 35 and 39).

[0805] DuoBody-PD-L1x4-1BB is a bispecific antibody based on the DuoBody technology platform (WO2011131746A2) that binds PD-L1 with one arm and 4-1BB with the other arm (WO2021 / 156326A1). DuoBody-PD-L1x4-1BB was generated using parental clones IgG1-CD137-009-H7 (HC SEQ ID NO:75; LC SEQ ID NO:76; HCDR1 SEQ ID NO:80, HCDR2 SEQ ID NO:81, HCDR3 SEQ ID NO:82, LCDR1 SEQ ID NO:84, LCDR2: GAS, LCDR3 SEQ ID NO:85) and IgG1-PD-L1-547 (HC SEQ ID NO:77; LC SEQ ID NO:78; HCDR1 SEQ ID NO:87, HCDR2 SEQ ID NO:88, HCDR3 SEQ ID NO:89, LCDR1 SEQ ID NO:91, LCDR2: DDN, LCDR3 SEQ ID NO:91). As a control antibody, the anti-HIV gp120 antibody IgG1-b12 (Barbas et al., J Mol Biol 1993 230: 812-823; VH: SEQ ID NO: 68, VL: SEQ ID NO: 72 of the present application) was used in the present application.

[0806] Table 1 - Amino Acid Sequence Listing

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834] Example 2: Agonist activity of anti-CD27 antibodies in CD27 activation reporter cell assay

[0835] The CD27 Thaw and Use Bioassay Kit (Promega, Custom Assay Services, CAS# CS1979A25) was used to measure the CD27 agonist activity of various anti-CD27 antibodies with and without the hexamerization-enhancing Fc mutations E345R or E430G. The kit contains NF-κB reporter-Jurkat recombinant cells expressing the firefly luciferase gene under the control of an NF-κB response element and constitutively expressing human CD27 and was used essentially according to the manufacturer's instructions. Briefly, Thaw-and-Use GloResponse NFκB-luc2 / CD27 Jurkat cells were thawed and incubated with a dilution series of antibody (final concentration range 0.04-20 μg / mL) in Bio-Glo luciferase assay buffer in a 96-well flat-bottom culture plate (PerkinElmer, Cat# 6005680) at 37°C, 5% CO2 for 6 hours. The anti-CD27 antibodies were wild-type (WT*) IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E, IgG1-CD27-F, and variants of each carrying either the E430G or E345R mutation. The anti-CD27 benchmark antibodies were IgG1-CD27-131A (WT and E430G variants), non-hexameric IgG1-CD27-15 (IgG1-CD27-15-P329R-E345R-K439E, which carries a combination of Fc mutations that prevent hexamerization; therefore, the mutations are functionally irrelevant in the context of this experiment and are therefore referred to as WT in the figure), and a hexameric variant of IgG1-CD27-15 containing the E345R mutation. An anti-HIV gp120 human antibody, IgG1-b12-E345R, was used as a non-binding negative control antibody (ctrl). After antibody incubation, Bio-Glo luciferase assay reagent (equilibrated to room temperature) was added to each well and incubated for 5-10 minutes at room temperature. Luminescence was measured using an EnVision Multilabel Reader (PerkinElmer) and presented as relative luminescence units (RLU) in a bar graph generated using GraphPad Prism software.

[0836] Compared with antibody clones IgG1-CD27-A to -E and the benchmark antibodies IgG1-CD27-131A (tested with E430G) and IgG1-CD27-15 (tested with E345R; Figure 1 ), the introduction of hexamerization-enhancing Fc mutations (E345R or E430G) resulted in enhanced CD27 agonism compared to the corresponding WT antibodies.

[0837] However, IgG-CD27-A, B, and C exhibited enhanced CD27 agonist activity at all tested concentrations after the introduction of E430G or E345R, while the IgG1-CD27-D and E variants containing hexamerization-enhancing mutations did not exhibit enhanced agonism at the lowest antibody concentration. The IgG1-CD27-F variant with the E430G or E345R mutations exhibited 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 exhibited higher or similar CD27 activation compared to IgG1-CD27-131A with the E430G mutation or CD27-15 with the E345R mutation, respectively.

[0838] *WT antibodies of IgG1-CD27-B and IgG1-CD27-F carry the F405L mutation in the IgG Fc domain, which is functionally irrelevant in the context of this experiment.

[0839] Example 3 Binding affinity of anti-human CD27 antibodies to recombinant human, mouse, and cynomolgus monkey CD27

[0840] The binding affinity of five anti-human CD27 IgG1 antibodies (IgG1-CD27-A, -B, -C, -D and -E) to recombinant human, cynomolgus monkey and mouse CD27 proteins was determined using label-free biolayer interferometry on an Octet HTX instrument (FortéBio, Portsmouth, UK). Experiments were performed using bispecific antibodies comprising one CD27-specific Fab arm and a non-binding Fab arm, such that the antibody is monovalent for CD27. These bispecific antibodies were generated by controlled Fab arm exchange between CD27 antibodies and non-binding antibodies (as described in Labrijn AF et al., Nat Protoc. 2014 Oct; 9(10): 2450-63).

[0841] To determine the affinity of CD27 antibodies to human and mouse CD27, 100 nM recombinant His-tagged mouse or human CD27 protein (Sino Biological, Cat#10039-H08B1 [human], Cat#50110-M08H [mouse]) was loaded onto a pre-set anti-penta-HIS (HIS1K) biosensor (FortéBio, Cat#18-5120) for 600 sec.

[0842] To assess the affinity of CD27 antibodies to cynomolgus CD27, 5 μg / mL recombinant cynomolgus CD27-Fc fusion protein (R&D Systems, Cat#9904-CD-100) was loaded onto activated amine-reactive second generation (AR2G) biosensors (FortéBio, Cat#18-5092).

[0843] After a 300-second baseline measurement in sample diluent (FortéBio, Cat# 18-1104), the association (200 seconds) and dissociation (1,000 seconds) of the CD27 antibody were determined for a concentration series of 0.78-800 nM antibody in two-fold dilution steps in sample diluent. Calculations were performed using an antibody molecular weight of 150 kDa. A reference sensor was incubated with the sample diluent.

[0844] Data were collected using Data Acquisition Software v11.1.1.19 (FortéBio) and analyzed using Data Analysis Software v9.0.0.14 (FortéBio). Data traces for each antibody were corrected by subtracting the reference sensor. The Y axis was aligned to the last 10 sec of the baseline and the alignment was corrected between steps with dissociation and Savitzky-Golay filtering applied. Data traces were excluded from analysis when the response was <0.05 nm and the calculated equilibrium was close to saturation (Req / Rmax>95% using a dissociation time of 50 sec). The data were fit to a 1:1 model using an association window of interest set to 200 sec and a dissociation time set to 50 sec. The correlation coefficient (R) was used to determine the relative abundance of the antibody. 2 ) The dissociation time was chosen based on an estimate of the goodness of the curve fit (preferably >0.98), visual inspection of the curve, and at least 5% signal decay during the association step.

[0845] The affinity of three CD27 antibodies (IgG1-CD27-A, -B, -C) to human CD27 can be accurately determined, among which K D The values ​​were in the nanomolar range (Table 2). For IgG1-CD27-D and -E, biolayer interferometry experiments confirmed that their binding affinities to human CD27 were in a similar range, although suboptimal curve fitting did not allow calculation of the exact K D values ​​(as shown in Table 2).

[0846] IgG1-CD27-A and -B also showed binding to recombinant cynomolgus monkey CD27, with K D The values ​​are in the same range as binding to human CD27. The results obtained with IgG1-CD27-C, -D, and -E also confirmed binding to cynomolgus monkey CD27 with affinities in a similar range, although suboptimal curve fitting did not allow calculation of the exact K Dvalues ​​(as shown in Table 2).

[0847] For antibody IgG1-CD27-C, binding was only observed to recombinant mouse CD27.

[0848] Table 2. Binding affinity of IgG1-CD27-A to -E antibodies to CD27 from the indicated species.

[0849]

[0850] *: Binding was observed, but KD, k on and k dis is a less reliable value, making the interpretation using the 1:1 model unreliable.

[0851] nb: no binding observed.

[0852] Example 4: Binding of anti-CD27 antibodies to human and cynomolgus monkey CD27 expressed on the cell surface

[0853] Binding of anti-CD27 antibodies IgG1-CD27-A to E* and prior art IgG1-CD27-131A* to cell surface-expressed human and cynomolgus monkey CD27 was analyzed by flow cytometry using transiently transfected HEK293F cells and primary T cells endogenously expressing CD27. A non-binding control antibody, IgG1-b12-FEAR, was used as a negative control.

[0854] FreeStyle 293-F suspension cells (HEK293F; ThermoFisher, Cat# R79007) were transiently transfected with mammalian expression vector pSB encoding full-length human or cynomolgus CD27 using 293fectin transfection reagent (ThermoFisher, Cat# 12347019) according to the manufacturer's instructions.

[0855] Human and cynomolgus monkey PBMCs were purified from buffy coats obtained from healthy human donors (Sanquin Blood Bank, The Netherlands) or cynomolgus monkeys (BPRC, The Netherlands, Cat# S-1135) by low-density gradient centrifugation using lymphocyte separation medium (LSM; Corning, Cat# 25-072CV) according to the manufacturer's instructions.

[0856] Cells were seeded in 96-well plates (100,000 cells per well; Greiner Bio-one, Cat#650180) for sequential incubations with wash steps in FACS buffer consisting of PBS (Lonza, Cat#BE17-517Q) + 1% BSA (Roche, Cat#10735086001) + 0.02% sodium azide (Bio-World, Cat#41920044-3). The following incubations were applied: antibody concentration series (0.0001-10 μg / mL final concentration) at 4°C for 30 minutes; live / dead marker FVS510 (BD, Cat#564406, diluted 1:1,000 in PBS) at room temperature for 20 minutes; PE-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, Cat#109-116-098, diluted 1:500) at 4°C for 30 minutes; and anti-CD3 antibodies for T cell identification (anti-human CD3: BD, Cat#555335, diluted 1:10; anti-cyno CD3: Miltenyi, Cat#130-091-998, diluted 1:10) at 4°C for 30 minutes. All samples were analyzed on a FACSCelesta flow cytometer (BD) and FlowJo software. Data were processed and visualized using GraphPad Prism.

[0857] All antibodies tested showed dose-dependent binding to human CD27 on human T cells and transfected HEK293F cells ( Figure 2 A, B). The highest maximum binding was observed for IgG1-CD27-B and IgG1-CD27-C, compared to moderate binding for IgG1-CD27-A and IgG1-CD27-131A, and low binding for IgG1-CD27-D and IgG1-CD27-E, with the differences being most pronounced using human T cells. For binding to cynomolgus macaque CD27 T cells, the highest binding was observed for IgG1-CD27-B, followed by IgG1-CD27-131A and IgG1-CD27-A. Lower binding was observed for IgG1-CD27-D and -E, while IgG1-CD27-C showed minimal binding to cynomolgus macaque T cells. All CD27 antibodies showed dose-dependent binding to HEK cells transfected with cynomolgus macaque CD27. The highest maximum binding was observed for IgG1-CD27-B and IgG1-CD27-131-A, with slightly lower binding observed for IgG1-CD27-A, -D, and -E. IgG1-CD27-C showed minimal binding to HEK cells transfected with cynomolgus CD27 ( Figure 2 C, D).

[0858] In summary, IgG1-CD27-A and IgG1-CD27-B showed dose-dependent binding to human and cynomolgus CD27 endogenously expressed on human or cynomolgus T cells, as well as transiently expressed in transfected HEK cells. IgG1-CD27-A and IgG-CD27-131A showed comparable binding to human T cells, whereas IgG1-CD27-B showed higher maximal binding.

[0859] *NBIgG1-CD27-A, -B, -C, -D, and -E carry the mutations F405L-L234F-L235E-D265A in the IgG Fc domain, which are functionally irrelevant in the context of this experiment. IgG1-CD27-131A carries the functionally irrelevant F405L mutation in the IgG1 Fc domain.

[0860] Example 5: Binding of anti-CD27 antibodies to native human CD27-A59T variant

[0861] Approximately 19% of the human population expresses a naturally occurring CD27 variant carrying the A59T mutation in the extracellular domain (SEQ ID NO. 2). Anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C*, and a benchmark IgG1-CD27-131A were tested for binding to human CD27-A59T by flow cytometry. Non-binding antibody IgG1-b12-FEAL was used as a negative control antibody. HEK293F cells transiently transfected with human CD27-A59T expressing cells (15,000 cells / well) were incubated with a concentration series (0.0001-10 μg / mL, using 10-fold dilution steps) of primary test antibodies IgG1-CD27-A to -C, non-binding control antibody IgG1-b12 (ctrl), and prior art benchmark IgG-CD27-131A (which has been previously described as binding to CD27-A59T (WO2018 / 058022)). After incubation, the antibodies were PE-labeled with polyclonal goat anti-human IgG. Binding was analyzed on a FACSCelesta flow cytometer (BD) and FlowJo software. Data were processed and visualized using GraphPad Prism v.8.

[0862] The tested anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, and IgG1-CD27-131A showed dose-dependent binding to CD27-A59T transfected HEK293F cells, with similar binding curves among the different antibodies ( Figure 3 ).

[0863] *NBIgG1-CD27-A, -B, and -C carry the mutations F405L-L234F-L235E-D265A in the IgG Fc domain, which are functionally irrelevant in the context of this experiment. IgG1-CD27-131A carries the functionally irrelevant F405L mutation in the IgG1 Fc domain.

[0864] Example 6: Anti-CD27 Antibody Induces Human T Cell Proliferation

[0865] Since enhanced IgG hexamerization through Fc-Fc interactions upon introduction of the E345R or E430G mutations enhances the CD27 agonist activity of anti-CD27 antibodies (Example 2), the ability of IgG1-CD27-A, IgG1-CD27-B, and IgG1-CD27-C antibody variants carrying the E430G or E345R mutations to increase the proliferation of TCR-activated T cells was tested in vitro.

[0866] In addition, Fc mutations that reportedly reduce binding to C1q and FcγR (G237A or P329R) or enhance binding to C1q (K326A / E333A double mutation) were introduced to test their potential impact on the CD27 agonist activity of CD27 antibodies carrying E345R or E430G mutations. The K326A / E333A double mutation was previously shown to enhance C1q binding and contribute to the enhanced agonistic activity of a DR5-specific human IgG1 antibody that includes mutations that enhance Fc-Fc interactions (WO2018 / 146317A1). In addition to E430G or E345R, 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 PBMCs obtained from healthy donors (Sanquin Blood Bank, The Netherlands).

[0867] Table 3. Mutations in the Fc domain of antibodies IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C and their biological effects

[0868]

[0869] *X in IgG1-CD27-X refers to the IgG1-CD27 clones IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C.

[0870] PBMCs were cultured at 5 x 10 6Cells were resuspended in PBS at a density of 10 cells / mL and labeled with CFSE using the CellTrace CFSE Cell Proliferation Kit (Invitrogen, Cat#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, Cat#60011) in T cell activation medium (ATCC, Cat#80528190) supplemented with 5% normal human serum (NHS; Sanquin, Cat#B0625) in 96-well round-bottom plates (Greiner Bio-one, Cat#650180) to activate T cells and incubated with CD27 antibody (final concentration 1 μg / mL) at 37°C / 5% CO2 for 96 h. For identification of CD4 + and CD8 + For living cells in T cell subsets, the cells were continuously incubated with the live / dead marker FVS510 (1:1,000) at room temperature for 20 minutes and incubated with a lymphocyte marker staining mixture in the dark at 4°C for 30 minutes. The staining mixture contained APC-eFluor780-labeled anti-human CD4 antibody (Invitrogen, Cat#47-0048-42, 1:50), AlexaFluor700-labeled anti-human CD8a antibody (BioLegend, Cat#301028; 1:100), PE-Cy7-labeled mouse anti-human CD14 antibody (BD Biosciences, Cat#557742; 1:50) and BV785-labeled anti-human CD19 antibody (BioLegend, Cat#363028; 1:50). Samples were measured on a FACSCelesta (BD Biosciences) flow cytometer and analyzed for viable CD4 + and CD8 + CFSE dilution peak in T cell subsets (FVS510 - CD14 - CD19 - CD4 + and FVS510 - CD14 - CD19 - CD8 +) as a readout of T cell proliferation. T cell proliferation was expressed as the percentage of proliferating cells or the division index, both of which were calculated using FlowJo software (version 10). The percentage of proliferating (dividing) cells was determined by gating on cells that had undergone CFSE dilution (CFSE 低峰 The division index is the average number of divisions a cell undergoes. Heatmaps were generated using GraphPad Prism version 8. Proliferation assays were performed using PBMCs from four different healthy donors.

[0871] IgG1-CD27-A, -B, and -C variants carrying the E430G or E345R mutations induced a small increase in CD8+ T cell proliferation in two of the four donors tested compared to the control antibody. Introducing additional mutations (P329R, G237A, or K326A / E333A) into the IgG1-CD27-A, -B, or -C variants carrying the E430G mutation showed a small increase in CD8+ T cell proliferation in four PBMC donors. + Different effects on T cell proliferation. In contrast, introduction of the P329R mutation into IgG1-CD27-A and IgG1-CD27-C variants carrying the E345R mutation consistently increased their enhanced activation of CD8 + This applies in particular to IgG1-CD27-A: whereas for IgG-CD27-A-E345R, IgG1-CD27-B-E345R and IgG1-CD27-C-E345R, the measured CD8 + T cell proliferation was comparable in each donor, and introduction of the additional P329R mutation consistently resulted in higher CD8 T cell proliferation in clones IgG1-CD27-A-E345R compared to IgG1-CD27-B-E345R or IgG1-CD27-C-E345R. + T cell proliferation. Therefore, upon TCR activation, CD8 + Regarding T cell proliferation, the effect of the combination of the E345R mutation and the P329R mutation was consistently greater in clones IgG1-CD27-A than in IgG1-CD27-B and IgG1-CD27-C. Among all antibody variants tested, IgG1-CD27-A-E345R-P329R induced CD8 T cell proliferation in all donors. + The maximum increase in T cell proliferation ( Figure 4A ).

[0872] Compared to antibodies containing the single mutation E345R, addition of mutations G237A or K326A-E333A to CD27 antibody variants carrying the E345R mutation did not or only minimally increase CD8 + T cell proliferation ( Figure 4A ).

[0873] Also in CD4 + Among 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 Although IgG1-CD27-A, -B, and -C variants carrying only E430G or E345R mutations were + T cell proliferation was generally comparable, but introduction of the additional P329R mutation resulted in a decrease in CD4 T cell proliferation in the IgG1-CD27-A variant carrying the E345R variant. + There was a greater increase in T cell proliferation than the IgG1-CD27-A-E430G or IgG1-CD27-B or -C variants carrying the E430G or E345R mutations. This effect was observed in three of the four donors tested. In donor 1, mutations other than E430G or E345R had no effect on CD4 T cell proliferation. + The effects on T-cell proliferation were generally minimal, and the effects observed in this donor were not reproduced in the other three donors.

[0874] For IgG1-CD27-C, the combination of E345R and P329R mutations also consistently increased CD4 + T cell proliferation was observed, 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 decrease in CD4 T cell proliferation compared to IgG1-CD27-B-E345R was observed in two of four donors. + T cell proliferation was moderately increased.

[0875] Introduction of P329R, G327A, or K326A / E333A mutations into IgG1-CD27-A, -B, or -C variants carrying the E430G mutation did not induce or did not consistently induce CD4 + Similarly, no effect or sustained effect was observed after the introduction of G327A or K326A / E333A into IgG1-CD27-A, -B, or -C variants carrying the E345R mutation.

[0876] In conclusion, IgG1-CD27-A-E345R-P329R continuously induced activated CD8 + T cells and CD4 +The highest increase in T cell proliferation indicates that IgG1-CD27-A-E345R-P329R induces the most effective CD27 agonism. Previous studies have shown that DR5-specific, hexamer-enhanced antibodies with P329R mutations show reduced ability in inducing DR5 agonism compared to DR5-specific hexamer-enhanced antibodies without P329R mutations (Overdijk et al, Mol Canc Ther 2020). Therefore, it is considered surprising that in IgG1-CD27-A, in addition to the E345R mutation, the introduction of the P329R mutation enhances CD27 agonist activity. In addition, it is unknown why the combined effect of E345R+P329R mutations for IgG1-CD27-A is always greater than the combined effect for IgG1-CD27-B or IgG1-CD27-C.

[0877] Example 7: Induction of human T cell proliferation by anti-CD27 antibody IgG1-CD27-A-P329R-E345R

[0878] The increase of TCR-stimulated human CD4 by IgG1-CD27-A-P329R-E345R was analyzed in a CSFE dilution assay using PBMC from healthy human donors. + and CD8 + The ability of the T cells to proliferate was compared with prior art anti-CD27 clones IgG1-CD27-131A*, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215*. 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 without anti-CD3 stimulated T cells were included to test the potential CD27 agonist activity of the antibodies in the absence of T cell receptor activation ( Figure 5 A and 5B). This activity is undesirable because it would pose a safety risk if the antibody were able to induce proliferation of resting T cells.

[0879] The percentage of proliferating T cells ( Figure 5 A, B, C, D) was calculated as the percentage of cells with reduced CFSE fluorescence, which indicates cell division. Figure 5 E and 5F ) The fold increase of cells in the wells was identified and calculated using the proliferation modeling tool in FlowJo version 10. Peaks were manually adjusted when necessary to more consistently determine the number of peaks present.

[0880] Neither the CD27 antibodies of the invention nor the prior art antibodies tested herein induced proliferation of unstimulated T cells (i.e., in the absence of CD3 cross-linking) ( Figure 5 A and B).

[0881] Most CD27 antibodies induced some activation of CD4 at the highest antibody concentration tested. + and CD8 + T cell proliferation ( Figure 5 C and D). Based on this, the amplification index ( Figure 5 E and F). Compared with the prior art anti-CD27 clones IgG1-CD27-131A, IgG1-CD27-CDX1127 and IgG1-CD27-BMS986215, the antibody IgG1-CD27-A-P329R-E345R of the present invention enhanced CD4 + and CD8 + Proliferation of T cells.

[0882] *For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants carrying the F405L mutation were used, which is functionally irrelevant in the context of this experiment.

[0883] Example 8: Binding of C1q to membrane-bound CD27 antibodies

[0884] The P329R mutation has been previously 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 vitro in a cellular C1q binding assay using human healthy donor T cells. Anti-HIV gp120 human antibody, IgG1-b12-F405L, was used as a non-binding isotype negative control antibody (ctrl). T cells were enriched from human healthy donor PBMCs using RosetteSep human T cell enrichment cocktail (Stemcell, Cat#15061) and resuspended in culture medium (RPMI 1640 [Gibco, Cat#A10491-01] supplemented with 0.1% BSA and 1% Pen / Strep [Lonza, Cat#DE17-603E]). T cells (2×10 6Cells (100 cells / well) were preincubated with an antibody dilution series (8 x 5-fold dilutions starting from a final assay concentration of 15 μg / mL) in a polystyrene 96-well round-bottom plate for 15 minutes at 37°C to allow antibody binding to T cells. The cells were then cooled on ice, supplemented with NHS as a source of human C1q (20% NHS final assay concentration), and incubated on ice for 45 minutes. The cells were then incubated with FITC-labeled rabbit anti-human C1q antibody (DAKO, Cat#F0254; 20 μg / mL) on ice for 30 minutes and resuspended in FACS buffer containing TO-PRO-3 (ThermoFisher, Cat#T3605; 1:5,000 dilution). C1q binding was determined by measuring the FITC signal on live cells by flow cytometry.

[0885] Membrane-bound WT IgG1-CD27-A antibody showed no C1q binding ( Figure 6 Introduction of the hexamerization-enhancing mutations E430G or E345R (IgG1-CD27-A-E430G and IgG1-CD27-A-E345R) resulted in binding of C1q to the CD27 antibody on the T cell surface, consistent with the increased binding affinity of the hexameric C1q protein to the hexameric antibody ring structure on the cell surface ( Figure 6 Introduction of the P329R mutation into IgG1-CD27-A-E345R (IgG1-CD27-A-P329R-E345R) resulted in loss of C1q binding ( Figure 6 ), which indicates that IgG1-CD27-A-P329R-E345R cannot bind to C1q.

[0886] These data show that IgG1-CD27-A-P329R-E345R is unable to bind C1q when bound to CD27 on the cell surface of T cells. This suggests that C1q binding does not contribute to the antibody-induced CD27 agonist activity of IgG1-CD27-A-P329R-E345R. This is in contrast to other previously described hexamerization-enhanced agonistic antibodies. Furthermore, the lack of C1q binding suggests that IgG1-CD27-A-P329R-E345R is unable to activate the classical pathway of complement activation. Therefore, IgG1-CD27-A-P329R-E345R is not expected to induce complement activation and CDC on T cells, an activity that is not expected.

[0887] Example 9: Binding of anti-CD27 antibodies to human Fc receptors

[0888] The binding of IgG1-CD27-A-P329R-E345R to human FcγR variants was analyzed using a Biacore surface plasmon resonance (SPR) system and compared with the anti-HIV gp120 antibody IgG1-b12 (ctrl). Biacore series S sensor chips CM5 (Cytiva, Cat# 29104988) were covalently coated with anti-His antibodies using an amine coupling and His capture kit (Cytiva, Cat# BR100050 and Cat# 29234602) according to the manufacturer's instructions. Then, 125nM Fcγ receptors FcγRIa, FcγRIIa (167-His[H] and 167-Arg[R]), FcγRIIb or FcγRIIIa (176-Phe[F] and 176-Val[V]) (Sino Biological, Cat#10256-H08S-B, Cat#10374-H27H, Cat#10374-H27H1-B, Cat#10259-H27H1-B, Cat#10389-H27H-B and Cat#10389-H27H1-B) in HBS-P+ (Cytiva, Cat#BR100827) were captured onto the surface. After three rounds of buffer rinses, the antibody samples were injected for 36 cycles using antibodies to other FcγRs in the range of 0-3,000nM and 0-10,000nM to generate binding curves. Each sample analyzed on the surface (active surface) coated with FcR was also analyzed on a parallel flow chamber without FcR (reference surface) for background correction. Dissociation from the surface coated with anti-His was performed by regenerating the surface using 10mM glycine-HCl pH1.5 (Cytiva, Cat#BR100354). Sensorgrams were generated using Biacore Insight evaluation software (Cytiva), and four-parameter logic (4PL) fitting was applied to calculate the relative binding of IgG1-CD27-A-P329R-E345R to the reference sample (ctrl).

[0889] Although some binding was observed at higher antibody concentrations, binding of IgG1-CD27-A-P329R-E345R to the high-affinity receptor FcγRIa was significantly reduced compared to the ctrl antibody ( Figure 7 A). IgG1-CD27-A-P329R-E345R does not bind to the human low affinity receptor FcγRIIa ( Figure 7 B and C), FcγRIIb ( Figure 7 D) and FcγRIIIa ( Figure 7 E and F) combined.

[0890] In summary, IgG1-CD27A-P329R-E345R showed minimal binding (FcγRIa) or no binding (FcγRIIa, FcγRIIb, and FcγRIIIa) to human IgG Fc receptors.

[0891] Example 10: Binding of anti-CD27 antibody IgG1-CD27-A-E345R-P329R to human T cells

[0892] Flow cytometry was used to characterize the binding of IgG1-CD27-A-P329R-E345R to CD27 on human healthy donor T cells in more detail. The anti-HIV gp120 antibody variant, IgG1-b12-P329R-E345R, was used as a non-binding control antibody (ctrl). Human PBMCs were isolated from buffy coats obtained from human healthy donors. PBMCs (1 x 10 5Cells were added to a polystyrene 96-well round-bottom plate (Greiner bio-one, Cat#650101) and precipitated by centrifugation at 300 x g for 3 minutes at 4 ° C. The cells were resuspended in 50 μL / well serial antibody dilutions (in 3-fold dilution steps, ranging from 0.0015 to 10 μg / mL) in FACS buffer and incubated at 4 ° C for 30 minutes. The cells were pelleted, washed twice with FACS buffer, and incubated with 50 μL / well FITC-conjugated secondary antibody (FITC AffiniPure F (ab') 2 fragment goat anti-human IgG, F (ab') 2 fragment specific, Jackson ImmunoResearch, Cat#109-096-097, 1:100 dilution) in the dark at 4 ° C for 30 minutes. The cells were pelleted again, washed twice with FACS buffer, and incubated in 50 μL / well of a lymphocyte marker staining mixture containing BV711-labeled anti-human CD19 antibody (BioLegend, Cat#302246, 1:50), AlexaFluor700-labeled anti-human CD8a antibody (BioLegend, Cat#301028, 1:100), APC-eFluor780-labeled anti-human CD4 antibody (Invitrogen, Cat#47-0048-42, 1:50), PE-CF594-labeled mouse anti-human CD56 antibody (BD Biosciences, Cat#564849, 1:100), PE-Cy7-labeled mouse anti-human CD14 antibody (BD Biosciences, Cat#564849, 1:100), and 1:50 μL / well of a 1:50 μL / well lymphocyte marker staining mixture in the dark at 4°C. Biosciences, Cat#557742, 1:50) and eFluor450 labeled anti-human CD3 antibody (Invitrogen, Cat#48-0037-42, 1:50). The cells were precipitated again, washed twice with FACS buffer, and resuspended in 80 μL of FACS buffer containing the dead cell marker 7-amino-actinomycin D (7-AAD; BD Biosciences, Cat#51-68981E, 1:240 dilution). Samples were measured by flow cytometry on an LSRFortessa (BD) flow cytometer and analyzed using FlowJo software. Binding curves were analyzed using GraphPad Prism 8 software using nonlinear regression (S-shaped dose response with variable slope).

[0893] Anti-CD27 antibody IgG1-CD27-A-P329R-E345R showed dose-dependent binding to healthy donor T cells and CD4 + and CD8 + T cells have similar binding characteristics ( Figure 8 ).

[0894] Example 11: FcγR-independent induction of CD27 cell signaling by anti-CD27 antibody IgG1-CD27-A-P329R-E345R

[0895] CD27-specific monoclonal antibodies that can induce CD27 signaling independent of secondary FcγR-mediated cross-linking may be immunostimulatory in the absence of FcγR-positive cells, which is advantageous in tumors where the frequency of FcγR-bearing cells is low.

[0896] The CD27 agonist activity of IgG1-CD27-A-P329R-E345R was tested in the presence or absence of FcγR-bearing cells and compared to the corresponding WT antibody IgG1-CD27-A and prior art antibodies IgG1-CD27-131A*, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215*. The non-binding antibody IgG1-b12-P329R-E345R was used as a negative control (ctrl). The CD27 reporter gene assay was performed essentially as described in Example 2, except that in the current example, Thaw-and-Use GloResponse NFκB-Luc2 / CD27 Jurkat cells were cultured in the presence of cells expressing human FcγRIIb to promote FcγR-mediated cross-linking of membrane-bound antibodies.

[0897] Thaw-and-Use effector FcγRIIb CHO-K1 cells (Promega, Cat#JA2251) were plated in 96-well flat-bottom culture plates (PerkinElmer, Cat#0815) either undiluted or at three increasing dilutions (1 / 3, 1 / 9, 1 / 27) and incubated overnight at 37°C / 5% CO. The supernatant of adherent FcγRIIb-expressing cells was replaced with a fixed cell concentration of Thaw-and-Use NFκB-Luc2 / CD27 Jurkat cell suspension in Bio-Glo Luciferase Assay Buffer (starting with a 1:1 NFκB-Luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratio for undiluted FcγRIIb CHO-K1 cells) containing serial dilutions of antibody (final concentration range 0.0002-10 μg / mL). After incubation at 37° C. / 5% CO 2 for 6 h, the plates were equilibrated to room temperature as described in Example 2, and bioluminescence was measured and expressed as RLU.

[0898] IgG1-CD27-A-P329R-E345R induced a dose-dependent CD27 activation that was independent of FcγRIIb-expressing cells ( Figure 9 In contrast, the corresponding WT antibody IgG1-CD27-A, which does not contain the E345R hexamerization-enhancing mutation and the P329R mutation, only exhibited CD27 agonism in the presence of FcγRIIb-expressing cells ( Figure 9 Similarly, CD27 activation by prior art antibodies IgG1-CD27-131A, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 also depended on the presence of FcγRIIb-expressing cells and gradually decreased with decreasing NFκB-Luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratios ( Figure 9 FJ).

[0899] Together, these data demonstrate that IgG1-CD27-A-P329R-E345R can induce CD27 agonism independent of secondary FcγR-mediated cross-linking, in contrast to prior art anti-CD27 antibodies that rely on the presence of FcγR-bearing cells to induce CD27 agonism.

[0900] *For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants carrying the F405L mutation were used, which is functionally irrelevant in the context of this experiment.

[0901] Example 12: Pharmacokinetic (PK) Analysis of Anti-CD27 Antibody IgG1-CD27-A-P329R-E345R in the Absence of Target Binding Studied in Mice

[0902] In the absence of target binding, the pharmacokinetic characteristics of the anti-CD27 antibody IgG1-CD27-A-P329R-E345R* were analyzed in mice and compared with the corresponding WT antibody IgG1-CD27-A*. IgG1-CD27-A does not bind to mouse CD27 (Example 3, Table 2), so the experiment was designed to test the pharmacokinetic behavior of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R in vivo in the absence of target binding. This study was performed by qualified personnel at Crown Bioscience (China) according to approved IACUC protocols and Crown Bioscience, Inc. standard operating procedures. 11-12 week old female SCID mice (CB-17, Vital River Laboratory Animal Technology Co., Ltd. (VR, Beijing, China; 3 mice per group) were injected intravenously with 500 μg of antibody (25 mg / kg) in an injection volume of 200 μL. 40 μL blood samples were collected at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration, and plasma was collected from the blood samples and stored at -80 ° C until the total human IgG concentration was determined by ELISA. 96-well ELISA plates (Greiner, Cat#655092) were coated overnight at 4 ° C with 2 μg / mL anti-human IgG (Sanquin, the Netherlands, product number M9105, batch number 8000260395) and subsequently washed with PBSA (supplemented with 0.2% bovine serum albumin [BSA, Roche, Cat#10 735086001] in PBS) 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, Cat# P1379]) for 1 h at room temperature, with polyclonal peroxidase-conjugated goat anti-human IgG secondary antibody (Jackson, Cat# 109-035-098) for 1 h at room temperature, and finally with 2,2′-amino-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS; Roche, Cat# 11112422001) for 1 h at room temperature on a plate shaker. The plates were incubated for 1 h at room temperature by adding 2% oxalic acid (Riedel The reaction was terminated with 4% paraformaldehyde (5% paraformaldehyde, ...

[0903] There was no substantial difference in the PK profiles of IgG1-CD27-A-P329R-E345R and the corresponding WT antibody IgG1-CD27-A ( Figure 10 ), as determined by measuring plasma IgG levels at different time points after intravenous injection in mice.

[0904] Although 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 predictions for human IgG1 in mice, the terminal elimination of both antibodies was consistent with the predicted rates for human wild-type IgG1 based on a 2-compartment model (Bleeker WK, Teeling JL, Hack CE. Blood. 2001 Nov 15;98(10):3136-42).

[0905] Together, this demonstrates that the introduction of the P329R and E345R mutations does not affect the pharmacokinetic properties of IgG1-CD27-A in the absence of target binding.

[0906] NB The experiments described in this example used variants of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R carrying the F405L mutation, which are functionally irrelevant in the context of this experiment.

[0907] Example 13: Induction of Antibody-Dependent Cellular Phagocytosis by Antibody-Dependent Cellular Phagocytosis with Antibody-Dependent Cellular Phagocytosis by Antibody-Dependent Cellular Phagocytosis

[0908] Antibody-dependent cellular cytotoxicity (ADCC) is mainly mediated by FcγRIIIa expressed on NK cells, while antibody-dependent cellular phagocytosis (ADCP) can be mediated by monocytes, macrophages, neutrophils and dendritic cells via FcγRI, FcγRIIa and FcγRIII (Hayes, JM et al 2016). In order to understand the effect of the residual binding of anti-CD27 antibody IgG1-CD27-A-P329R-E345R to FcγRIa (Example 9) on the effector function of immune cells expressing FcγRIa, CTV-labeled CD27 was used to detect the effect of FcγRIa on the effector function of immune cells expressing FcγRIa. + The ability of IgG1-CD27-A-P329R-E345R to induce ADCP was analyzed in vitro using Burkitt lymphoma Daudi cells as target cells and human monocyte-derived macrophages (hMDM) as effector cells (E:T=2:1).

[0909] hMDM were isolated from PBMC by positive selection using CD14 microbeads (Miltenyi Biotec, cat. no. 130-050-201) according to the manufacturer's instructions. PBMC were centrifuged (1,200 RPM, 5 min, RT) and washed with 1.25 × 10 7 The density of PBMC / mL was resuspended in ice-cold monocyte separation buffer (PBS, 0.5% BSA, 2mM EDTA). 20 μL CD14 microbeads were added to every 80 μL PBMC suspension and incubated on a roller mixer at 4°C for 15 minutes. 30 mL ice-cold monocyte separation buffer was added, and the PBMC / CD14 microbead mixture was centrifuged (300 × g, 10 minutes, 4°C) and resuspended in 6 mL ice-cold monocyte separation buffer. LS columns (Miltenyi Biotec, cat.no.130-042-401) were rinsed with 3 mL ice-cold monocyte separation buffer, and each column was loaded with 3 mL PBMC / CD14 microbead mixture. - After cells flowed through and the column was washed three times with ice-cold monocyte isolation buffer, CD14 was recovered in 3 mL of ice-cold monocyte isolation buffer using a plunger. + Monocytes. Using ViaStain TM The viability dye acridine orange / propidium iodide (AOPI; Nexcelom Bioscience, cat. no. CS2-0106) was used to measure CD14 cells on a Cellometer Auto 2000 cell viability counter (Nexcelom Bioscience). + The cells were counted and the 6 The cells were suspended at a density of 10 cells / mL. GMP DC culture medium (CellGenix, cat. no. 20801-0500) was supplemented with macrophage colony-stimulating factor (M-CSF; Gibco, cat. no. PH9501; 50 ng / mL final concentration) and 3 mL of monocyte suspension (i.e., 2.4 × 10 6 monocytes) in 100 mm 2 With UpCell TM Surface Nunc TMThe cells were harvested in a culture dish that allowed the plates to be placed at room temperature (Thermo Fisher Scientific, cat. no. 174902). After three days of incubation, 2 mL of fresh culture medium containing 5× M-CSF was added to the plates. After 7 days of incubation (37°C, 5% CO2), the macrophages were detached from the surface by placing the plates at room temperature for 1 to 1.5 hours. The detached macrophages were pelleted by centrifugation, counted using AOPI, and counted at 1×10 6 The cells were resuspended in culture medium (RPMI 1640 containing 10% DBSI) at a density of 10 cells / mL.

[0910] CellTrace was used according to the manufacturer's instructions. TM Violet Cell Proliferation Kit (Thermo Fisher Scientific, cat. no. C34557) was used to culture human Burkitt lymphoma Daudi cells ( CCL-213 TM Briefly, cell tracking violet (CTV) was added to a medium containing 1×10 6 Daudi cells / mL in PBS and incubated at 37°C in the dark for 20 minutes (15 mL incubation volume). 10 mL of DBSI was added to inactivate unbound dye. Cells were pelleted by centrifugation (300 x g, 5 minutes), washed in PBS, and counted using AOPI. CTV-labeled Daudi cells were plated at 0.5 × 10 6 Resuspend in culture medium at a density of 10 cells / mL.

[0911] For ADCP assay, hMDM (50,000 cells / well) and CTV-labeled Daudi cells (25,000 cells / well) were seeded together in 96-well plates (E:T = 2:1) on ice in a final volume of 150 μl of culture medium and incubated with anti-CD27 antibody IgG1-CD27-A-P329R-E345R or anti-CD20 antibody IgG1-CD20 (0.000001 to 10 μg / mL concentration range, 10-fold dilution) for 4 h (37°C, 5% CO2). After incubation, 100 μL of human BD Fc Block was added. TM(BD Biosciences, cat. no. 564220; 1: 100 in FACS buffer) and incubated at 4°C for 10 minutes. The cells were pelleted by centrifugation (300 × g, 5 minutes), resuspended in FACS buffer containing PE-Cy7-conjugated anti-human CD11b antibody (BioLegend, cat. no. 301322; 1: 80) and TO-PRO-3 (Thermo Fisher Scientific, cat. no. T3605; 1: 25,000), and incubated at 4°C for 30 minutes. The cells were washed, resuspended in FACS buffer, collected, and analyzed on a FACSymphony. TM The cells were analyzed on an A3 cell analyzer (BD Biosciences). Data were analyzed using FlowJo software to measure the number of viable target cells and phagocytic hMDMs, and processed and visualized using GraphPad Prism software.

[0912] The percentage of viable Daudi cells in each condition was calculated according to the following formula:

[0913] Live Daudi

[0914] The amount of phagocytic hMDM in each condition was determined as TO-PRO-3 - CD11b + CTV + cell%.

[0915] Using hMDM from four different human healthy donors, IgG1-CD27-A-P329R-E345R did not increase the percentage of phagocytic hMDM or decrease the percentage of viable Daudi cells in a phagocytosis assay. This demonstrates that residual FcγRIa binding by IgG1-CD27-A-P329R-E345R does not result in FcγRIa-mediated effector function (from Figure 11 Data from a representative human healthy donor are shown.) The positive control antibody IgG1-CD20 effectively induced phagocytosis of Daudi cells expressing high levels of CD20, as demonstrated by an increase in the percentage of phagocytic hMDMs and a decrease in the percentage of viable Daudi cells.

[0916] In conclusion, residual binding to FcγRIa is insufficient to induce CD27 + IgG1-CD27-A-P329R-E345R-dependent ADCP of cells.

[0917] Example 14: Fluid-Phase Target-Independent Complement Activation by Anti-CD27 Antibody IgG1-CD27-A-P329R-E345R as Determined by Measuring C4d Deposition

[0918] In the case of an active Fc region, antibodies with enhanced Fc-Fc interactions typically exist as monomeric IgG1 molecules in solution and hexamerize on the cell surface upon target binding to form a C1q docking site (Diebolder, CA et al 2014; de Jong, RN et al 2016). The IgG Fc domain of the anti-CD27 antibody IgG1-CD27-A-P329R-E345R was silenced by introducing the P329R mutation, which resulted in a lack of C1q binding to membrane-bound IgG1-CD27-A-P329R-E345R ( Figure 6 To confirm that IgG1-CD27-A-P329R-E345R was unable to activate complement in solution in the absence of target binding, target-independent complement activation in the fluid phase was investigated by determining C4d deposition, which is considered a measure of activation of the classical complement pathway. TM The C4d enzyme immunoassay (EIA; Quidel, cat. no. A008) was performed to analyze the liquid-phase C4d fragment deposition of IgG1-CD27-A-P329R-E345R by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol. Heat-aggregated gamma globulin (HAGG; a complement activator; Quidel, cat. no. 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 into IgG1 antibodies has been described to induce hexamer formation in solution, leading to liquid-phase complement activation (Diebolder, C.A et al, 2014; Wang, G., RN et al, 2016; de Jong, RN et al, 2016). IgG1-b12-P329R-E345R was included as an isotype control antibody.

[0919] Antibody dilutions were prepared in phosphate-buffered saline (PBS) to a concentration of 1 mg / mL, except for HAGG, which was diluted to a concentration of 10 mg / mL. The test samples were then further diluted in 90% (final concentration) normal human serum (NHS) (CompTech, Lot. No. 42a) to a concentration of 100 μg / mL (for monoclonal IgG) or 1,000 μg / mL (for HAGG) and incubated at 37°C for 1 h. In parallel, a "no antibody" sample (no antibody, 90% NHS) and a "PBS only" sample (no antibody, no NHS) were included as negative controls. The samples were then diluted 1:250 in the cold complement sample diluent provided by the kit. Simultaneously, a strip coated with mouse anti-human C4d antibody was placed in a 96-well plate, and the assay wells were washed three times with 250 to 300 μL of wash buffer, with a 1-minute wait step after the first wash. The test sample is added to the well (100 μL / well), and as a negative control, only the complement sample diluent (blank) is used in the ELISA. In parallel, 100 μL standards (standard AE) and internal controls provided by the test kit are added to separate wells. The flat plate is incubated at room temperature for 30 minutes. Then, the flat plate is washed five times with wash buffer as described above. 50 μL C4d conjugates (peroxidase-conjugated goat anti-human C4d) are added to the well, and the flat plate is incubated at room temperature for 30 minutes. After five washing steps with wash buffer as described above, 100 μL C4d substrates [0.7% 2-2'-nitrogen-two-(3-ethylbenzthiazoline sulfonic acid diammonium salt] are added, and the flat plate is incubated at room temperature for 30 minutes again. Finally, the stop solution provided by the 50 μL test kit is added, and within 1 hour, the optical density is measured at 405 nm using an ELISA plate reader (EL808 BioSPX, BioTek).

[0920] IgG1-CD27-A-P329R-E345R and the control antibody IgG1-b12-P329R-E345R (which has 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 a control antibody with a wild-type Fc domain (IgG1-b12) and a 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 to the same level as HAGG.

[0921] These data show that IgG1-CD27-A-P329R-E345R does not induce target-independent fluid-phase complement activation in vitro.

[0922] Example 15: Ability of anti-CD27 antibody IgG1-CD27-A-P329R-E345R to compete with CD70 for ligand binding

[0923] 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 saturating concentrations 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.

[0924] Daudi cells were cultured in RPMI 1640 medium (Gibco, cat. no. A10491-01) supplemented with 10% donor bovine serum and iron (DBSI; Gibco, cat. no. 20731-030). CCL-213 TM ) were seeded at 50,000 cells / well in a round-bottom 96-well plate (Greiner Bio One, cat. no. 650261). Cells were pelleted by centrifugation (300 × g, 3 minutes at 4°C) and resuspended in FACS buffer (PBS, 1% BSA [Roche, cat. no. 1073508600]) containing anti-CD27 or control antibodies (50 μg / mL final concentration). Biotinylated recombinant human CD70 ECD (Abcam, cat. no. ab271443) was added at a saturating concentration (6 μg / mL) and the cells were incubated at 4°C for 30 minutes.

[0925] The cells were washed twice and resuspended in 4°C containing brilliant violet (BV) 421 TM The cells were washed twice and resuspended in FACS buffer containing TO-PRO-3 iodide (Thermo Fisher Scientific, cat. no. T3605; 1:25,000) and analyzed on a BD FACSymphony. TMData were collected on an A3 flow cytometer (BD Biosciences) and analyzed using FlowJo software. For compensation, one drop of UltraComp eBeads was added to each well. TM Compensation beads (Life Technologies, cat. no. 01-2222-42) were added. 2 μL of each antibody was added, and the mixture was incubated for 20 minutes. The plate was centrifuged, and 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 live cells. Cells were centrifuged and resuspended in TO-PRO-3 diluted in FACS buffer. Data were processed and visualized using GraphPad Prism.

[0926] IgG1-CD27-A-P329R-E345R or IgG1-CD27-A does not block the interaction of CD70 ECD with CD27 + Binding of Daudi cells was not significantly affected, as the CD70 binding levels were comparable to those of Daudi cells incubated with nonbinding isotype control antibodies IgG1-b12-P329R-E345R or IgG1-b12, or cells without antibody ( Figure 13 ). In addition, 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, in the prior art anti-CD27 antibody IgG1-CD27-CDX1127 ( Figure 13 ), CD70 cannot bind to surface CD27 on Daudi cells. The anti-CD27 antibody IgG1-CD27-CDX1127 was previously reported to block ligand binding (Vitale et al, 2012).

[0927] In conclusion, IgG1-CD27-A-P329R-E345R binding did not block the binding of CD27 by its natural ligand CD70 on Daudi cells.

[0928] Example 16: Expression of T cell activation markers after polyclonal stimulation of human PBMCs incubated with anti-CD27 antibodies

[0929] The effect of IgG1-CD27-A-P329R-E345R on the expression of T cell activation markers in polyclonal activated T cells was investigated using PBMCs obtained from three different healthy human donors. The expression of HLA-DR, CD25, CD107a, and 4-1BB was analyzed after 2 and 5 days of incubation of PBMCs with IgG1-CD27-A-P329R-E345R or prior art anti-CD27 antibodies.

[0930] 75,000 freshly isolated PBMCs / well were seeded in cell culture medium in a 96-well U-bottom plate (Greiner Bio-One). Repeated wells were incubated with anti-CD3 antibodies (UCHT1 clone; stem cells; 0.1 μg / mL); and IgG1-CD27-A-P329R-E345R (0.0005 to 30 μg / mL, three-fold dilution); or prior art anti-CD27 antibodies IgG1-CD27-CDX1127, IgG1-CD27-131A, and IgG1-CD27-BMS986215 (30 μg / mL); or non-binding control antibody IgG1-b12-P329R-E345R (10 μg / mL). To determine the expression of each activation marker in the absence of treatment, replicate control wells with untreated (no anti-CD3 or anti-CD27 antibodies) cells were supplemented with culture medium only. In order to set the gate for identifying activation marker-positive cells, fluorescence minus one (FMO) control was used. For FMO control, all antibodies used in the experiment were added to 75,000 PBMC / well from a donor activated with anti-CD3 antibody, except for the antibodies corresponding to activation markers in duplicate wells. Untreated cells from each donor in a single well without staining antibody were included as a negative control. In order to detect viable cells, untreated cells from each donor were stained with 4',6-diamidino-2-phenylindole (DAPI) in a single well.

[0931] After two or five days of incubation (37°C, 5% CO2), the plates were washed once with FACS buffer and resuspended in an antibody cocktail in FACS buffer containing antibodies against T cell activation markers 4-1BB, CD25, CD107a, human leukocyte antigen (HLA)-DR; and CD4 for gating in flow cytometry. + and CD8 + 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 CD4 + and CD8 + The median fluorescence intensity (MFI) and percentage of positive cells for each T cell activation marker on T cells were calculated. Changes in the expression levels of T cell activation markers induced by anti-CD27 antibodies were expressed as the fold change in the MFI of the anti-CD27 antibody sample relative to the non-binding control antibody IgG1-b12-P329R-E345R. FlowJo software was used on a BD LSRFortessa TMSamples were analyzed on a cell analyzer (BD Biosciences).

[0932] IgG1-CD27-A-P329R-E345R increases activated CD4 + Expression of CD25, CD107a and 4-1BB on T cells ( Figure 14A These effects were more pronounced after 2 days of incubation than after 5 days of incubation. + On T cells, incubation with IgG1-CD27-A-P329R-E345R resulted in increased expression of HLA-DR, CD107a, and 4-1BB after 2 and 5 days of incubation ( Figure 14B ).

[0933] The expression of T cell activation markers was also assessed after 2 and 5 days of incubation with three prior art antibodies. IgG1-CD27-131A and IgG1-CD27-BMS986215 induced CD4 + and CD8 + Comparable increases in HLA-DR, 4-1BB, CD25, and CD107a expression on T cells were observed, whereas incubation with IgG1-CD27-CDX1127 for 2 or 5 days had less pronounced effects on the expression of T cell activation markers.

[0934] In conclusion, incubation of polyclonal activated PBMC with IgG1-CD27-A-P329R-E345R resulted in the proliferation of CD4 + and CD8 + The expression of activation markers HLA-DR, CD25, CD107a, and 4-1BB on T cells was increased.

[0935] Example 17: After injection of anti-CD27 antibody in human CD27-KI mouse model, OVA-specific CD8 + Percentage of T cells

[0936] The effect of IgG1-CD27-A-P329R-E345R treatment on the expansion of antigen-specific T cells in splenocytes in the hCD27 KI OVA model was analyzed by flow cytometry.

[0937] Homozygous human CD27 (hCD27)-KI mice on a C57BL / 6 background (hCD27 KI mice) were obtained from Beijing Biocytogen Co., Ltd. (strain name C57BL / 6-Cd27tm1 (CD27) / Bcgen, stock number 110006). This strain is a hybrid of Crown Bioscience's HuGEMM TMThe platform was developed collaboratively and features 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 by subcutaneous (sc) injection of the immunogen ovalbumin (OVA) in hCD27-KI mice, and the agonist effect of IgG1-CD27-A-P329R-E345R was tested by simultaneously treating the mice with the antibody intravenously (iv).

[0938] On day 0, mice were injected subcutaneously with 5 mg of OVA (InvivoGen, cat. no. vac-pova-100, lot. no. EFP-42-04) and treated with IgG1-CD27-A-P329R-E345R (30 mg / kg), IgG1-CD27-CDX1127 (30 mg / kg), or IgG1-b12-P329R-E345R (30 mg / kg) via intravenous injection into the tail vein. On days 12 and 21, mice were boosted with OVA and treated with antibodies as on day 0. On days 10, 19, and 24, blood was collected via the cheek pouch or saphenous vein in BD containing dipotassium EDTA. Blood was collected in tubes (K2-EDTA; BD, cat. no. 365974) and used immediately for further analysis. On day 28, mice were euthanized and the spleens were removed under sterile conditions.

[0939] The spleen tissues were excised in RPMI1640 medium (Thermo Fisher Scientific, cat. no. C22400500BT) and transferred to gentleMACs. TM C tubes (Miltenyi Biotec, cat. no. 130-093-237) and gentleMACs according to the manufacturer's instructions. TM The cells were mechanically dissociated into a single cell suspension using a dissociator (Miltenyi, cat. no. 130-093-235). After dissociation, the cell suspension was filtered through a 70 μm cell strainer (Falcon, cat. no. 352350). Next, the sample was washed twice by resuspending in 3 mL of wash buffer (sterile PBS [Hyclone, SH0256.01B] supplemented with 4% FBS [Gibco, cat. no. 10099141]). The cells were counted on a Cellometer Auto T4 (Nexcelom Bioscience) and the cell number was adjusted to 2 × 10 per tube. 6splenocytes.

[0940] 2×10 6 Splenocytes were transferred to FACS tubes (Falcon, cat. no. 352052) and resuspended in wash buffer (sterile PBS [Hyclone, SH0256.01B] supplemented with 4% FBS [Gibco, cat. no. 10099141]) supplemented with 1 μg / mL purified rat anti-mouse CD16 / CD32 (mouse BD Fc Block TM , BD Biosciences, cat.no.553141). After pre-incubation for 10 minutes at 2-8°C in the dark, 10 μL of PE-labeled OVA-tetramer (MBL Life science, cat.no.TS 50011C) was added and the sample was gently vortexed and then further incubated for 30-60 minutes at 2-8°C in the dark. Without washing, labeled antibodies and compounds for flow cytometry gating of T cell subsets were added. The samples were gently vortexed and incubated for another 30 minutes at 2-8°C in the dark. Next, the samples were washed twice by resuspending in 2 mL of wash buffer and centrifuged at 300 x g for 5 minutes. Finally, the cells were resuspended in 250 μL of wash buffer and centrifuged on a BD LSRFortessa TM The cells were analyzed on an X-20 cell analyzer (BD Biosciences) and the data were processed using Kaluza analysis software (Beckman Coulter).

[0941] IgG1-CD27-A-P329R-E345R increased OVA-specific CD8 in the spleen of mice injected with OVA protein vaccine. + Percentage of OVA-specific CD8 T cells in mice treated with 30 mg / kg IgG1-CD27-CDX1127 + The percentage of T cells was lower than that in the IgG1-CD27-A-P329R-E345R-treated group and comparable to that in the IgG1-b12-P329R-E345R-treated group ( Figure 15 ). Similar observations were made in peripheral blood samples.

[0942] Example 18: OVA-specific CD8 from the spleen of OVA-immunized mice injected with anti-CD27 antibody + IFNγ secretion by T cells

[0943] The spleen tissue excised in RPMI1640 medium (see Example 17) was gently triturated on a 70 μm cell strainer (Falcon, cat. no. 352350), pelleted by centrifugation (1,500 rpm, 5 minutes), and resuspended in 10 mL of ammonium-chloride-potassium (ACK) lysis buffer (Invitrogen, cat. no. A1049201). After incubation at room temperature 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 solution supplemented with 50 U / mL penicillin and 50 μg / mL streptomycin (pen / strep, Gibco, cat. no. 15070-063). TM The collected spleen cells were filtered again through a 70 μm cell strainer and cultured in Vi-CELL TM The cells were counted on an XR cell viability analyzer (Beckman Coulter) and the concentration was adjusted to 3.125 × 10 6 cells / mL.

[0944] IFNγ production by splenocytes was analyzed using the Mouse IFN-γ ELISpotPLUS Kit (Mabtech, cat. no. 3321-4HPW-2) essentially as described by the manufacturer. Pre-coated MultiScreen HTS IP Filter (MSIP) white plates (mAb AN18) were washed four times with 200 μL sterile PBS per well and conditioned with 200 μL CTL assay medium containing pen / strep (RT, 30 min). The medium was removed and 5×10 5 splenocytes / well were inoculated with 2 μg / mL OVA in duplicate 257-264The peptide SIINFEKL (Invivogen, cat. no. vac-sin) or the scrambled control peptide FILKSINE (SB-PEPTIDE, cat. no. SB073-1MG) was incubated in a humidified incubator (37°C, 5% CO2) for 20 hours in a total volume of 180 μL / well. As a positive control for IFNγ production, splenocytes were incubated in parallel with a cell stimulation mixture consisting of 500 ng / mL phorbol ester (PMA) and 10 μg / mL ionomycin (PMA+ionomycin, Dakewe Biotech, cat. no. DKW ST PI). Cultures of splenocytes without peptide were included as negative controls. After incubation, the cells were removed and the plates were washed five times with PBS. Next, the plate was incubated sequentially with biotinylated detection mAb (R4-6A2; RT, 2 hours), streptavidin-horseradish peroxidase (HRP; RT, 1 hour), and finally with 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (all provided by the kit), with five wash steps in between with PBS. When obvious spots appeared, the reaction was stopped by washing extensively 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 and presented as bar graphs and presented as the average number of spots per well ± SEM for all mice from each treatment group (n = 5).

[0945] Splenocytes from all IgG1-CD27-A-P329R-E345R treated animal groups showed an increase in IFNγ production in response to treatment with OVA peptide, as demonstrated by ELISpot analysis ( Figure 16 Stimulation of splenocytes with a scrambled control peptide induced little or no IFNγ production, suggesting that IFNγ is produced by OVA-specific T cells. In contrast, no IFNγ production was observed in splenocytes from mice treated with 30 mg / kg IgG1-CD27-CDX1127.

[0946] Example 19: Effect of IgG1-CD27-A-P329R-E345R Treatment on T Cell Activation in OVA-Immunized Mice

[0947] By analyzing the expression of PD-1 in CD8 + The expression of IgG1-CD27-A-P329R-E345R on CD8 T cells was studied in vivo. +Effect of T Cell Activation Mice were treated as described in Example 17. In addition, methods for obtaining splenocytes and analyzing splenocytes by FACS are described in Example 17.

[0948] On day 28, IgG1-CD27-A-P329R-E345R induced CD8 + Increased percentage of T cells. CD8 + PD-1 + T cell percentages were low in animals treated with IgG1-CD27-CDX1127 or the control antibody IgG1-b12-P329R-E345R ( Figure 17 ).

[0949] Example 20: Effect of IgG1-CD27-A-P329R-E345R Treatment on In Vivo Induction of T Cell Subpopulations in OVA-Immunized Mice

[0950] The effect of IgG1-CD27-A-P329R-E345R on the expansion of T cell subsets was investigated 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. + T cells. Memory T cells are 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. In addition, methods for obtaining splenocytes and analyzing splenocytes by FACS are described in Example 17.

[0951] IgG1-CD27-A-P329R-E345R (30 mg / kg) induced pro-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. + The percentage of T cells increased ( Figure 18 ). In CD45 + In the population, the percentages of pre-effector T cells and effector memory T cells induced by IgG1-CD27-A-P329R-E345R were higher than those of IgG1-CD27-CDX1127 (30 mg / kg), while the percentages of CD8 + In part, the mean percentages of these T cell populations induced by the two anti-CD27 antibodies were comparable.

[0952] Example 21: Effect of IgG1-CD27-A-P329R-E345R Treatment on the In Vivo Expansion of T Cells in OVA-Immunized Mice

[0953] The effect of IgG1-CD27-A-P329R-E345R on T cell expansion was investigated by analyzing CD3 expression in splenocytes and blood samples from OVA-treated hCD27-KI mice. Mice were treated as described in Example 17. In addition, methods for obtaining splenocytes and blood samples and analyzing them by flow cytometry are described in Example 17.

[0954] Treatment of OVA-immunized hCD27-KI mice with 30 mg / kg IgG1-CD27-A-P329R-E345R did not increase CD3 + The percentage of T cells ( Figure 19 In contrast, treatment with the benchmark antibody IgG1-CD27-CDX1127 (30 mg / kg) resulted in a decrease in CD3 + Similar observations were made in peripheral blood samples.

[0955] Example 22: Effect of IgG1-CD27-A-P329R-E345R on T cell cytokine production in antigen specificity studies

[0956] The ability of IgG1-CD27-A-P329R-E345R to increase cytokine production was investigated using T cells that had been stimulated with their cognate antigen.PBMCs were isolated from buffy coats obtained from healthy human donors by Ficoll-Paque density gradient separation (GE Healthcare, cat. no. 17 144 0 03) according to the manufacturer's instructions.

[0957] Human magnetic CD14 and CD8 microbeads (Miltenyi Biotec, cat. no. 130 050 201 and 130 045201, respectively) were used for CD14 from human PBMCs. + Positive selection of monocytes and CD14 - Negative selection of PBL, and CD8 + Positive selection of T cells. The cell suspension was centrifuged and diluted to 1 × 10 cells / 80 μL MACS buffer. 7Each 1×10 living cells were resuspended in magnetic activated cell sorting (MACS) buffer (Dulbecco's phosphate buffered saline [DPBS] containing 5 mM EDTA and 1% human albumin). 7 12 μL of CD14 or CD8 microbeads were added to each cell. Subsequent MACS separation was performed using an automated magnetic cell separation instrument or by manual separation. Automated MACS separation was performed using a Pro separator (Miltenyi Biotec). + Monocytes and CD8 + T cells were centrifuged (8 min, 300 x g, room temperature), resuspended in X-VIVO 15 medium (Lonza), and counted with erythrosine B solution for further use; that is, monocytes were differentiated into iDCs or CD8 T cells with PD-1 and / or CLDN6-specific T cell receptor (TCR) mRNA. + Electroporation of T cells.

[0958] For the generation of monocyte-derived iDCs, up to 40 × 10 cells were cultured in DC culture medium (RPMI 1640, 5% pooled human serum [PHS; OneLambda, cat. no. A25761], 1× Minimum Essential Medium Non-Essential Amino Acids Solution [MEM NEAA, Life Technologies, cat. no. 11140 035], 1 mM sodium pyruvate [Life Technologies, cat. no. 11360039]) in T175 flasks at 37°C, 5% CO2. 6 PBMC-derived CD14 +After 5 days of incubation, the iDC culture medium was supplemented with 100 ng / mL human granulocyte / macrophage colony stimulating factor (GM-CSF; Miltenyi Biotec, cat. no. 130-093-868) and 50 ng / mL human IL-4 (Miltenyi Biotec, cat. no. 130093 924). After 3 days of incubation, half of the culture medium was replaced in each flask. Since the culture medium removed from the flask contained non-adherent monocytes, it was centrifuged (8 minutes, 300 x g, RT), the supernatant was discarded, the cell pellet was resuspended in fresh DC culture medium, and then returned to the starting flask together with 200 ng / mL GM-CSF and 200 ng / mL IL-4 (final concentration). After incubation for 5 days, iDCs adhered to the culture flask were isolated using 10 mL DPBS containing 2 mM EDTA (37° C., 10 minutes). Isolated iDCs were washed, pelleted (8 min, 300 xg, room temperature) and used for electroporation with CLDN6 mRNA.

[0959] Human CD8 T cells were electroporated with RNA encoding the α and β chains of mouse TCR specific for human CLDN6, either alone or together with RNA encoding PD-1. + T cells and human monocyte-derived iDCs were electroporated with RNA encoding human C...

Claims

1. A method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor.

2. The method of claim 1, wherein the binding agent comprises heavy chain variable (VH) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, and light chain variable (VL) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

3. The method of claim 1 or 2, wherein the binding agent comprises two binding regions capable of binding to human CD27, wherein the antibody comprises heavy chain variable (VH) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, and light chain variable (VL) region CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

4. The method of any of the preceding claims, wherein the binding agent comprises a VH region comprising the sequence shown in SEQ ID NO:

4.

5. The method of any of the preceding claims, wherein the binding agent comprises a VL region comprising the sequence shown in SEQ ID NO:

8.

6. The method of any of the preceding claims, wherein the binding agent comprises a VH region and a VL region comprising the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 8, respectively.

7. The method of any of the preceding claims, wherein the binding agent is an antibody, preferably a human antibody or a humanized antibody.

8. The method of any 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).

9. The method of claim 8, wherein the light chain constant region is human κ.

10. The method of claim 8, wherein the light chain constant region is human lambda.

11. The method of any of the preceding claims, wherein the binding agent further comprises a heavy chain constant region that is of the human IgG isotype, optionally a modified human IgG.

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

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

16. The method of any one of claims 11 to 15, wherein the substitutions in the heavy chain constant region induce increased CD27 agonism compared to the same antibody except comprising a wild-type IgGl antibody heavy chain constant region.

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

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

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

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

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

22. The method of any one of claims 11 to 21, wherein the binding agent has a pharmacokinetic profile as a parent antibody comprising a wild-type IgGl heavy chain constant region.

23. The method of any of the preceding claims, wherein the binding agent comprises a heavy chain constant region comprising a 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.

24. The method of any of the preceding claims, wherein the binding agent comprises a heavy chain constant region comprising the sequence shown in SEQ ID NO:

15.

25. The method of any of the preceding claims, wherein the binding agent comprises a heavy chain constant region that is modified such that the binding agent induces one or more Fc-mediated effector functions to a lesser extent relative to a parent antibody.

26. The method of 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 of claim 25 or 26, wherein the binding agent does not induce one or more Fc-mediated effector functions.

28. The method of any one of claims 25 to 27, wherein the one or more Fc-mediated effector functions are selected from the group consisting of 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.

29. The method of any one of claims 25 to 28, wherein the binding agent does not induce CIq binding when measured by the method of Example 8.

30. The method of any of the preceding claims, wherein the binding agent is a monovalent antibody.

31. The method of any of the preceding claims, wherein the binding agent is a bivalent antibody.

32. The method of any of the preceding claims, wherein the binding agent is a monospecific antibody.

33. The method of any of the preceding claims, wherein the binding agent is a bispecific antibody comprising a first antigen binding region capable of binding to human CD27 according to any of the preceding claims and comprising a second antigen binding region capable of binding to a different epitope on human CD27 or capable of binding to a different target.

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

2.

35. The method of any of the preceding claims, wherein the binding agent 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. CL region, which comprises the amino acid sequence shown in SEQ ID NO: 17; 36. The method of any of the preceding claims, wherein the binding agent 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 of any of the preceding claims, wherein PD-L1 is human PD-L1, in particular human PD-L1 comprising the sequence shown in SEQ ID NO:

98.

38. The method of any of the preceding claims, wherein PD1 is human PD1, preferably, the PD1 has or comprises the amino acid sequence as shown in SEQ ID NO: 58 or SEQ ID NO: 59, or the amino acid sequence of the PD1 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity with the amino acid sequence as shown in SEQ ID NO: 58 or SEQ ID NO: 59, or is an immunogenic fragment thereof.

39. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1 or PD-L1, preferably an antibody that is an antagonist of the PD1 / PD-L1 interaction and / or a PD1 or PD-L1 blocking antibody.

40. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is an antibody of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, such as an antibody of the IgG1 isotype.

41. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is a full-length antibody or an antibody fragment, such as a full-length IgG1 antibody.

42. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is a monospecific antibody.

43. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 99, 100, and 101, respectively, and a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 102, LAS, and SEQ ID NOs: 103, respectively.

44. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, the antibody that binds to PD1 comprises a VH region and a VL region, the VH region comprises the amino acid sequence of SEQ ID NO: 104, and the VL region comprises the amino acid sequence of SEQ ID NO:

105.

45. The method of any of the preceding claims, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, the antibody that binds to PD1 comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 106, and the light chain comprising the amino acid sequence of SEQ ID NO:

107.

46. ​​The method of any one of the preceding claims, wherein a) The binding agent is an antibody, which 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 PD1 / PD-L1 inhibitor is pembrolizumab or a biosimilar thereof.

47. The method of any one of claims 1-42, wherein a) The binding agent is an antibody, which 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 PD1 / PD-L1 inhibitor is nivolumab or its biosimilar.

48. The method of any one of claims 1-42, wherein a) The binding agent is an antibody, which 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 PD1 / PD-L1 inhibitor is atezolizumab or its biosimilar.

49. The method of any one of claims 1-42, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, or an antigen-binding fragment thereof, wherein the antibody that binds to PD1 comprises VH region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 49, 46, and 45, respectively, and VL region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 52, QAS, and SEQ ID NO: 50, respectively.

50. The method of claim 49, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH), which comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of the VH sequence shown in SEQ ID NO:

56.

51. The method of claim 50, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH), wherein the VH comprises the sequence shown in SEQ ID NO:

56.

52. The method of any one of claims 49-51, wherein the antibody that binds to PD1 comprises a light chain variable region (VL), wherein the light chain variable region (VL) comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of the VL sequence shown in SEQ ID NO:

57.

53. The method of claim 52, wherein the antibody that binds to PD1 comprises a light chain variable region (VL), wherein the VL comprises the sequence shown in SEQ ID NO:

57.

54. The method of any one of claims 49-53, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or has the sequence shown in SEQ ID NO: 56, and the VL comprises or has the sequence shown in SEQ ID NO:

57.

55. The method of any one of claims 49-54, wherein the antibody that binds to PD1 comprises a heavy chain constant region, wherein the heavy chain constant region comprises an aromatic or non-polar amino acid at a position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering and an amino acid other than glycine at a position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering.

56. The method of claim 55, wherein the amino acid at the position corresponding to position 236 is a basic amino acid.

57. The method of claim 56, wherein the basic amino acid is selected from the group consisting of lysine, arginine, and histidine.

58. The method of claim 56 or 57, wherein the basic amino acid is arginine (G236R).

59. The method of any one of claims 55-58, wherein the amino acid at the position corresponding to position 234 is an aromatic amino acid.

60. The method of claim 59, wherein the aromatic amino acid is selected from the group consisting of phenylalanine, tryptophan, and tyrosine.

61. The method of any one of claims 55-58, wherein the amino acid at the position corresponding to position 234 is a non-polar amino acid.

62. The method of claim 61, wherein the non-polar amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

63. The method of claim 61 or 62, wherein the non-polar amino acid is selected from the group consisting of isoleucine, proline, phenylalanine, methionine, and tryptophan.

64. The method of any one of claims 55-63, wherein the amino acid at position corresponding to position 234 is phenylalanine (L234F).

65. The method of any one of claims 55-64, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acid at the position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering is an acidic amino acid.

66. The method of claim 65, wherein the acidic amino acid is aspartic acid or glutamic acid.

67. The method of any one of claims 55-66, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acid at the position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering is glutamic acid (L235E).

68. The method of any one of claims 55-67, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acids at positions corresponding to positions 234, 235, and 236 are a nonpolar or aromatic amino acid at position 234, an acidic amino acid at position 235, and a basic amino acid at position 236.

69. The method of any one of claims 55-68, wherein in the heavy chain constant region of the antibody that binds to PD1, the amino acid corresponding to position 234 is phenylalanine, the amino acid corresponding to position 235 is glutamic acid, and the amino acid corresponding to position 236 is arginine (L234F / L235E / G236R).

70. The method of any one of claims 49-69, wherein the heavy chain constant region of the antibody that binds to PD1 comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to the amino acid sequence of the HC sequence shown in SEQ ID NO:

38.

71. The method of any one of claims 49-70, wherein the heavy chain constant region of the antibody that binds to PD1 comprises the sequence shown in SEQ ID NO:

38.

72. The method of any one of claims 49-71, wherein the isotype of the heavy chain constant region of the antibody that binds PD1 is IgG1.

73. The method of any one of claims 49-72, wherein the antibody that binds to PD1 comprises a heavy chain having the sequence shown in SEQ ID NO: 139 and a light chain having the sequence shown in SEQ ID NO:

140.

74. The method of any one of claims 49-73, wherein the antibody that binds to PD1 is a monoclonal, chimeric, or humanized antibody or a fragment of such an antibody.

75. The method of any one of claims 49-74, wherein the antibody that binds PD1 has reduced or depleted Fc-mediated effector function.

76. The method of any one of claims 49-75, wherein binding of complement protein C1q to the constant region of the antibody that binds to PD1 is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

77. The method of any one of claims 49-76, wherein binding of one or more IgG Fc-γ receptors to the antibody that binds to PD1 is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, compared to a wild-type antibody.

78. The method of claim 77, wherein the one or more IgG Fc-γ receptors are selected from at least one of Fc-γRI, Fc-γRII, and Fc-γRIII.

79. The method of claim 77 or 78, wherein the IgG Fc-γ receptor is Fc-γRI.

80. The method of any one of claims 49-79, wherein the antibody that binds to PD1 is unable to induce Fc-γRI-mediated effector function, or wherein the induced Fc-γRI-mediated effector function is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

81. The method of any one of claims 49-80, wherein the antibody that binds to PD1 is unable to induce at least one of complement dependent cytotoxicity (CDC)-mediated lysis, antibody dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis, or wherein at least one of complement dependent cytotoxicity (CDC)-mediated lysis, antibody dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis is induced to a reduced extent, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

82. The method of any one of claims 49-81, wherein binding of the neonatal Fc receptor (FcRn) to the PD1 binding antibody is not affected compared to a wild-type antibody.

83. The method of any one of claims 49-82, wherein the antibody that binds to PD1 binds to a native epitope of PD1 present on the surface of living cells.

84. The method of any one of claims 49-83, wherein the antibody that binds to PD1 is a multispecific antibody comprising a first antigen binding region that binds PD1 and at least one additional antigen binding region that binds another antigen. The method of claim 84 , wherein the antibody that binds to PD1 is a bispecific antibody comprising a first antigen-binding region that binds to PD1 and a second antigen-binding region that binds to another antigen.

86. The method of claim 84 or 85, wherein the first antigen-binding region that binds to PD1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown in any one of claims 50 to 54.

87. The method of any one of claims 49-86, wherein a) the binding agent comprises a VH region and a VL region, the VH region comprises the amino acid sequence shown in SEQ ID NO:4, and the VL region comprises the amino acid sequence shown in SEQ ID NO:8; b) The antibody that binds to PD1 comprises a VH region and a VL region, wherein the VH comprises or has the sequence shown in SEQ ID NO:56, and the VL comprises or has the sequence shown in SEQ ID NO:

57.

88. The method of any one of claims 49-87, wherein a) the binding agent is an antibody comprising a VH region, a VL region, a CH region and a CL region, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO:4, the VL region comprises the amino acid sequence shown in SEQ ID NO:8, the CH region comprises the amino acid sequence shown in SEQ ID NO:15, and the CL region comprises the amino acid sequence shown in SEQ ID NO:17; b) The antibody that binds to PD1 comprises a VH region, a VL region, a CH region and a CL region, the VH region comprises the amino acid sequence shown in SEQ ID NO: 56, the VL region comprises the amino acid sequence shown in SEQ ID NO: 57, the CH region comprises the amino acid sequence shown in SEQ ID NO: 38, and the CL region comprises the amino acid sequence shown in SEQ ID NO:

42.

89. The method of any one of claims 1-41, wherein the PD1 / PD-L1 inhibitor is a multispecific antibody, such as a bispecific antibody.

90. The method of claim 89, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor comprising a first binding region that binds CD137 and a second binding region that binds PD-L1.

91. The method of claim 90, wherein CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO:

97.

92. The method of claim 90 or 91, wherein a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 79, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 83; and b) The second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 86, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:

90.

93. The method of any one of claims 90-92, wherein a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 80, 81, and 82, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 84, GAS, and SEQ ID NOs: 85, respectively; and b) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 87, 88, and 89, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 91, DDN, and SEQ ID NOs: 92, respectively.

94. The method of any one of claims 90-93, wherein a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 79, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 83; and b) The second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:

90.

95. The method of any one of claims 90-94, wherein the PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises i) a polypeptide comprising said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising said first light chain variable region (VL) and a first light chain constant region (CL); and the second binding arm comprises iii) a polypeptide comprising said second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) a polypeptide comprising said second light chain variable region (VL) and a second light chain constant region (CL).

96. The method of any one of claims 90-95, wherein the PD-L1 inhibitor comprises i) a first heavy chain and a first light chain comprising said antigen binding region capable of binding to CD137, said first heavy chain comprising a first heavy chain constant region, and said first light chain comprising a first light chain constant region; and ii) a second heavy chain and a second light chain, which comprise the antigen binding region capable of binding to PD-L1, the second heavy chain comprises a second heavy chain constant region, and the second light chain comprises a second light chain constant region.

97. The method of claim 95 or 96, wherein (i) in the first heavy chain constant region (CH), the amino acid in the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L, and in the second heavy chain constant region (CH), the amino acid in the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, or (ii) in the first heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, and in the second heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L.

98. The method of any one of claims 95-97, wherein in the first and second heavy chains, the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering are F and E, respectively.

99. The method of any of claims 95-98, wherein in the first and second heavy chain constant regions (HC), the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively.

100. The method of any of claims 95-99, wherein the positions of both the first and second heavy chain constant regions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively, and wherein (i) the position of the first heavy chain constant region corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L, and the position of the second heavy chain corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, or (ii) the position of the first heavy chain constant region corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L.

101. The method of any of claims 95-100, wherein the positions of both the first and second heavy chain constant regions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering are F, E and A, respectively, and wherein (i) the position of the first heavy chain constant region corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L, and the position of the second heavy chain constant region corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, or (ii) the position of the first heavy chain corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R, and the position of the second heavy chain corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L.

102. The method of any one of Es 95-101, wherein the constant region of the first and / or second heavy chain (e.g., the second heavy chain) comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of: a) the sequence shown in SEQ ID NO:94 or 96 [IgG1-Fc_FEAL]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) sequences 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 sequences defined in a) or b).

103. The method of any one of claims 95-102, wherein the constant region of the first and / or second heavy chain (e.g., the first heavy chain) comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of: a) the sequence shown in SEQ ID NO:93 or 95 [IgG1-Fc_FEAR]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) sequences 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 sequences defined in a) or b).

104. The method of any one of Es 95-103, wherein the PD-L1 inhibitor comprises a kappa (κ) light chain constant region.

105. The method of any one of Es 95-104, wherein the PD-L1 inhibitor comprises a lambda (λ) light chain constant region.

106. The method of any one of Es 95-105, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region. E 107. The method of any of E 95-106, wherein the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

108. The method of any one of claims 95-107, 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.

109. The method of any one of E104-108, wherein the kappa (κ) light chain comprises an amino acid sequence selected from the group consisting of: 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) sequences having up to 10 substitutions, such as up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in a) or b).

110. The method of any one of claims 105-109, wherein the lambda (λ) light chain comprises an amino acid sequence selected from the group consisting of: a) the sequence set forth in SEQ ID NO: 17, 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) sequences having up to 10 substitutions, such as up to 9 substitutions, up to 8 substitutions, up to 7 substitutions, up to 6 substitutions, up to 5 substitutions, up to 4 substitutions, up to 3 substitutions, up to 2 substitutions or up to 1 substitution compared to the amino acid sequence defined in a) or b).

111. The method of any one of claims 90-110, wherein the PD-L1 inhibitor is an antibody of the IgG1m(f) allotype.

112. The method of any one of claims 90-111, wherein the PD-L1 inhibitor is a bispecific antibody that binds to CD137 and PD-L1, the bispecific antibody having i) a first heavy chain comprising the amino acid sequence shown in SEQ ID NO: 75 and a first light chain comprising the amino acid sequence shown in SEQ ID NO: 76, and ii) a second heavy chain comprising the amino acid sequence shown in SEQ ID NO: 77 and a second light chain comprising the amino acid sequence shown in SEQ ID NO:

78.

113. The method of any one of claims 90-112, wherein the PD-L1 inhibitor is acalabrutinib or a biosimilar thereof.

114. The method of any one of claims 90-113, wherein a) the binding agent comprises heavy chain variable (VH) region CDR1, CDR2 and CDR3 and light chain variable (VL) region CDR1, CDR2 and CDR3, wherein the heavy chain variable (VH) region CDR1, CDR2 and CDR3 comprise the sequences shown in SEQ ID NOs: 5, 6 and 7, respectively, and the light chain variable (VL) region CDR1, CDR2 and CDR3 comprise the sequences shown in SEQ ID NOs: 9, 10 and 11, respectively; b) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 80, 81 and 82, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 84, GAS and SEQ ID NOs: 85, respectively; and c) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 87, 88 and 89, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 91, DDN and SEQ ID NOs: 92, respectively.

115. The method of any one of claims 90-114, wherein a) the binding agent comprises a VH region and a VL region, the VH region comprises the amino acid sequence shown in SEQ ID NO:4, and the VL region comprises the amino acid sequence shown in SEQ ID NO:8; b) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 79, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 83; and c) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:

90.

116. The method of any one of claims 90-115, wherein a) the binding agent is an antibody comprising a VH region, a VL region, a CH region and a CL region, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO:4, the VL region comprises the amino acid sequence shown in SEQ ID NO:8, the CH region comprises the amino acid sequence shown in SEQ ID NO:15, and the CL region comprises the amino acid sequence shown in SEQ ID NO:17; b) the PD-L1 inhibitor 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 PD-L1 inhibitor comprises a VH region, a VL region, a CH region and a CL region, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 79, the VL region comprises the amino acid sequence shown in SEQ ID NO: 83, the CH region comprises the amino acid sequence shown in SEQ ID NO: 95, and the CL region comprises the amino acid sequence shown in SEQ ID NO: 16; and d) the second binding arm of the PD-L1 inhibitor comprises a VH region, a VL region, a CH region and a CL region, the VH region comprises the amino acid sequence shown in SEQ ID NO: 86, the VL region comprises the amino acid sequence shown in SEQ ID NO: 90, the CH region comprises the amino acid sequence shown in SEQ ID NO: 96, and the CL region comprises the amino acid sequence shown in SEQ ID NO:

17.

117. The method of any one of claims 90-116, wherein a) the binding agent comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 25; d) the PD-L1 inhibitor is a bispecific antibody that binds to CD137 and PD-L1, the bispecific antibody having i) a first heavy chain comprising the amino acid sequence shown in SEQ ID NO: 75 and a first light chain comprising the amino acid sequence shown in SEQ ID NO: 76, and ii) a second heavy chain comprising the amino acid sequence shown in SEQ ID NO: 77 and a second light chain comprising the amino acid sequence shown in SEQ ID NO:

78.

118. The method of any one of claims 90-117, wherein a) the binding agent comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 25; d) The PD-L1 inhibitor is acalabrutinib or a biosimilar thereof.

119. The method of any one of claims 1-38, wherein the PD1 / PD-L1 inhibitor is a PD1 inhibitor selected from the following: pembrolizumab, nivolumab, cemiprilimab, dotalimumab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-224, AMP-514, or their respective biosimilars.

120. The method of any one of claims 1-38, wherein the PD1 inhibitor is selected from pembrolizumab, nivolumab, cemiplizumab, dotalimumab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-514, or their respective biosimilars.

121. The method of any one of claims 1-38, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor selected from the group consisting of atezolizumab, avelumab, durvalumab, KN035, CK-301, acamprosate, AUNP12, CA-170, BMS-986189, or their respective biosimilars.

122. The method of any one of claims 1-38, wherein the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, aclidiniumumab, or a biosimilar thereof of each.

123. The method of any of the preceding claims, wherein the subject is a human subject.

124. The method of any of the preceding claims, wherein the tumor or cancer is a solid tumor.

125. The method of any of the preceding claims, wherein the tumor is a PD-L1 positive tumor.

126. The method of any 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.

127. The method of claim 126, wherein the HNSCC is recurrent, unresectable, or metastatic.

128. The method of any one of claims 1-125, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), e.g., squamous or non-squamous NSCLC.

129. The method of claim 128, wherein the NSCLC is recurrent, unresectable, or metastatic.

130. The method of claim 128 or 129, wherein the NSCLC does not have an epidermal growth factor (EGFR) sensitizing mutation and / or anaplastic lymphoma (ALK) translocation and / or ROS1 rearrangement.

131. The method of any one of claims 128-130, wherein the NSCLC is NTRK1 / 2 / 3 (neurotrophic receptor tyrosine kinase 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 previously been treated with the corresponding targeted therapy.

132. The method of any of the preceding claims, wherein the subject has previously received treatment with a PD1 inhibitor or a PD-L1 inhibitor (eg, an anti-PD1 antibody or an anti-PD-L1 antibody), preferably at least two doses of the PD1 inhibitor or the PD-L1 inhibitor.

133. The method of any of the preceding claims, wherein the subject has previously been treated with a platinum-based therapy or an alternative chemotherapy if platinum is ineligible, such as a gemcitabine-containing regimen.

134. The method of any of the preceding claims, wherein the tumor or cancer recurs and / or progresses following treatment (e.g., systemic treatment with a checkpoint inhibitor).

135. The method of any of the preceding claims, wherein the subject has received at least one prior line of systemic therapy, e.g., systemic therapy comprising a PD1 inhibitor or a PD-L1 inhibitor, e.g., an anti-PD1 antibody or an anti-PD-L1 antibody.

136. The method of any of the preceding claims, wherein the cancer or tumor has relapsed and / or is refractory, or the subject has progressed following treatment with a PD1 inhibitor or PD-L1 inhibitor (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody), administered as a monotherapy or as part of a combination therapy.

137. The method of any of the preceding claims, wherein the last prior treatment was with a PD1 inhibitor or a PD-L1 inhibitor, e.g., an anti-PD1 antibody or an anti-PD-L1 antibody, administered as a monotherapy or as part of a combination therapy.

138. The method of any of the preceding claims, wherein the time to progression from the last treatment with a PD1 inhibitor or a PD-L1 inhibitor (eg, an anti-PD1 antibody or an anti-PD-L1 antibody) is 6 months or less.

139. The method of any of the preceding claims, wherein the time from the last administration of a PD1 inhibitor or PD-L1 inhibitor (e.g., an anti-PD1 antibody or an anti-PD-L1 antibody) as part of the last prior treatment is 6 months or less.

140. The method of any of the preceding claims, wherein the cancer or tumor has relapsed and / or is refractory, or the subject has progressed during or after i) platinum-doublet chemotherapy following treatment with anti-PD1 antibody or anti-PD-L1 antibody, or ii) Treatment with anti-PD1 antibody or anti-PD-L1 antibody after platinum doublet chemotherapy.

141. A kit comprising i) a binding agent comprising at least one binding region that binds CD27, and ii) PD1 / PD-L1 inhibitors.

142. The kit of claim 141, wherein the binding agent is as defined in any one of claims 1-140 and / or the PD1 / PD-L1 inhibitor is as defined in any one of claims 1-140.

143. The kit according to claim 141 or 142, wherein the binding agent, the PD1 / PD-L1 inhibitor and (if present) one or more additional therapeutic agents are for systemic administration, in particular for injection or infusion, such as intravenous injection or infusion.

144. A kit according to any one of claims 141-143, for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject.

145. A kit for use according to claim 144, wherein the tumor or cancer is as defined in any one of claims 1-140, and / or the subject is as defined in any one of claims 1-140, and / or the method is as defined in any one of claims 1-140.

146. A pharmaceutical composition comprising i) a binding agent comprising at least one binding region that binds CD27; ii) PD1 / PD-L1 inhibitors; and iii) optionally a pharmaceutically acceptable carrier.

147. The kit of claim 146, wherein the binding agent is as defined in any one of claims 1-140 and / or the PD1 / PD-L1 inhibitor is as defined in any one of claims 1-140.

148. A pharmaceutical composition according to claim 146 or 147 for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject.

149. A pharmaceutical composition for use according to claim 148, wherein the tumor or cancer is as defined in any one of claims 1-140, and / or the subject is as defined in any one of claims 1-140, and / or the method is as defined in any one of claims 1-140.

150. A binding agent for use in a method of reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) the binding agent comprising at least one binding region that binds CD27; and ii) a PD1 / PD-L1 inhibitor.

151. A kit for use according to claim 150, wherein the method is as defined in any one of claims 1-140, and / or the binding agent is as defined in any one of claims 1-140, and / or the PD1 / PD-L1 inhibitor is as defined in any one of claims 1-140.

152. A PD1 / PD-L1 inhibitor for use in a method for reducing tumor progression or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject i) a binding agent comprising at least one binding region that binds CD27; and ii) the PD1 / PD-L1 inhibitor.

153. A PD1 / PD-L1 inhibitor for use according to claim 152, wherein the method is as defined in any one of claims 1-140, and / or the binding agent is as defined in any one of claims 1-140, and / or the PD1 / PD-L1 inhibitor is as defined in any one of claims 1-140.

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