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

Abstract

The present invention provides a combination therapy using a binder comprising at least one binding region that binds to CD27 in combination with a PD1 / PD-L1 inhibitor for reducing or preventing tumor progression or treating cancer. TIFF2025516631000048.tif66170

Description

[Technical field]

[0001] The present invention relates to combination therapies using binding agents that comprise at least one binding region that binds to CD27 in combination with PD1 / PD-L1 inhibitors to reduce or prevent tumor progression or to treat cancer. [Background technology]

[0002] background 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 costimulates T cell activation after binding to its ligand CD70. In humans, it is expressed on the plasma membrane of T, B, and 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 the plasma membrane of resting αβ CD4 + (Treg and conventional T cells), CD8 + It 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 quite restrictively and only transiently on activated immune cells, including T, B, NK, 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 signaling resulting from CD27 engagement.

[0005] To release their effector functions, T cells require T cell antigen receptor-mediated recognition of their cognate antigen in the context of major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs), and activation of costimulatory receptors. CD27 and CD28 are thought to be the most important costimulatory receptors expressed on T cells.

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

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

[0008] In the treatment of cancer, the engagement and stimulation of immune response has been shown to induce and / or enhance antitumor immunity, resulting in clinical responses, as exemplified by the clinical success of immune checkpoint inhibitors (CPIs). Active immune response and / or existing antitumor immunity can be increased by providing costimulatory signaling, for example CD27 costimulatory signaling.

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

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

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

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

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

[0014] WO2019 / 195452 (Patent Document 4) discloses a non-ligand blocking agonist anti-human CD27 antibody designated BMS-986215, which is reported to have a higher affinity for human and cynomolgus CD27 than the CD27 antibody 1F5 described above. It is disclosed that in the presence of BMS-986215, CD27 costimulation of T cells by binding to its ligand CD70 occurs. BMS-986215 inhibits CD4+ expression by regulatory T cells (Tregs). + It is further disclosed that BMS-986215 reduces the suppression of responder T cells, and that BMS-986215 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 FcyR and in the absence of sCD70.

[0015] Cancer cells can evade and suppress immune responses through upregulation of 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, and thus restoring T cell function, has provided a breakthrough therapy for cancer.

[0016] PD-1 (also known as CD279) is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. The protein PD-1 has two naturally occurring ligands known as PD-L1 (also known as CD274) and PD-L2 (also known as CD273). A variety of cancers express PD-L1, including melanoma, lung, kidney, bladder, esophagus, stomach, and other cancers. Thus, upon interaction of PD-L1 with PD-1 in cancer, the PD-1 / PD-L1 system can inhibit T lymphocyte proliferation, cytokine release, and cytotoxicity, thereby providing cancer cells with an opportunity to evade T cell-mediated immune responses.

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

[0018] Anti-CD27 antibodies must induce CD27 clustering on the plasma membrane to induce CD27 agonism. In the case of wild-type IgG1 antibodies, CD27 clustering can be achieved through the interaction of membrane-bound CD27 antibodies with FcyR-bearing cells, such as monocytes, macrophages, B cells and other immune cells. As a consequence, anti-CD27 IgG1 molecules may be less efficient when the number of FcyR-expressing cells is limited. Optimizing effector function by modifying the Fc region of antibodies can improve the effectiveness of therapeutic antibodies for treating cancer or other diseases, for example, improving the ability of the antibody to induce an immune response against antigen-expressing cells.Such efforts are described, for example, in WO 2013 / 004842 A2 (Patent Document 5); WO 2014 / 108198 A1 (Patent Document 6); WO2018 / 146317 (Patent Document 7); WO2018 / 083126 (Patent Document 8); WO 2018 / 031258 A1 (Patent Document 9); Dall'Acqua, Cook et al. J Immunol 2006, 177(2): 1129-1138 (Non-Patent Document 7); Moore, Chen et al. MAbs 2010 2(2): 181-189 (Non-Patent Document 8); Desjarlais and Lazar, Exp Cell Res 2011, 317(9): 1278-1285 (Non-Patent Document 9); Kaneko and Niwa, BioDrugs 2011,25(1):1-11(Non-patent document 10);Song, Myojo et al.,Antiviral Res 2014,111:60-68(Non-patent document 11);Brezski and Georgiou,Curr Opin Immunol 2016,40:62-69(Non-patent document 12);Sondermann and Szymkowski,Curr Opin Immunol 2016,40:78-87(Non-patent document 13);Zhang, Armstrong et al.MAbs 2017,9(7):1129-1142.(Non-patent document 14);Wang,Mathieu et al.Protein&Cell 2018,9(1):63-73(Non-patent document 15);Diebolder FJ et al.,Science.2014 Mar 14;343(6176):1260-3 (Non-Patent Document 16)).

[0019] By activating the immune system, immune CPIs may also cause autoimmune side effects in some patients. Additionally, engagement of the Fc domain with Fc receptors or components of the complement system may also result in undesirable effector functions, such as activation of ADCC, ADCP, and CDC, which may cause undesirable depletion of CD27-positive T cells. Thus, activation of Fc-mediated effector functions may be undesirable in the context of monoclonal antibodies that block PD-1 / PD-L1 interaction. A wide range of IgG antibody formats have been developed that contain Fc domains that do not engage Fc receptors and / or the complement system, 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 (Non-Patent Document 17); Liu et al., Antibodies, 2020 Nov 17;9(4):64;29(10):457-66 (Non-Patent Document 18)). Examples of such substitutions include the introduction of an L234A-L235A-P329G inactivating mutation (Schlothauer et al., Protein Eng. Design and Selection 2016;29(10):457-66 (Non-Patent Document 19)), or an L234F-L235E-D265A inactivating mutation (also referred to herein as FEA or FEA-style; Engelberts et al., EBioMedicine 2020;52:102625 (Non-Patent Document 20); US10590206B2 (Patent Document 10)).Other deactivating modes have been developed using one of the human IgG subclasses with reduced effector function, human IgG4, in combination with amino acid substitutions in the constant heavy chain region of the antibody to further eliminate Fc-mediated effector functions (e.g. introduction of the E233P-F234V-L235A-G236del deactivating mutations described in WO2015 / 143079 (Patent Document 11) or introduction of the F234A-L235A deactivating mutations described by Vafa et al. Methods 2014;65:114-126 (Non-Patent Document 21)).

[0020] Among others, Garber et al. have discussed the opportunity for combination therapy consisting of agonistic antibodies targeting costimulatory receptors on T cells, such as 4-1BB (CD137), OX40, glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR) and independent co-stimulation (ICOS), as well as monoclonal antibodies blocking the PD-1 / PD-L1 axis (Garber et al. Nat Rev Drug Discov. 2020 Jan;19(1):3-5 (Non-Patent Document 22)). Azpilikueta et al. (J Thorac Oncol 2016;11:524-36) have published preclinical data from a combination therapy including a PD-1 blocking antibody and a 4-1BB targeting antibody in a mouse lung cancer model showing that the combination therapy outperformed single agent treatment.

[0021] WO2008 / 051424A2 (Patent Document 12) provides methods that include the administration of a CD27-targeting agonist antibody alone or in combination with other immunomodulatory agents, such as antibodies targeting CD40, OX40, 4-1BB or CTLA-4.

[0022] US10668152B2 (Patent Document 13) 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 (Non-Patent Document 24)).

[0024] WO2018 / 127916 (Patent Document 14) provides a PD1-CD70 dual signal fusion protein based on MIRP technology (Multifunctional Immune Recruitment Protein) (DSP-106).

[0025] WO2015 / 016718A1 (Patent Document 15) describes a method for preventing CD27 by administering an anti-CD27 antibody in combination with an antibody that blocks the PD1 / PD-L1 interaction. + Treatment of any condition known or predicted to be ameliorated by stimulation of immune cells or by inhibition of one or more immune checkpoint proteins is provided.

[0026] Despite these and other efforts in the art, however, there remains a need for improved antibody-based immunotherapies with increased agonism and / or increased potency for engaging CD27, provided together as combination therapies with other immune-modulating antibodies or antibodies that block immune checkpoints. [Prior art documents] [Patent documents]

[0027] [Patent Document 1] WO2012 / 004367 [Patent Document 2] WO2011 / 130434 [Patent Document 3] WO2018 / 058022 [Patent Document 4] WO2019 / 195452 [Patent Document 5] WO 2013 / 004842 A2

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Non-licensed literature

[0028] [Non-licensed document 1] Carr JM et al,Proc Natl Acad Sci USA 2006 Dec 19;130(51):19454-9 [Non-licensed document 2] Van de Ven K,Borst J.Immunotherapy 2015;7(6):655-67 [Non-licensed document 3] Oosterwijk et al,Int Immunol.2007 Jun;19(6):713-8

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[0029] The present invention relates to binding agents capable of binding to CD27 in combination therapy.

[0030] In a first aspect, the disclosure provides a method for reducing or preventing tumor progression or treating cancer in a subject comprising administering to the subject: i) a binding agent comprising at least one binding region that binds to CD27; and ii) a PD1 / PD-L1 inhibitor.

[0031] In a second aspect, the disclosure provides a kit comprising i) a binding agent comprising at least one binding region that binds to CD27 and ii) a PD1 / PD-L1 inhibitor.

[0032] In a third aspect, the present disclosure provides a kit for use in a method for reducing or preventing the progression of a tumor or treating cancer in a subject, the kit comprising i) a binding agent comprising at least one binding region that binds to CD27 and ii) a PD1 / PD-L1 inhibitor.

[0033] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising: i) a binding agent comprising at least one binding region that binds to CD27; ii) a PD1 / PD-L1 inhibitor; and iii) optionally, a pharma- ceutically acceptable carrier.

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

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

[0036] In a seventh aspect, the present disclosure provides a PD1 / PD-L1 inhibitor for use in a method for reducing or preventing the progression of a tumor or treating cancer in a subject, the method comprising the steps of: i) a binding agent comprising at least one binding region that binds to CD27; and ii) administering to the subject the PD1 / PD-L1 inhibitor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description of the Invention definition The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, having the ability to specifically bind to an antigen. The antibody of the present invention comprises an Fc domain and an antigen-binding region of an immunoglobulin. An antibody generally contains two CH2-CH3 regions and a connecting region, e.g., a hinge region, e.g., at least an Fc domain. Thus, the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant or "Fc" region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, e.g., C1q, the first component in the classical pathway of complement activation. As used herein, unless inconsistent with the context, the Fc region of an immunoglobulin typically contains at least the CH2 and CH3 domains of an immunoglobulin CH, and may include a connecting region, e.g., a hinge region. The Fc region is typically in a dimerized form, for example, via a disulfide bridge connecting the two hinge regions and / or a non-covalent interaction between the two CH3 regions. The dimer may be a homodimer (the amino acid sequences of the two Fc region monomers are identical) or a heterodimer (the amino acid sequences of the two Fc region monomers differ in one or more amino acids). As is well known in the art, Fc region fragments of full-length antibodies can be generated, for example, by digestion of full-length antibodies with papain. In addition to the Fc region and the antigen-binding region, an antibody as defined herein may further comprise one or both of the immunoglobulin CH1 and CL regions. The antibody may also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody that has specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. As indicated above, not otherwise stated,Or, unless the context clearly contradicts, the term antibody in this specification includes fragments of antibodies that contain at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "Ab" or "antibody" are monovalent antibodies (described by Genmab in WO2007059782); heavy chain antibodies, which consist of only two heavy chains and occur naturally, for example in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMab, Roche, WO2011069104); strand-exchange engineered domain (SEED or Seed-body), which is an asymmetric and bispecific antibody-like molecule (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792); Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768); mAb-Fv (Xencor, WO2011 / 028952); Xmab (Xencor); Dual variable domain immunoglobulins (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); Dual domain dual head antibodies (Unilever; Sanofi Aventis, WO20100226923); Di-diabody (ImClone / Eli Lilly); Knob-into-hole antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545); DuetMab (MedImmune, US2014 / 0348839); electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai,US201000155133;Oncomed, WO2010129304A2;Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545);CrossMAb (Roche, WO2011117329);LUZ-Y (Genentech);Biclonic (Merus, WO2013157953);Dual targeting domain antibodies (GSK / Domantis);Two-in-one antibodies or dual acting Fabs recognizing two targets (Genentech, NovImmune, Adimab);Crosslinked Mabs (Karmanos Cancer Center);Covalently fused mAbs (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); Zybodies (Zyngenia); approaches using common light chains (Crucell / Merus, US7262028) or common heavy chains (κλBodies by NovImmune, WO2012023053), as well as fusion proteins comprising polypeptide sequences fused to antibody fragments containing Fc region-like scFv fusions, e.g. BsAb by ZymoGenetics / BMS,HERCULES by Biogen Idec (US007951918); SCORPIONS (Emergent BioSolutions / Trubion and Zymogenetics / BMS); Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol,2009.393(3):p.672-92);scFv 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. The term antibody, unless otherwise specified, should be understood to include 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; antibody mixtures (recombinant polyclonal), such as those produced by the technology utilized 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 formats are generally included within the meaning of antibody, they are unique features of the present invention collectively and each independently and exhibit different biological properties and utility.

[0038] An "agonist antibody" for a natural receptor is a compound that binds to the receptor to form a receptor-antibody complex and activates the receptor, thereby initiating pathway signaling and further biological processes.

[0039] The terms "agonism" and "agonistic" are used interchangeably herein to refer to or describe an antibody that has the ability to substantially induce, promote, or enhance, directly or indirectly, the biological activity or activation of CD27. Optionally, an "agonistic CD27 antibody" is an antibody that has the ability to activate the CD27 receptor by a mechanism similar to that of the ligand of CD27, known as CD70 (tumor necrosis factor superfamily member 7, TNFSF7; CD27 ligand, CD27L), resulting in the activation of one or more intracellular signaling pathways, which may include activation of the NF-KB and MAPK8 / JNK pathways. "Agonism" as defined herein may be determined according to Example 2 herein.

[0040] A "CD27 antibody" or "anti-CD27 antibody" as described herein is an antibody that specifically binds to the protein CD27, in particular human CD27.

[0041] "Variant" as used herein refers to a protein or polypeptide sequence that differs from a parent or reference sequence in one or more amino acid residues. A variant may, for example, have at least 80%, for example 90%, or 95%, or 97%, or 98%, or 99% sequence identity to a parent or reference sequence. Additionally or alternatively, a variant may differ from a parent or reference sequence by 12 or less, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, for example substitution, insertion, or deletion of amino acid residues. Thus, as used interchangeably herein, "variant antibody" or "antibody variant" refers to an antibody that differs in one or more amino acid residues compared to a parent or reference antibody, for example in the antigen binding region, Fc region, or both. Similarly, a "variant Fc region" or "Fc region variant" refers to an Fc region that differs in one or more amino acid residues compared to a parent or reference Fc region, optionally differing from the parent or reference Fc region amino acid sequence by 12 or fewer, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, e.g., substitution, insertion, or deletion of amino acid residues. The parent or reference Fc region is typically the Fc region of a human wild-type antibody, which may be of a particular isotype depending on the context. The variant Fc region may be in a dimerized form, homodimer or heterodimer, e.g., one of the amino acid sequences of the dimerized Fc region contains a mutation and the other is identical to the parent or reference wild-type amino acid sequence. Examples of wild-type (typically parent or reference sequence) IgG CH and variant IgG constant region amino acid sequences, including Fc region amino acid sequences, are shown in Table 3.

[0042] The term "immunoglobulin heavy chain" or "heavy chain of immunoglobulin" as used herein is intended to refer to one of the heavy chains of immunoglobulins. Heavy chains typically include 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 typically includes three domains, CH1, CH2, and CH3. The term "immunoglobulin" as used herein is intended to refer to a class of structurally related glycoproteins that consists of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four potentially 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 through disulfide bonds in the so-called "hinge region". Like the heavy chain, each light chain typically 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. Furthermore, the VH and VL regions can be further divided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or shape of structurally defined loops), also referred to as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences herein are defined according to IMGT unless otherwise stated or contradicted by context (see Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)).

[0043] As used herein, the terms "half molecule", "Fab arm" and "arm" refer to one of the heavy-light chain pairs. When a bispecific antibody is described as comprising a half molecule antibody "derived from" a first antibody and a half molecule antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody was generated by recombining the half molecules from each of the first and second antibodies into the resulting bispecific antibody by any known method. In this context, "recombining" is not intended to be limited by any particular method of recombination, and thus includes all of the methods for producing bispecific antibodies described herein below, including recombination at the nucleic acid level and / or through co-expression of two half molecules in the same cell, in addition to recombination by "half molecule exchange", also described in the art as "Fab arm exchange" and the DuoBody® method.

[0044] The term "antigen-binding region" or "binding region" or antigen-binding domain, as used herein, refers to the region of an antibody that has the ability to bind to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which can be further divided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or shape of structurally defined loops), also referred to as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). The antigen can be any molecule, e.g., a polypeptide, present, for example, on a cell, bacteria, or virion. The terms "antigen-binding region" and "antigen-binding site" and "antigen-binding domain" may be used interchangeably in the context of the present invention, unless the context is inconsistent.

[0045] The terms "antigen" and "target" may be used interchangeably in the context of the present invention, unless the context is contradictory.

[0046] The term "binding" as used herein refers to a binding that is typically greater than 1E when determined by biolayer interferometry using an antibody as the ligand and an antigen as the analyte. 6 M or less, e.g. 5E 7 M or less, 1E 7 M or less, e.g. 5E 8 M or less, e.g. 1E 8 M or less, e.g. 5E 9 M or less, or for example 1E 9 M or less K D and which is at least 10-fold lower, e.g., at least 100-fold lower, e.g., at least 1,000-fold lower, e.g., at least 10,000-fold lower, e.g., at least 100,000-fold lower, e.g., at least 100,000-fold lower, than its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). D The antibody binds to a predetermined antigen with an affinity corresponding to

[0047] The term “K D " (M) as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, k d k a It is obtained by dividing by

[0048] The term "k" d " (sec -1 ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. The value is also referred to as k off This is referred to as the off-value or off-rate.

[0049] The term "k" a " " -1 ×sec -1 ) as used herein refers to the association rate constant of a particular antibody-antigen interaction. The value is also referred to as k on This is referred to as the on-rate or on-value.

[0050] The term "CD27" as used herein refers to a human protein designated CD27, also known as tumor necrosis factor receptor superfamily member 7 (TNFRSF7). In the amino acid sequence shown in SEQ ID NO:1 (Uniprot ID P26842), amino acid residues 1-19 are the signal peptide and amino acid residues 20-240 are the mature polypeptide. Unless contradicted by the context, CD27 may also refer to variants of CD27, its isoforms and orthologs. A naturally occurring variant of human CD27 containing the A59T mutation is shown in SEQ ID NO:2.

[0051] In the cynomolgus monkey (Macaca fascicularis), the CD27 protein has the amino acid sequence shown in SEQ ID NO:3 (Genbank XP_005569963). No signal peptide is defined in the 240 amino acid sequence shown in SEQ ID NO:3.

[0052] The term "antibody binding region" refers to the region of an antigen that contains the epitope to which the antibody binds. The antibody binding region may be determined by epitope binding using biolayer interferometry, by alanine scanning, or by shuffle assays (using an antigen construct in which a region of the antigen has been exchanged with that of another species and determining whether the antibody still binds to the antigen). Amino acids within the antibody binding region involved in interaction with the antibody may be determined by hydrogen / deuterium exchange mass spectrometry and by crystallography of the antibody bound to the antigen.

[0053] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids, sugar side chains or combinations thereof and usually have specific charge characteristics as well as specific three-dimensional structural features. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents while the binding to the latter is not. Epitopes may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked or covered by an antibody when it is bound to an antigen (in other words, amino acid residues that are within or closely adjacent to the footprint of a unique antibody).

[0054] The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", or "mAb", as used herein, refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies may be produced by hybridomas comprising B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse or rat, whose genome includes a human heavy chain transgene and a light chain transgene, fused to an immortalized cell. Monoclonal antibodies may also be produced from recombinantly engineered host cells or from systems using cell extracts that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

[0055] The term "isotype" as used herein refers to an immunoglobulin class (e.g., IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotypes thereof, such as IgG1m(za) and IgG1m(f), encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be paired with either a kappa (κ) or lambda (λ) light chain.

[0056] The term "full-length antibody" as used herein indicates that the antibody contains all of the domains of a particular isotype that are normally found for that isotype in nature, not fragments, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains for an IgG1 antibody. In a full-length variant antibody, the heavy and light chain constant and variable domains may contain amino acid substitutions that improve the functional properties of the antibody when compared to the full-length parent or wild-type antibody. A full-length antibody according to the invention may be produced by a method comprising (i) cloning the CDR sequences into a suitable vector containing the complete heavy and light chain sequences, and (ii) expressing the complete heavy and light chain sequences in a suitable expression system. It is within the knowledge of the skilled artisan to produce a full-length antibody when starting from either the CDR sequences or the entire variable region sequences. Thus, the skilled artisan knows how to generate a full-length antibody according to the invention.

[0057] The term "human antibody" as used herein is intended to include antibodies that contain variable and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibody of the present invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as mice, have been grafted onto human framework sequences.

[0058] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen-binding site onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). To fully reconstitute the binding affinity and specificity of the parent antibody, substitution (backmutation) of framework residues from the parent antibody (i.e., non-human antibody) into the human framework region may be required. Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily back mutations, may be applied to obtain a humanized antibody with favorable characteristics, such as affinity and biochemical properties.

[0059] The terms "Fc region" or "Fc domain" as used herein may be used interchangeably and refer to a region of the heavy chain constant region that includes, from the N-terminus to the C-terminus of an antibody, at least the hinge, CH2 and CH3 regions. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0060] The term "parent polypeptide" or "parent antibody" should be understood as a polypeptide or antibody that is identical to the polypeptide or antibody of the present invention, except that the parent polypeptide or antibody does not have the mutation, unless otherwise stated or clearly contradicted by the context. For example, the antibody IgG1-CD27-A of the present invention is a parent antibody of IgG1-CD27-A-P329R-E345R.

[0061] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the Eu numbering (Eu index) as set forth 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 of any of the other subtypes described herein.

[0062] The term "CH1 region" or "CH1 domain" as used herein refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering set forth in Kabat, supra. However, the CH1 region may also be of any of the other subtypes described herein.

[0063] The term "CH2 region" or "CH2 domain" as used herein refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering set forth in Kabat, supra. However, the CH2 region may also be of any of the other subtypes described herein.

[0064] The term "CH3 region" or "CH3 domain" as used herein refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering set forth in Kabat, supra. However, the CH3 region may also be of any of the other subtypes described herein.

[0065] The term "Fc-mediated effector function" or "Fc effector function", as used herein, is used interchangeably and is intended to refer to a function that is a consequence of binding of a polypeptide or antibody to a target or antigen on a cell membrane, where the Fc-mediated effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc gamma receptor (FcγR) binding, (vi) antibody-dependent, FcγR-mediated antigen cross-linking, (vii) antibody-dependent cellular phagocytosis (ADCP), (viii) complement-dependent cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x) antibody-mediated binding of opsonized antibodies to complement receptors, (xi) opsonization, and (xii) any combination of (i)-(xi).

[0066] The terms "reduced Fc effector function" or "reduced Fc-mediated effector function," as used herein, are used interchangeably and are intended to refer to an Fc effector function that is reduced for an antibody when directly compared to the Fc effector function of a parent polypeptide or antibody in the same assay.

[0067] The term "inertness", "inert" or "non-activating" as used herein refers to an Fc region that at least cannot bind to any FcγR, cannot induce Fc-mediated cross-linking of FcγR, cannot induce FcγR-mediated cross-linking of target antigens through two Fc regions of individual antibodies, or cannot bind to C1q.Therefore, in certain embodiments of the present invention, the Fc region is inactive.Therefore, in certain embodiments, some or all of the Fc-mediated effector functions are attenuated or completely absent.

[0068] The term "oligomerization" as used herein is intended to refer to the process of converting monomers to a finite degree of polymerization. The antibodies according to the invention can form oligomers, e.g., hexamers, via non-covalent association of Fc regions after target binding, e.g., at the cell surface. Oligomerization of anti-CD27 antibodies upon cell surface binding through Fc:Fc interactions may increase CD27 clustering, which results in activation of CD27 intracellular signaling. The ability of antibodies comprising E345R or E430G mutations to form oligomers, e.g., hexamers, upon 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 after target binding on the (cell) surface through intermolecular association of Fc regions between adjacent antibodies, and is increased by the introduction of E345R or E430G mutations (numbering according to the Eu index).

[0069] The term "clustering," as used herein, refers to the oligomerization of antibodies through non-covalent interactions.

[0070] The term "Fc-Fc enhancing" as used herein is intended to refer to increasing the binding strength between the Fc regions of two Fc region-containing antibodies or stabilizing the interaction between the Fc regions such that the antibodies form oligomers, e.g., hexamers, on the cell surface. This enhancement can be obtained by certain amino acid mutations in the Fc region of the antibody, e.g., E345R or E430G. The term "monovalent antibody" in the context of the present invention refers to an antibody molecule that can interact with a specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm). In the context of a bispecific antibody, "monovalent antibody binding" refers to the binding of the bispecific antibody to one specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm).

[0071] The term "monospecific antibody" in the context of the present invention refers to an antibody that has binding specificity for only one epitope. The antibody may be a monospecific, monovalent antibody (i.e., having only one antigen-binding region) or a monospecific, bivalent antibody (i.e., having two identical antigen-binding regions).

[0072] The term "bispecific antibody" refers to an antibody that comprises two non-identical antigen-binding domains, such as two non-identical Fab arms or two Fab arms with non-identical CDR regions. In the context of the present invention, a bispecific antibody has specificity for at least two different epitopes. Such epitopes may be on the same or different antigens or targets. When the epitopes are on different antigens, such antigens may be on the same cell or different cells, cell types or structures, such as extracellular matrix or vesicles and soluble proteins. A bispecific antibody may therefore have the ability to crosslink multiple antigens, such as two different cells. A particular bispecific antibody of the present invention has the ability to bind to CD27 and a second target.

[0073] The term "bivalent antibody" refers to an antibody that has two antigen-binding regions that bind to epitopes on one or two targets or antigens, or that bind to one or two epitopes on the same antigen. Thus, a bivalent antibody may be a monospecific bivalent antibody or a bispecific bivalent antibody.

[0074] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds that contain amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) that is specific to each amino acid. In the context of the present invention, amino acids can be classified based on structure and chemical characteristics. Thus, the classes of amino acids can be reflected in one or both of the following tables:

[0075] Table 20. Main classifications based on structure and general chemical characterization of the R group TIFF2025516631000002.tif47128

[0076] Table 21. Alternative physical and functional classifications of amino acid residues TIFF2025516631000003.tif106128

[0077] The substitution of one amino acid for another amino acid may be classified as conservative or non-conservative substitution.In the context of the present invention, "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics, for example, the substitution of one amino acid residue with another amino acid residue of the same class as defined in either of the above two tables: for example, leucine may be substituted with isoleucine, because they are both aliphatic, branched, hydrophobic substances.Similarly, aspartic acid may be substituted with glutamic acid, because they are both small, negatively charged residues.

[0078] In the context of the present invention, substitutions in an antibody include Original amino acid - position - substituted amino acid Pointed out as; Reference is made to the well-recognized notation, three-letter code, or one-letter code for amino acids, including the code "Xaa" or "X" to indicate any amino acid residue. Thus, Xaa or X can typically represent any of the 20 naturally occurring amino acids. The term "naturally occurring" as used herein refers to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Thus, the notation "K409R" or "Lys409Arg" means that the antibody comprises a substitution of lysine with arginine at amino acid position 409.

[0079] Substitution of an amino acid at a given position with any other amino acid is the original amino acid-position; or for example "K409" It is called.

[0080] For modifications in which the original and / or substituted amino acids may include more than one, but not all, amino acids, the more than one amino acids may be separated by "," or " / ". For example, substitution of lysine at position 409 with arginine, alanine, or phenylalanine is "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409→R, A, or F".

[0081] Such designations may be used interchangeably in the context of the present invention and may have the same meaning and purpose.

[0082] Moreover, the term "substitution" encompasses substitutions to any one or the other of the 19 natural amino acids or to other amino acids, e.g., unnatural amino acids. For example, substitution of the 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. Note that this is equivalent to the designation 409X, where X designates any amino acid other than the original amino acid. These substitutions may also be designated K409A, K409C, etc., or K409A,C, etc., or K409A / C / , etc. The same applies to each and every position mentioned herein, and any such substitutions are specifically included herein.

[0083] Antibodies according to the invention may also include deletions of amino acid residues. Such deletions may be designated as "del", and include, for example, the designation K409del. Thus, in such embodiments, the lysine at position 409 is deleted from the amino acid sequence.

[0084] The term "host cell" as used herein is intended to refer to a cell into which an expression vector is introduced. It should be understood that such a term is intended to refer not only to a specific target cell, but also to the descendants of such a cell. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such descendants may not actually be identical to the parent cell, but still fall within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytic cells, as well as prokaryotic cells, such as E. coli, and other eukaryotic hosts, such as plant cells and fungi.

[0085] The term "transfectoma" as used herein includes recombinant eukaryotic host cells expressing an antibody or a target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungi, including yeast cells.

[0086] For the purposes of the present invention, sequence identity between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled as "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (identical residues × 100) / (length of alignment − total number of gaps in alignment) It is calculated as follows:

[0087] Retention of similar residues may additionally or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through NCBI using standard settings BLOSUM62, open gap = 11 and 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 higher (e.g., about 99%) similarity to the parent sequence.

[0088] The term "internalized" or "internalization," as used herein, refers to the biological process by which a molecule, such as an antibody according to the present invention, is engulfed by the cell membrane and directed to the interior of the cell. Internalization may also be referred to as "endocytosis."

[0089] As used herein, the term "effector cell" refers to immune cells that participate in the effector phase of 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 (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcgR) or complement receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, have the ability to induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells that express FcgR are involved in the specific killing of target cells and / or the presentation of antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, ADCC can be further enhanced by antibody-driven classical complement activation, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote the enhancement of Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products can promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells, which depends on antibody binding and can be mediated by FcγRs expressed by effector cells. The expression of certain FcRs or complement receptors on effector cells can be regulated by humoral factors, such as cytokines. For example, expression of FcγRI has been found to be upregulated by interferon gamma (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells.These C3 cleavage products can promote direct phagocytosis by effector cells or indirectly by enhancing antibody-mediated phagocytosis. In certain embodiments herein where the antibody has an inactive Fc region, the antibody does not induce Fc-mediated effector function.

[0090] "Effector T cells" or "Teffs" or "Teff" as used herein refers to T lymphocytes that carry out functions of the immune response, such as activating an anti-tumor immune response that can result in the killing of tumor cells and / or the clearance of tumor cells from the body. An example of a Teff phenotype is the CD3 + CD4 + and CD3 + CD8 + Teffs may secrete, contain, or express markers such as IFNγ, Granzyme B, and ICOS. It is understood that Teffs may not be entirely restricted to these phenotypes.

[0091] "Memory T cells", as used herein, refer to T lymphocytes that remain in the body for a long period of time after infection has been cleared. Examples of memory T cells include central memory T cells (CD45RA-CCR7+) and effector memory T cells (CD45RA-CCR7-). It is understood that memory T cells may not be completely restricted to these phenotypes.

[0092] "Regulatory T cells" or "Tregs" or "Treg" as used herein refers to T lymphocytes that regulate the activity of other T cells and / or other immune cells, usually by suppressing their activity. An example of a Treg phenotype is the CD3 + CD4 + CD25 + CD127dim. Tregs may further express Foxp3. It is understood that Tregs may not be completely restricted to this phenotype.

[0093] As used herein, the term "complement activation" refers to the activation of the classical complement pathway, which is initiated by a large macromolecular complex called C1, which binds to an antibody-antigen complex on a surface. C1 is a complex consisting of six recognition proteins C1q and a heterotetramer of serine protease, C1r2C1s2. C1 is the first protein complex in the early events of the classical complement cascade, with a series of cleavage reactions beginning with the cleavage of C4 into C4a and C4b, and C2 into C2a and C2b. C4b is deposited and forms an enzymatically active convertase called C3 convertase with C2a, which cleaves the complement component C3 into C3b and C3a, which allows the formation of C5 convertase. This C5 convertase splits C5 into C5a and C5b, the last component is deposited on the membrane, and then triggers 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 the cell membrane, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). In certain embodiments herein where the antibody has an inactive Fc region, the antibody does not induce complement activation.

[0094] Complement activation can be assessed by using C1q binding efficiency, CDC kinetics, CDC assays (described in WO2013 / 004842, WO2014 / 108198), or by the method of cellular deposition of C3b and C4b described in Beurskens et al., J Immunol April 1, 2012 vol.188 no.7, 3532-3541.

[0095] The term "C1q binding" as used herein is intended to refer to the binding of C1q in the context of the binding of C1q to an antibody bound to its antigen. Antibody bound to its antigen should be understood to occur both in vivo and in vitro in the context described herein. As described in Example 8 herein, C1q binding can be assessed, for example, by using an antibody immobilized on an artificial surface, or by using an antibody bound to a predetermined antigen on a cell or virion surface. The binding of C1q to an antibody oligomer should be understood herein as a multivalent interaction that results in high avidity binding. The reduction in C1q binding, for example resulting from the introduction of a mutation in an antibody of the present invention, may be measured by comparing the C1q binding of the mutated antibody with the C1q binding of its parent antibody (the antibody of the present invention without the mutation in the same assay).

[0096] The term "treatment" refers to the administration of an effective amount of a therapeutically active antibody of the invention with the goal of alleviating, ameliorating, arresting, or eradicating (curing) the symptoms or disease state.

[0097] The term "effective amount" or "therapeutically effective amount" refers to an amount effective for the dosage and time period necessary to achieve the desired therapeutic result. The therapeutically effective amount of an antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody variant are outweighed by the therapeutically beneficial effects.

[0098] The term "pharmacokinetic profile" as used herein may be determined as plasma IgG levels over time as described in Example 12 herein.

[0099] The term "CD137" as used herein refers to CD137(4-1BB), also referred to as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the receptor for the ligand TNFSF9 / 4-1BBL. CD137(4-1BB) is believed to be involved in T cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CD137, T cell antigen 4-1BB homologue, and T cell antigen ILA. In one embodiment, CD137(4-1BB) is human CD137(4-1BB), which has 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, i.e., amino acids 187-213 and 214-255 of SEQ ID NO:130, are the transmembrane and cytoplasmic domains, respectively.

[0100] "Programmed Death-1 (PD-1) receptor" refers to an immunoinhibitory receptor belonging to the CD28 family.

[0101] The term "PD-L1", as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, such as macaque (Macaca fascicularis), African elephant, wild boar, and mouse PD-L1 (see, for example, Genbank Accession Nos. NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), and analogs that share at least one epitope in common with hPD-L1. The sequence of human PD-L1 is also shown in SEQ ID NO:98 (mature sequence), and SEQ ID NO:129, with amino acids 1-18 predicted to be a signal peptide. The term "PD-L2" as used herein includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs that have 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. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 can switch off T cells expressing PD-1, resulting in suppression of anti-cancer immune responses. The interaction between PD-1 and its ligands results in a reduction in tumor-infiltrating lymphocytes, a reduction in T cell receptor-mediated proliferation, and immune escape by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is also blocked.

[0102] The term "PD-1" refers to programmed cell death-1 and includes any variant, conformation, isoform and species homologue of PD-1 expressed naturally by cells or expressed by cells transfected with the PD-1 gene. Preferably, "PD-1" refers to human PD-1, in particular the protein having the amino acid sequence set forth in SEQ ID NO:58 of the Sequence Listing (NCBI Reference Sequence: NP_005009.2), or more preferably encoded by the nucleic acid sequence set forth in SEQ ID NO:60 of the Sequence Listing (NCBI Reference Sequence: NM_005018.2). Alternative names for "PD-1" include CD279 and SLEB2.

[0103] 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.

[0104] The term "PD-1 variants" encompasses (i) PD-1 splice variants, (ii) PD-1 post-translational modification variants, including in particular variants with different N-glycosylation states, and (iii) PD-1 conformational variants. Such variants may include soluble forms of PD-1.

[0105] PD-1 is a type I membrane protein belonging to the immunoglobulin superfamily (The EMBO Journal (1992), vol. 11, issue 11, p. 3887-3895). Human PD-1 protein includes an extracellular domain consisting of amino acids 24-170 of the sequence shown in SEQ ID NO: 58 in the sequence listing, a transmembrane domain (amino acids 171-191 of the sequence shown in SEQ ID NO: 58), and a cytoplasmic domain (amino acids 192-288 of the sequence shown in SEQ ID NO: 58). The term "PD-1 fragment" as used herein includes any fragment of the PD-1 protein, preferably an immunogenic fragment. The term also includes, for example, the above domains of the full-length protein or any fragment of these domains, particularly an immunogenic fragment. A preferred amino acid sequence of the extracellular domain of human PD-1 protein is shown in SEQ ID NO: 59 in the sequence listing.

[0106] Fc regions may have a lysine at their C-terminus. The source of this lysine is the naturally occurring sequence found in the human from which these Fc regions are derived. During cell culture production of recombinant antibodies, this terminal lysine can be cleaved off by proteolytic hydrolysis by endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking the C-terminal lysine. For the purpose of antibody production, the DNA encoding this terminal lysine can be omitted from the sequence such that the antibody is produced without the lysine. Antibodies produced from either a nucleic acid sequence that encodes or does not encode a terminal lysine are substantially identical in sequence and function, since the degree of processing of the terminal lysine is typically high, for example, when using antibodies produced in a CHO-based production system (Dick, LWet al. Biotechnol. Bioeng. 2008;100:1132-1143). It is therefore understood that the proteins, e.g., antibodies, of the present invention can be produced with or without encoding or carrying a terminal lysine. It is also understood in accordance with the present invention that a sequence having a terminal lysine, e.g., a constant region sequence having a terminal lysine, can be understood as the corresponding sequence without the terminal lysine, and that a sequence without a terminal lysine can also be understood as the corresponding sequence with a terminal lysine.

[0107] Aspects and embodiments of the present disclosure In a first aspect, the disclosure provides a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject: i) a binding agent that comprises at least one binding region that binds to CD27; and ii) a PD1 / PD-L1 inhibitor.

[0108] Binding agents that bind to CD27 In one embodiment of the invention, the binding agent comprises at least one antigen-binding region capable of binding to human CD27, the binding agent comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:9, 10, and 11, respectively.

[0109] In a further embodiment of the invention, the binding agent comprises two of said antigen binding regions comprising VH regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and VL regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively. According to the invention there is provided an anti-CD27 antibody capable of binding to human CD27 and further binding to a variant of human CD27 comprising the A59T mutation.

[0110] In one embodiment of the present invention, the binding agent binds to CD27, for example, on T cells, and is agonistic in binding to its target.The present invention provides a binding agent that stimulates T cell activation and proliferation.The binding agent may further stimulate T cell memory formation and survival.Such a binding agent is useful, for example, in the treatment of cancer.The binding agent further has the ability to bind to cynomolgus monkey CD27, which is useful for toxicology studies of the binding agent.

[0111] In one embodiment, the binding agent is an isolated antibody.

[0112] 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.

[0113] In a preferred embodiment, the binding agent is a full-length antibody. Thus, the binding agent of the present invention may further comprise a light chain constant region (CL) and a heavy chain constant region (CH). The CH preferably comprises a CH1 region, a hinge region, a CH2 region and a CH3 region.

[0114] It is well known in the art that mutations in the VH and VL of an antibody can be made, for example, to increase the affinity of the antibody for its target antigen, to reduce its potential immunogenicity, and / or to increase the yield of the antibody expressed by a host cell. Thus, in some embodiments, variants of the CDR, VH and / or VL sequences of the binding agents of the invention are also envisaged, in particular binding agents comprising functional variants of the VH and / or VL regions shown in SEQ ID NO:4 and SEQ ID NO:8, respectively. Functional variants may, for example, differ in one or more amino acids 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 proportion (at least about 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent or higher) or 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 variants that differ from the respective parent VH or VL regions by 12 or less, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, e.g., substitution, insertion, or deletion of amino acid residues. Exemplary variants include variants that differ from the VH and / or VL and / or CDR regions of the parent sequence mainly by conservative amino acid substitutions; e.g., 12, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the amino acid substitutions in the variant can be conservative. In a further embodiment of the invention, the binding agent may include up to 1, 2 or 3 mutations in the VH CDR region and / or the VL CDR region, respectively. Such mutations may be substitutions. It is preferred that 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 present invention, which have 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.

[0115] In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 80% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 85% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 90% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 95% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 96% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 97% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 98% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 99% identical to the VH region shown in SEQ ID NO:4. In another embodiment of the invention, the binding agent comprises a VH region comprising the sequence shown in SEQ ID NO:4.

[0116] In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 80% identical to the VH region shown in SEQ ID NO:8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 85% identical to the VH region shown in SEQ ID NO:8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 90% identical to the VH region shown in SEQ ID NO:8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 95% identical to the VH region shown in SEQ ID NO:8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 96% identical to the VH region shown in SEQ ID NO:8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 97% identical to the VH region shown in SEQ ID NO:8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 98% identical to the VH region shown in SEQ ID NO:8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence at least 99% identical to the VH region shown in SEQ ID NO: 8. In another embodiment of the invention, the binding agent comprises a VH region comprising a sequence shown in SEQ ID NO: 8.

[0117] In another embodiment of the invention, 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.

[0118] The binding agent used in the methods of the present invention may comprise a light chain constant region that is a human kappa light chain, hi another embodiment, it may comprise a human lambda light chain constant region.

[0119] The binding agent may preferably further comprise a heavy chain constant region that is a human IgG isotype. It may optionally comprise a modified human IgG constant region. Such a human IgG comprises an Fc region that includes CH2 and CH3 regions. By modifying the IgG constant region in the Fc region, it is possible, for example, to modulate the Fc effector function of the antibody or to increase the Fc-Fc interaction and thereby the tendency of the antibody to form clusters, e.g., 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 one particular embodiment, the IgG is a modified human IgG that comprises one or more amino acid substitutions in the Fc region. In one embodiment, it may be a modified human IgG1 that comprises one or more amino acid substitutions in the Fc region. In a further embodiment of the invention, the IgG1 comprises two or more amino acid substitutions in the Fc region. In one embodiment, the IgG1 Fc region has two amino acid substitutions.

[0120] 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.

[0121] Mutations in amino acid residues at positions corresponding to E430, E345 and S440 in the human IgG1 heavy chain, numbered according to the EU index, can improve the ability of an antibody to induce CDC. Without being bound by theory, it is believed that substituting one or more amino acids at these positions can stimulate antibody oligomerization, thereby modulating Fc-mediated effector functions, for example increasing C1q binding, complement activation, CDC, ADCP, internalization, or other related functions that may provide in vivo efficacy.

[0122] In a further embodiment of the invention, the binding agent is a variant antibody comprising an antigen-binding region and a variant Fc region.

[0123] In certain embodiments, the antibody variant that binds to human CD27 is (a) a heavy chain comprising a 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, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region comprising mutations at one or more of E430, E345 and S440, wherein amino acid residues are numbered according to the EU index; (b) a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11. Includes.

[0124] In certain other embodiments, the antibody variant that binds to human CD27 is (a) a heavy chain comprising a VH region comprising SEQ ID NO:4 and a human IgG1 CH region comprising a mutation at one or more of E430, E345 and S440, wherein the amino acid residues are numbered according to the EU index; and (b) a light chain comprising a VL region comprising SEQ ID NO:8 Includes.

[0125] The variant antibodies of the invention that bind to human CD27 comprise a variant Fc region or a variant human IgG1 CH region comprising mutations at one or more of P329, E430 and E345. Hereinafter, references to mutations in the Fc region may equally apply to mutations in the human IgG1 CH region and vice versa.

[0126] As described herein, the position of the amino acid to be mutated in the Fc region may be given with respect to (i.e., "corresponding to") its position in a naturally occurring (wild-type) human IgG1 heavy chain when numbered according to the Eu index. Thus, if the parent Fc region already contains one or more mutations and / or is, for example, an IgG2, IgG3 or IgG4 Fc region, the position of the amino acid corresponding to an amino acid residue, such as E430, in the human IgG1 heavy chain numbered according to the Eu index may be determined by alignment. In particular, the parent Fc region is aligned with the wild-type human IgG1 heavy chain sequence to identify the residue at the position corresponding to E430 in the human IgG1 heavy chain sequence. Any wild-type human IgG1 constant region amino acid sequence may be useful for this purpose, including any one of the different human IgG1 allotypes shown in Table 3.

[0127] In one embodiment of the invention, the modification in the IgG Fc region induces increased CD27 agonism compared to an identical antibody except comprising a wild-type IgG Fc region of the same isotype, e.g. IgG1. This may be obtained, for example, by introducing an amino acid other than E at the amino acid positions corresponding to positions E345 and / or E430 in the human IgG1 heavy chain according to the Eu numbering. In one embodiment of the invention, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to the Eu numbering is selected from the group comprising 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 the human IgG1 heavy chain according to the Eu numbering is selected from the group comprising A, C, D, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W.

[0128] In a preferred embodiment, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to Eu numbering is R. Thus, the 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 according to Eu numbering is G. Thus, the 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 comprising E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y.

[0129] According to the invention, binding agents are provided in the form of one or more antibodies that have enhanced Fc-Fc interactions that can lead to antibody-dependent clustering of CD27 on the cell surface upon antibody binding, thereby increasing the agonism of the binding agents of the invention.

[0130] In another embodiment of the binding agent used 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 comprising A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y. Thus, the binding agent used according to the invention may further comprise a mutation at position 329.

[0131] In a further embodiment of the invention, the binding agent has an amino acid residue R at a position corresponding to position P329 in a human IgG1 heavy chain according to Eu numbering. Thus, the binding agent of the invention may have a P329R substitution in the Fc region. Without being bound by theory, it is believed that binding agents comprising an E345R mutation in the Fc region (e.g. as shown in SEQ ID NO:13) have increased serum clearance. The inventors have found that further introducing a mutation at position 329, e.g. P329R (e.g. as shown in SEQ ID NO:15), restores the clearance of the binding agent to the level of a binding agent comprising wt IgG1, e.g. as shown in SEQ ID NO:12.

[0132] In another preferred embodiment, the amino acid residues at positions corresponding to positions P329 and E345 in a human IgG1 heavy chain according to Eu numbering are both R. The invention provides binding agents that have increased CD27 receptor agonism and comparable pharmacokinetic properties, such as serum clearance, when compared to binding agents that comprise the same VH and VL regions and that comprise the same IgG1 heavy chain constant region with the exception of the wild-type amino acid P at position 329 and the wild-type amino acid E at position 345.

[0133] Thus, in one embodiment, the binding agent has increased receptor agonism in binding to CD27 and further has pharmacokinetic properties that are comparable, e.g., similar or identical, when compared to the pharmacokinetic properties of a binding agent that comprises the same VH and VL regions but that comprises a wild-type IgG1 heavy chain constant region, such as that shown in SEQ ID NO: 12. In other words, the binding agent may have pharmacokinetic properties that do not differ significantly from the pharmacokinetic properties of an identical binding agent except that it comprises a wild-type IgG1 heavy chain constant region.

[0134] In another embodiment of the invention, the binding agent comprises a variant Fc region according to any one of the preceding sections, wherein the variant Fc region is 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 mutations at one or more of the amino acid residues corresponding to E430 and E345 and P329 are made in a parent Fc region which is 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. Thus, the variant Fc region may be of human IgG1, IgG2, IgG3 or IgG4 isotype, or a mixed isotype thereof, except for the mutations described (at one or more of the amino acid residues selected from E430 and E345 and P329).

[0135] In one embodiment, the parent Fc region and / or human IgG1 CH region is of the wild-type human IgG1 isotype.

[0136] Thus, the variant Fc region may be a human IgG1 Fc region, except for the mutations described (at E430 or E345 or P329).

[0137] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(f) isotype.

[0138] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(z) isotype.

[0139] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(a) isotype.

[0140] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(x) isotype.

[0141] In specific embodiments, the parent Fc region and / or human IgG1 CH region is a human wild-type IgG1 of mixed allotypes, such as IgG1m(za), IgG1m(zax), or IgG1m(fa).

[0142] As such, the variant Fc region and / or human IgG1 CH region may be of the human IgG1m(f), IgG1m(a), IgG1m(x), IgG1m(z) allotype or a mixed allotype of any two or more thereof, except for the mutations described (at E430 or E345 or P329).

[0143] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(za) isotype.

[0144] In a specific embodiment, the parent Fc region is a human wild-type IgG2 isotype.

[0145] In a specific embodiment, the parent Fc region is a human wild-type IgG3 isotype.

[0146] In a specific embodiment, the parent Fc region is a human wild-type IgG4 isotype.

[0147] The CH region amino acid sequences of specific examples of wild-type human IgG isotypes and IgG1 allotypes are shown in Table 3.

[0148] In another embodiment, the binding agent comprises a heavy chain constant region comprising an amino acid sequence selected from the group comprising SEQ ID NO:12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and 36. In one 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.

[0149] In one embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:15; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0150] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:12; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0151] In another embodiment, the first bond is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:13; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0152] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:14; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0153] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:18; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0154] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:19; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0155] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:20; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0156] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:21; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0157] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:22; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0158] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:23; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0159] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:27; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0160] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:28; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0161] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:29; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0162] In another embodiment, the binder according to the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:30; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0163] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:31; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0164] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:32; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0165] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:33; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0166] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:34; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0167] In another embodiment, the binder is a. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 b. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID NO:36; and d. A CL region comprising the amino acid sequence set forth in SEQ ID NO:16 Includes.

[0168] In an alternative embodiment, the CL region may be the amino acid sequence shown in SEQ ID NO:17.

[0169] In one embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:15; h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0170] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:12; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0171] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:13; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0172] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:14; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0173] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:18; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0174] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:19; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0175] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:20; h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0176] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:21; h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0177] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:22; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0178] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:23; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0179] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:27; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0180] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:28; h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0181] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:29; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0182] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:30; h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0183] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:31; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0184] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:32; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0185] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:33; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0186] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:34; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0187] In another embodiment, the binder is e. A VH region comprising the amino acid sequence shown in SEQ ID NO:4 f. A VL region comprising the amino acid sequence shown in SEQ ID NO:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:36; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. Includes.

[0188] 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.

[0189] 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.

[0190] In yet another embodiment, the binding agent comprises a heavy chain constant region that is modified such that the binding agent induces Fc-mediated effector functions to a lesser extent compared to the same binding agent except for the modification. An example is a CD27-binding antibody of the invention that comprises a P329R and E345R substitution. Such an antibody induces one or more Fc-mediated effector functions to a lesser extent compared to an antibody that comprises the same sequence except for the P329R substitution, and also compared to the same antibody that comprises the same sequence except for the P329R and E345R substitution, e.g., a wild-type IgG1 heavy chain. 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 Fc-mediated effector function is reduced by at least 80%. In another embodiment, the Fc-mediated effector function is reduced by at least 90%. In another embodiment, the binding agent does not induce one or more Fc-mediated effector functions. The one or more Fc effector functions that are reduced or not induced at all may be selected from the following group: complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding. Thus, in one embodiment, the binding agent induces CDC to an extent that is 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%, compared to an identical binding agent except for the wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce CDC.

[0191] In another embodiment, the binding agent induces CDCC to an extent that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced compared to an identical binding agent except having a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce CDCC.

[0192] In another embodiment, the binding agent induces ADCC to an extent that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced compared to an identical binding agent except having a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce ADCC.

[0193] In another embodiment, the binding agent induces ADCP to a degree that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced compared to an identical binding agent except having a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce ADCP.

[0194] In another embodiment, the binding agent induces C1q binding to an extent that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced compared to an identical binding agent except 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 in Example 8.

[0195] In another embodiment, the binding agent induces FcγR binding to a degree that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced, compared to an identical binding agent except that it has a wild-type IgG1 HC constant region. In another embodiment, the binding agent does not induce FcγR binding. Preferably, FcγR binding is determined as in Example 9.

[0196] In one embodiment, the binding agent has reduced C1q binding and reduced FcγR binding compared to a binding agent comprising the same amino acid sequence except that it does not contain the P329R substitution.

[0197] In one embodiment, the binding agent used in any aspect or embodiment herein is a human antibody, except for the mutations described.

[0198] In one embodiment of the invention, the binding agent is a monovalent antibody.

[0199] In another embodiment, the binding agent is a bivalent antibody.

[0200] Furthermore, a binding agent of the invention may be a monospecific antibody.

[0201] In one embodiment, the binding agent used in any aspect or embodiment herein is a monoclonal antibody, such as a human monoclonal antibody, such as a human bivalent monoclonal antibody, such as a human bivalent full length monoclonal antibody.

[0202] In a preferred embodiment, the binding agent used in any aspect or embodiment herein is an IgG1 antibody, such as a full length IgG1 antibody, such as a human full length IgG1 antibody, optionally a human monoclonal full length bivalent IgG1,κ antibody, such as a human monoclonal full length bivalent IgG1m(f),κ antibody, except for any described mutations in the Fc region.

[0203] The binding agent used in the present invention is advantageously a bivalent monospecific format, comprising two antigen-binding regions that bind to the same epitope.However, a bispecific format is also envisaged, in which one of the antigen-binding regions binds to a different epitope.Therefore, the binding agent used according to any aspect or embodiment herein can be either a monospecific antibody or a bispecific antibody, unless the context is inconsistent.

[0204] Thus, in another embodiment, the binding agent is a bispecific antibody comprising a first antigen-binding region capable of binding to human CD27 as described herein and a second antigen-binding region 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 capable of binding to human CD27 as described herein and a second antigen-binding region capable of binding to a different target. Such a target may be on a different cell than CD27 or on the same cell.

[0205] In one embodiment of the invention, the binding agent has the ability to bind to human CD27 having the sequence shown in SEQ ID NO:1. However, human CD27 may be expressed as variants thereof in some individuals. Thus, in another embodiment, the binding agent further has the ability to bind to a human CD27 variant, such as the human CD27 variant shown in SEQ ID NO:2. In another embodiment, the binding agent further has the ability to bind to cynomolgus monkey CD27, such as that shown in SEQ ID NO:3.

[0206] In a further embodiment of the invention, the binding agent has the capacity of binding to CD27-expressing human T cells.

[0207] In another embodiment of the invention, the binding agent is capable of binding to CD27-expressing cynomolgus monkey T cells.

[0208] In one embodiment of the invention, the full length IgG1 antibody has the C-terminal lysine of the HC truncated. Such an antibody is also considered a "full length antibody."

[0209] In another embodiment of the invention, the binding agent is a binding agent for human T cells, e.g., CD4 + and CD8 + It has the ability to induce proliferation of T cells, such as helper T cells and cytotoxic T cells. Such activity may be assayed as described in Examples 6 or 7 herein.

[0210] In another embodiment of the invention, the binding agent has the ability to induce activation of human CD27-expressing Jurkat reporter T cells, such as those described in Example 2 herein.

[0211] In another embodiment of the invention, the binding agent has the ability to induce activation of human CD27-expressing Jurkat reporter T cells in the absence of Fcγ receptor IIb cross-linking, such as that described in Example 11 herein.

[0212] In another embodiment of the invention, the binding agent is a CD4+ and CD8 T cell with a central memory T cell phenotype. + It has the ability to induce T cell proliferation.

[0213] In another embodiment of the invention, the binding agent is capable of inducing the production of IFN gamma.

[0214] In another embodiment of the invention, the binder is in a composition or formulation comprising acetate, sorbitol, polysorbate 80 and has a pH of 5-6, preferably 5.5.

[0215] PD1 / PD-L1 inhibitors In one embodiment, the PD1 / PD-L1 inhibitor prevents inhibitory signals associated with PD-1. In one embodiment, the PD1 / PD-L1 inhibitor is an antibody, or a fragment thereof, that blocks or inhibits inhibitory signaling associated with PD-1. In one embodiment, the PD1 / PD-L1 inhibitor is a small molecule inhibitor that blocks or inhibits inhibitory signaling. In one embodiment, the PD1 / PD-L1 inhibitor is a peptide-based inhibitor that blocks or inhibits inhibitory signaling. In one embodiment, the PD1 / PD-L1 inhibitor is an inhibitory nucleic acid molecule that blocks or inhibits inhibitory signaling.

[0216] As described herein, inhibition or blocking of PD-1 signaling results in prevention or reversal of immune suppression and establishment or enhancement of T cell immunity against cancer cells. In one embodiment, inhibition of PD-1 signaling as described herein reduces or inhibits immune system dysfunction. In one embodiment, inhibition of PD-1 signaling as described herein makes dysfunctional immune cells less dysfunctional. In one embodiment, inhibition of PD-1 signaling as described herein makes dysfunctional T cells less dysfunctional.

[0217] In one embodiment, the PD-L1 is human PD-L1, in particular a human PD-L1 comprising the sequence shown in SEQ ID NO:98.

[0218] In one embodiment, the PD1 is human PD1. Preferably, the PD1 has or comprises the amino acid sequence set forth in SEQ ID NO:58 or SEQ ID NO:59, or the amino acid sequence of 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 to the amino acid sequence set forth in SEQ ID NO:58 or SEQ ID NO:59, or is an immunogenic fragment thereof.

[0219] In one embodiment, the PD1 / PD-L1 inhibitor prevents the interaction between PD-1 and PD-L1.

[0220] The PD1 / PD-L1 inhibitor may be an antibody, an antigen-binding fragment thereof, or a construct thereof comprising an antibody portion having an antigen-binding fragment of the required specificity. The antibody or antigen-binding fragment thereof is as described herein. Antibodies or antigen-binding fragments thereof that are PD1 / PD-L1 inhibitors include, in particular, antibodies or antigen-binding fragments thereof that bind to PD-1, and antibodies or antigen-binding fragments thereof that bind to PD-L1. The antibodies or antigen-binding fragments may also be conjugated to further moieties, as described herein. In particular, the antibodies or antigen-binding fragments thereof are chimerized, humanized or human antibodies.

[0221] In one embodiment, the antibody that is a PD1 / PD-L1 inhibitor is an isolated antibody.

[0222] 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.

[0223] The PD1 / PD-L1 inhibitor may be an inhibitory nucleic acid molecule, such as an oligonucleotide, an siRNA, an shRNA, an antisense DNA or RNA molecule, and an aptamer (e.g., a DNA or RNA aptamer), particularly an antisense-oligonucleotide. In one embodiment, the PD1 / PD-L1 inhibitor that is an siRNA interferes with mRNA and thus blocks translation, e.g., translation of PD-1 protein.

[0224] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody, antigen-binding portion thereof, or construct thereof that interferes with 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 interfere with or inhibit the interaction between PD-1 and one or more of its ligands are known in the art. In certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-1. In certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-L1.

[0225] In certain preferred embodiments, the PD1 / PD-L1 inhibitor is an antibody that binds to PD-1, e.g., a PD-1 blocking antibody. In certain preferred embodiments, the PD1 / PD-L1 inhibitor is an antibody that binds to PD-L1, e.g., a PD-L1 blocking antibody.

[0226] Exemplary PD1 / PD-L1 inhibitors include anti-PD-1 antibodies, such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606 and US 2015 / 0079109), lambrolizumab (disclosed, for example, as hPD109A and its humanized derivatives h409A1, h409A16 and h409A17 in WO2008 / 156712), AB137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; U.S. Patent No. 8,008,449; WO 2013 / 173223; see 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 (WO 2014 / 179664), cemiplimab (REGN-2810; Regeneron; H4H7798N; see US 2015 / 0203579 and WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70:136), AMP-224 (GSK-2661380; Li et al., 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. Pat. No. 9,834,606, and US 2015 / 0079109), BI 754091, SHR-1210 (see WO2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, as described in WO 2006 / 121168, INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; WO 2015 / 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 WO 2014 / 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; see JNJ-3283; Calvo et al., J. Clin. Oncol. 36, no. 5_suppl(2018) 58), genolimuzumab (CBT-501; see Patel et al., J. ImmunoTher. Cancer, 2017, 5(Suppl 2):P242), and sasunlimab (PF-06801591; Youssef et al., Proc. Am. Assoc. Cancer Res. Ann.Meeting 2017; Cancer Res 2017; 77(13 Suppl):Abstract), toripalimab (JS-001; see US 2016 / 0272708), camrelizumab (SHR-1210; INCSHR-1210; see US 2016 / 376367; Huang et al., Clin. Cancer Res. 2018; 24(6):1296-1304), spartalizumab (PDR001; see WO 2017 / 106656; Naing et al., J. Clin. Oncol. 34, no. 15_suppl(2016) 3060-3060), BCD-100 (JSC BIOCAD, Russia; WO 2018 / 103017), balstilimab (AGEN2034; see WO 2017 / 040790), sintilimab (IBI-308; see WO 2017 / 024465 and WO 2017 / 133540), ezabenlimab (BI-754091; 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 US 2017 / 210806), AK-103 (see WO 2017 / 071625, WO 2017 / 166804, and WO 2018 / 036472), retifanlimab (MGA-012; see WO 2017 / 019846), Sym-021 (see WO 2017 / 055547), CS1003 (see CN107840887), e.g., US 7,488,802, US 8,008,449, US 8,168,757, WO Nos. 03 / 042402, 2010 / 089411 (which further disclose anti-PD-L1 antibodies), WO 2010 / 036959, WO 2011 / 159877 (which further discloses antibodies to TIM-3), WO 2011 / 082400, WO 2011 / 161699, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2012 / 145493 (which further discloses antibodies to PD-L1), WO 2015 / 035606, WO 2014 / 055648 (which further discloses anti-KIR antibodies), US anti-PD-1 antibodies, such as those described in WO 2018 / 0185482 (which further discloses anti-PD-L1 and anti-TIGIT antibodies), US 8,008,449, US 8,779,105, US 6,808,710, US 8,168,757, US 2016 / 0272708, and US 8,354,509; small molecule antagonists against the PD-1 signaling pathway, such as those disclosed in Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497; siRNAs directed against PD-1, such as those disclosed in WO 2019 / 000146 and WO 2018 / 103501; Examples of suitable PD-1 proteins include, but are not limited to, the soluble PD-1 proteins disclosed in WO 2018 / 222711, as well as oncolytic viruses containing a soluble form of PD-1, for example, as described in WO 2018 / 022831.

[0227] 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.

[0228] In certain embodiments, the PD1 / PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody or antigen-binding fragment thereof that comprises the complementarity determining regions (CDRs) of one of the anti-PD1 or anti-PD-L1 antibodies or antigen-binding fragments described herein, such as the CDRs of one anti-PD1 or anti-PD-L1 antibody or antigen-binding fragment selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0229] In certain embodiments, the PD1 / PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody or antigen-binding fragment thereof comprising the heavy and light chain variable regions of one of the anti-PD1 or anti-PD-L1 antibodies or antigen-binding fragments described above, such as the heavy and light chain variable regions of one of the anti-PD1 or anti-PD-L1 antibodies or antigen-binding fragments selected from the group consisting of nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0230] 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, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, and CS1003.

[0231] In certain embodiments, the PD1 / PD-L1 inhibitor is an antibody that binds to PD1 or PD-L1. In some preferred embodiments, the PD1 / PD-L1 inhibitor is an antibody that is an antagonist of the PD1 / PD-L1 interaction. In some preferred embodiments, the PD1 / PD-L1 inhibitor is a PD1 blocking antibody or a PD-L1 blocking antibody.

[0232] 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, for example, 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.

[0233] In certain embodiments, the PD1 / PD-L1 inhibitor is a full length antibody or an antibody fragment, such as a full length IgG1 antibody.

[0234] In certain embodiments, the PD1 / PD-L1 inhibitor is a monospecific antibody.

[0235] In one embodiment, 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 set forth in SEQ ID NOs:99, 100 and 101, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:102, LAS and SEQ ID NO:103, respectively.

[0236] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising 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.

[0237] In one embodiment, the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising 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.

[0238] In a preferred embodiment, the PD1 / PD-L1 inhibitor is pembrolizumab or a biosimilar thereof.

[0239] In a preferred embodiment, the PD1 / PD-L1 inhibitor is nivolumab or a biosimilar thereof.

[0240] In a preferred embodiment, the PD1 / PD-L1 inhibitor is atezolizumab or a biosimilar thereof.

[0241] 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.

[0242] In certain embodiments, the PD1 / PD-L1 inhibitor is a PD1 inhibitor selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-224, AMP-514, or their respective biosimilars.

[0243] In certain embodiments, the PD1 inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-514, or their respective biosimilars.

[0244] In certain embodiments, the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, akasunlimab, AUNP12, CA-170, BMS-986189, or their respective biosimilars.

[0245] In certain embodiments, the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, akasunlimab, or their respective biosimilars.

[0246] 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 set forth 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 set forth in SEQ ID NO:52, QAS, and SEQ ID NO:50, respectively. A specific, non-limiting example of such an antibody is MAB-19-0202.

[0247] The terms "heavy chain variable region" (also referred to as "VH") and "light chain variable region" (also referred to as "VL") are used herein in their most general sense and include any sequence that may include complementarity determining regions (CDRs) interspersed with other regions, also referred to as framework regions (FRs). The framework regions position the CDRs such that, inter alia, they are capable of forming an antigen-binding site, particularly after folding and pairing of the VH and VL. Preferably, each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. That is, the terms "heavy chain variable region" and "light chain variable region" should not be construed as being limited to sequences as may be found in native antibodies or in the VH and VL sequences exemplified herein (SEQ ID NOs: 54-57 in the Sequence Listing). These terms include any sequence capable of containing and appropriately positioning the CDRs, such as sequences derived from the VL and VH regions of a native antibody or from the sequences shown in SEQ ID NOs:54-57 of the sequence listing. In particular, it will be understood by those skilled in the art that the sequences of the framework regions may be modified without losing the characteristics of the VH and VL, respectively (including both variants with respect to amino acid substitutions and variants with respect to sequence length, i.e., insertion or deletion variants). In a preferred embodiment, any modifications are limited to the framework regions. However, those skilled in the art are also well aware of the fact that the CDR, hypervariable and variable regions may also be modified without losing the ability to bind to PD-1. For example, the CDR regions are identical or highly homologous to the regions specified herein. By "highly homologous", it is envisaged that 1-5, preferably 1-4, such as 1-3 or 1 or 2 substitutions may be made in the CDRs. Additionally, the hypervariable and variable regions may be modified to show substantial homology with the regions specifically disclosed herein.

[0248] In the antibodies that bind to PD-1, the CDRs identified herein have been identified by using two different CDR identification methods. The first numbering scheme used herein is according to Kabat (Wu and Kabat, 1970; Kabat et al., 1991), and the second scheme is the IMGT numbering (Lefranc, 1997; Lefranc et al., 2005). In the third approach, the intersection of both identification schemes is used.

[0249] An antibody that binds to PD-1 may comprise one or more CDRs, and a set of CDRs or a combination of sets of CDRs described herein comprises said CDRs together with their intervening framework regions (also referred to herein as framing regions or FRs) or portions of said framework regions. Preferably, the portions comprise at least about 50% of either 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 made by recombinant DNA techniques may result in the introduction of residues at the N- or C-terminus of the encoded variable region by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers for joining the variable regions of the disclosure to additional protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or protein tags.

[0250] An antibody that binds to PD-1 may comprise a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of a VH sequence set forth in any one of SEQ ID NOs: 56. In one embodiment, the antibody comprises a heavy chain variable region (VH), wherein the VH comprises a sequence set forth in any one of SEQ ID NOs: 56. In one embodiment, the antibody comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of a VL sequence set forth in any one of SEQ ID NOs: 57. In one embodiment, the antibody comprises a light chain variable region (VL), wherein the VL comprises a sequence set forth in any one of SEQ ID NO:57.

[0251] An antibody that binds to PD-1 may comprise a heavy chain variable region (VH) and a light chain variable region (VL), where the VH comprises or has the sequence 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 may comprise a VH that comprises or has the sequence shown in SEQ ID NO:56, or a variant thereof, and a VL that comprises or has the sequence shown in SEQ ID NO:57, or a variant thereof. A specific, non-limiting example of such an antibody is MAB-19-0618. The antibody MAB-19-0618 is derived from MAB-19-0202. Variants of the heavy chain variable region (VH) and the light chain variable region (VL), and respective combinations of these variant VHs and VLs, are also encompassed by the present disclosure.

[0252] An antibody that binds to PD-1 may comprise a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region that comprises or has the sequence set forth in SEQ ID NO:38 or 128, and a heavy chain variable region (VH) that comprises or has the sequence set forth in SEQ ID NO:56, and the light chain comprises a light chain constant region that comprises or has the sequence set forth in SEQ ID NO:42, and a light chain variable region (VL) that comprises or has the sequence set forth in SEQ ID NO:57.

[0253] An antibody that binds to PD-1 may comprise a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region that comprises or has the sequence set forth in SEQ ID NO:38 or 128, and a heavy chain variable region (VH) that comprises the CDR1, CDR2 and CDR3 sequences of the sequence set forth in SEQ ID NO:56, and the light chain comprises a light chain constant region that comprises or has the sequence set forth in SEQ ID NO:42, and a light chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the sequence set forth in SEQ ID NO:57. For example, the CDR1, CDR2 and CDR3 sequences are as specified herein.

[0254] The antibody that binds to PD-1 may be a monoclonal, chimeric or monoclonal humanized antibody, or a fragment of such an antibody. The antibody can be, for example, a whole antibody or an antigen-binding fragment thereof, including a bispecific antibody.

[0255] In an antibody that binds PD-1, one or more, preferably both, heavy chain constant regions may be modified such that binding of C1q to the antibody is reduced compared to a wild type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%. In one embodiment, C1q binding may be determined by ELISA.

[0256] By "wild-type" or "WT" or "native" is meant an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally altered.

[0257] In an antibody that binds to PD-1, one or more, preferably both, heavy chain constant regions may be modified such that binding of one or more IgG Fc gamma 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 gamma receptors are selected from at least one of Fc gamma RI, Fc gamma RII, and Fc gamma RIII. In one embodiment, the IgG Fc gamma receptor is Fc gamma RI.

[0258] In one embodiment, the antibody that binds to PD-1 does not have the ability to induce FcgammaRI-mediated effector function, or the induced FcgammaRI-mediated effector function is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, compared to a wild-type antibody.

[0259] In one embodiment, an antibody that binds PD-1 does not have the ability to induce at least one of complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cellular lysis (ADCC) mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis, or at least one of complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cellular lysis (ADCC) mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis is induced to a reduced extent, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0260] Antibody-dependent cell-mediated cytotoxicity is also referred to herein as "ADCC." ADCC describes the cell-killing ability of effector cells, particularly lymphocytes, described herein, which preferably requires that the target cells be marked with an antibody.

[0261] ADCC preferably occurs when an antibody binds to an antigen on a tumor cell and the antibody Fc domain engages with the Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and certain cell populations characteristically express defined Fc receptors. ADCC can be seen as a mechanism for directly inducing variable degrees of immediate tumor destruction, which leads to antigen presentation and induction of tumor-directed T cell responses. Preferably, the in vivo induction of ADCC leads to tumor-directed T cell responses and host-derived antibody responses.

[0262] Complement-dependent cytotoxicity is also referred to herein as "CDC". CDC is another cell killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also both highly effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex is followed by the C-terminal end of the participating antibody molecules, e.g., IgG molecules. H This results in the exposure of multiple C1q binding sites in close proximity on the 2 domain (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low affinity C1q-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of effector cell chemotactic / activating factors C3a and C5a. Preferably, the complement cascade ends with the formation of a membrane attack complex, which creates pores in the cell membrane that allow the free passage of water and solutes in and out of the cell, and can lead to apoptosis.

[0263] In one embodiment, the antibody that binds to PD-1 has reduced or depleted effector function, hi one embodiment, the antibody does not mediate ADCC or CDC, or both.

[0264] In one embodiment, one or more, preferably both, heavy chain constant regions of an antibody that binds PD-1 are altered such that neonatal Fc receptor (FcRn) binding to the antibody is unaffected compared to the wild-type antibody.

[0265] In one embodiment, the PD-1 to which the antibody can bind is human PD-1. In one embodiment, the PD-1 has or comprises the amino acid sequence set forth in SEQ ID NO: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 set forth in SEQ ID NO:58 or SEQ ID NO:59, or is an immunogenic fragment thereof. In one embodiment, the antibody is capable of binding to a native epitope of PD-1 present on the surface of a living cell.

[0266] 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 nonpolar amino acid at a position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering and an amino acid other than glycine at a position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering.

[0267] The term "amino acid corresponding to position" and similar expressions, as used herein, refers to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is an amino acid or segment that is aligned with the other amino acid or segment using a standard sequence alignment program, such as ALIGN, ClustalW or similar, typically with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain. Methods for aligning sequences or segments in sequences and thereby determining positions in sequences that correspond to the amino acid positions according to the present disclosure are believed to be well known in the art.

[0268] For example, with respect to the amino acid sequence according to SEQ ID NO.38 of the sequence listing of the present disclosure, the amino acid positions corresponding to positions 234-236 in the human IgG1 heavy chain according to EU numbering are amino acid positions 117-119 of SEQ ID NO.38, with F at position 117 (corresponding to position 234 in the human IgG1 heavy chain according to EU numbering), E at position 118 (corresponding to position 235 in the human IgG1 heavy chain according to EU numbering), and R at position 119 (corresponding to position 236 in the human IgG1 heavy chain according to EU numbering). In the sequences shown below, the FER amino acid sequence is underlined and shown in bold. TIFF2025516631000004.tif61147

[0269] Unless otherwise indicated herein or otherwise clearly contradicted by context, all references throughout this disclosure to amino acid positions in antibody heavy chain constant regions refer to the positions corresponding to the respective positions in the human IgG1 heavy chain according to the EU numbering set out in Kabat (as described in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition-US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991)).

[0270] In one embodiment, an antibody that binds to PD-1 comprises a heavy chain constant region that has reduced or depleted Fc-mediated effector function, or induces Fc-mediated effector function to a lesser extent compared to another antibody that comprises the same antigen binding region and a heavy chain constant region (CH) that contains human IgG1 hinge, CH2 and CH3 regions.

[0271] In one specific embodiment, the heavy chain constant region (CH) in an antibody that binds PD-1 is modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an identical antibody except for that antibody comprising an unmodified heavy chain constant region (CH).

[0272] The term "Fc-mediated effector function" as used herein refers in particular to such functions selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC, CDC and any combination thereof.

[0273] In the context of the present disclosure, the term "having reduced or depleted Fc-mediated effector functions" as used in relation to antibodies, including multispecific antibodies, means that the antibody causes an overall reduction in Fc-mediated effector functions, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, most preferably 75% or more, at a level compared to a human IgG1 antibody comprising (i) the same CDR sequences as said antibody, in particular the same first and second antigen binding regions, and (ii) two heavy chains comprising the human IgG1 hinge, CH2 and CH3 regions, such functions being in particular selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC or CDC. "Depleted Fc-mediated effector functions" or similar phrases include complete or essentially complete inhibition, i.e. reduction to zero or essentially to zero.

[0274] In the context of the present disclosure, the term "induces Fc-mediated effector functions to a lesser extent" as used in relation to antibodies, including multispecific antibodies, 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 the same first and second antigen-binding regions, and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions, such functions being in particular selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC or CDC.

[0275] Fc-mediated effector function may be determined by measuring binding of a binding agent to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. In particular, Fc-mediated effector function may be determined by measuring binding of a binding agent to C1q and / or IgG Fc gamma RI.

[0276] In one embodiment relating to the use of an antibody that binds to PD-1, the amino acid at the position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering is a basic amino acid.

[0277] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds that contain an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain (R group) that is specific to each amino acid. In the context of this disclosure, amino acids can be classified based on structure and chemical characteristics.

[0278] In this disclosure, amino acid residues are represented by using the following abbreviations: and, unless expressly indicated otherwise, amino acid sequences of peptides and proteins are identified from N-terminus to C-terminus (left to right terminus), with the N-terminus identified as the first residue. Amino acids are designated by their three-letter abbreviations, one-letter abbreviations, or full names as follows: Ala: A: alanine; Asp: D: aspartic acid; Glu: E: glutamic acid; Phe: F: phenylalanine; Gly: G: glycine; His: H: histidine; Ile: I: isoleucine; Lys: K: lysine; Leu: L: leucine; Met: M: methionine; Asn: N: asparagine; Pro: P: proline; Gln: Q: glutamine; Arg: R: arginine; Ser: S: serine; Thr: T: threonine; Val: V: valine; Trp: W: tryptophan; Tyr: Y: tyrosine; Cys: C: cysteine.

[0279] Naturally occurring amino acids are also commonly divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

[0280] In one embodiment of the use of an antibody that binds to PD-1, the basic amino acid at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is selected from the group consisting of lysine, arginine and histidine. In one embodiment, the basic amino acid at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering is arginine (G236R). Such an amino acid substitution is also referred to herein as G236R. The term "G236R" indicates that the amino acid glycine (G) at position 236 in the human IgG1 heavy chain according to EU numbering is replaced by arginine (R). Similar terms are used for other amino acid positions and amino acids in this disclosure. Unless otherwise indicated, the amino acid position referred to in these terms is the amino acid position in the human IgG1 heavy chain according to EU numbering.

[0281] In one embodiment for use of an antibody that binds to PD-1, the amino acid at the position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering is an aromatic amino acid. In one embodiment, the aromatic amino acid at this position is selected from the group consisting of phenylalanine, tryptophan and tyrosine.

[0282] In one embodiment, for use of an antibody that binds to PD-1, the amino acid at the position corresponding to position 234 in a 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.

[0283] In one embodiment relating to the use of an antibody that binds to PD-1, the amino acid at the position corresponding to 234 in a human IgG1 heavy chain according to EU numbering is phenylalanine (L234F).

[0284] 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 shown below.

[0285] (Table 22) TIFF2025516631000005.tif219106

[0286] For example, at positions corresponding to positions 234 and 236 in a human IgG1 heavy chain according to EU numbering, the following amino acids in particular may be present in a heavy chain constant region of an antibody that binds PD-1: 234F / 236R, 234W / 236R, 234Y / 236R, 234A / 236R, 234L / 236R, 234F / 236K, 234W / 236K, 234Y / 236K, 234A / 236K, 234L / 236K, 234F / 236H, 234W / 236H, 234Y / 236H, 234A / 236H, or 234L / 236H.

[0287] The above-mentioned amino acids or amino acid substitutions at positions 234 and 236 may be present in only one heavy chain of an antibody that binds to PD-1 or in both heavy chains of an antibody that binds to PD-1. Each amino acid present in the first and second heavy chains of the antibody may be selected independently of each other.

[0288] For example, at least one heavy chain of an antibody that binds to PD-1 can comprise the following sequence (SEQ ID NO:38): TIFF2025516631000006.tif61147

[0289] In one embodiment regarding an antibody that binds to PD-1, wherein the amino acids in said heavy chain at positions corresponding to 234 and 236 in a human IgG1 heavy chain according to EU numbering are as specified above, furthermore the amino acid at position corresponding to 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 position corresponding to 235 in a human IgG1 heavy chain according to EU numbering is glutamic acid (L235E).

[0290] In one embodiment of 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 non-polar or aromatic amino acid at position 234, an acidic amino acid at position 235, and a basic amino acid at position 236.

[0291] 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 shown below.

[0292] (Table 23) TIFF2025516631000007.tif151163TIFF2025516631000008.tif236163

[0293] For example, at positions corresponding to positions 234, 235 and 236 in a human IgG1 heavy chain according to EU numbering, the following amino acids in particular may be present in a 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 / 235D / 236R, 234F / 235L / 236R, 234W / 235L / 236R, 234Y / 235L / 236R, 234A / 235L / 236R, 234L / 235L / 2 36R, 234F / 235A / 236R, 234W / 235A / 236R, 234Y / 235A / 236R, 234A / 235A / 236 R, 234L / 235A / 236R, 234F / 235E / 236K, 234W / 235E / 236K, 234Y / 235E / 236K, 234A / 235E / 236K, 234L / 235E / 236K, 234F / 235D / 236K, 234W / 235D / 236K, 23 4Y / 235D / 236K, 234A / 235D / 236K, 234L / 235D / 236K, 234F / 235L / 236K, 234W / 235L / 236K, 234Y / 235L / 236K, 234A / 235L / 236K, 234L / 235L / 236K, 234F / 2 35A / 236K, 234W / 235A / 236K, 234Y / 235A / 236K, 234A / 235A / 236K, 234L / 235 A / 236K, 234F / 235E / 236H, 234W / 235E / 236H, 234Y / 235E / 236H, 234A / 235E / 236H, 234L / 235E / 236H, 234F / 235D / 236H, 234W / 235D / 236H, 234Y / 235D / 23 6H, 234A / 235D / 236H, 234L / 235D / 236H, 234F / 235L / 236H, 234W / 235L / 236H , 234Y / 235L / 236H, 234A / 235L / 236H, 234L / 235L / 236H, 234F / 235A / 236H, 234W / 235A / 236H, 234Y / 235A / 236H, 234A / 235A / 236H, or 234L / 235A / 236H.

[0294] The above-mentioned amino acids or amino acid substitutions at positions 234, 235 and 236 may be present in only one heavy chain of the antibody or in both heavy chains of the antibody. Each amino acid present in the first and second heavy chains of the antibody may be selected independently of each other.

[0295] For example, at least one heavy chain of an antibody that binds to PD-1 can comprise the following sequence (SEQ ID NO:128 or 38): TIFF2025516631000009.tif61147

[0296] Unless the context indicates otherwise, any permutations and combinations of all described amino acid substitutions at positions 234, 236 and 235 in the present application, where applicable, as shown, for example, in Tables 22 and 23, should be considered as 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 234-236 in a human IgG1 heavy chain according to EU numbering, or the first heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:38, and the second heavy chain of said antibody comprises other amino acids, e.g., the amino acids AAG or LLG, at positions corresponding to positions 234-236 in a human IgG1 heavy chain according to EU numbering, or the second heavy chain of said antibody comprises, consists essentially of, or consists of the amino acid sequence set forth 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 234-236 in a human IgG1 heavy chain according to EU numbering, i.e. the same aromatic or non-polar amino acid, e.g. F, at the position corresponding to position 234 in a human IgG1 heavy chain according to EU numbering, and the same amino acid other than glycine, e.g. R, e.g. FER or FLR, in a specific combination, at the position corresponding to position 236 in a human IgG1 heavy chain according to EU numbering.

[0297] In one embodiment, an antibody that binds to PD-1 comprises at least one or two heavy chain constant regions, wherein the amino acid corresponding to position 234 is phenylalanine, the amino acid corresponding to position 235 is glutamic acid, and the amino acid corresponding to position 236 is arginine (L234F / L235E / G236R=FER).

[0298] In one embodiment, an antibody that binds to PD-1 comprises one or more heavy chain constant regions (CH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the heavy chain constant region sequence shown in SEQ ID NO:38.

[0299] In one embodiment, an antibody that binds to PD-1 comprises one or more, e.g., two, heavy chain constant regions (CH), wherein the heavy chain constant region comprises the sequence shown in SEQ ID NO:38.

[0300] In one embodiment, the antibody that binds to PD-1 comprises a heavy chain having the sequence set forth in SEQ ID NO:139, and a light chain having the sequence set forth in SEQ ID NO:140.

[0301] The antibody is preferably of the IgG1 isotype.

[0302] As used herein, the term "isotype" refers to the immunoglobulin class encoded by heavy chain constant region genes.When IgG1 isotype is referred to herein, the term is not limited to a specific isotype sequence, e.g., a specific IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgG1, than to other isotypes.Thus, for example, the IgG1 antibody disclosed herein may be a sequence variant of naturally occurring IgG1 antibody, including variations in constant region.

[0303] IgG1 antibodies can exist in multiple polymorphic variants, called allotypes (reviewed in Jefferis and Lefranc 2009.mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some of the embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z or combinations thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region that comprises a human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0304] There are two types of light chains in mammals: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within the different isotypes of immunoglobulins, while the variable portion is highly diversified and is responsible for antigen recognition.

[0305] For example or in one embodiment, the antibody, preferably a monoclonal antibody, used in accordance with the present invention is of the IgG1, κ or λ isotype, preferably comprising a human IgG1 / κ or human IgG1 / λ constant part, or the antibody, preferably a 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.

[0306] In one embodiment, an antibody that binds to PD-1 comprises a light chain having a light chain constant region (LC) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the LC sequence set forth in SEQ ID NO:42. In one embodiment, the antibody comprises a light chain having a light chain constant region (LC) comprising the sequence set forth in SEQ ID NO:42.

[0307] In one embodiment of the invention, the antibody that binds to PD-1 is a full length IgG1 antibody, such as, for example, IgG1, kappa. In one embodiment of the invention, the binding agent is a full length human IgG1 antibody, such as, for example, IgG1, kappa.

[0308] In one embodiment, an antibody that binds PD-1 may be derivatized, linked, or co-expressed with other binding specificities. In another embodiment, an antibody may be derivatized, linked, or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., a Fab' fragment). For example, it may be operatively linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, e.g., another antibody (e.g., to produce a bispecific or multispecific antibody).

[0309] An antibody that binds to PD-1 may be a human antibody. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. A human antibody that binds to PD-1 may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., where mutations have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0310] The present disclosure includes the use of bispecific and multispecific molecules that comprise at least one first binding specificity for PD-1 and a second binding specificity (or additional binding specificity) for a second target epitope (or additional target epitope).

[0311] 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 shown herein.

[0312] In one embodiment of the use of a multispecific antibody that binds to PD-1, the antibody comprises first and second binding arms derived from a full-length antibody, such as a full-length IgG1, lambda (lambda) or IgG1, kappa (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, kappa or lambda isotype, preferably comprising a human IgG1 / kappa or human IgG1 / lambda constant portion.

[0313] The first antigen-binding region that binds PD-1 of a multispecific or bispecific antibody used in accordance with the invention may comprise heavy and light chain variable regions of an antibody that competes with PD-1 for binding to PD-L1 and / or PD-L2. In one embodiment of the use of a multispecific or bispecific antibody, the first antigen-binding region that binds PD-1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown herein.

[0314] As used herein, the term "effector cell" refers to the immune cell that is involved in the effector phase of immune response, but not in the recognition and activation phase of 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, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

[0315] "Target cell" refers to any undesirable cell in a subject (e.g., a human or animal) that can be targeted by an antibody. In a preferred embodiment, the target cell is a tumor cell.

[0316] In another embodiment, the PD1 / PD-L1 inhibitor is a multispecific antibody, such as a bispecific antibody.

[0317] In a preferred embodiment, the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor that comprises a first binding domain that binds to CD137 and a second binding domain that binds to PD-L1.

[0318] In one embodiment, the PD-L1 is human PD-L1, particularly human PD-L1 comprising the sequence set forth in SEQ ID NO: 98. In one embodiment, the CD137 is human CD137, particularly human CD137 comprising the sequence set forth in SEQ ID NO: 97.

[0319] In one embodiment, 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.

[0320] In one embodiment, a) a first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:79 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO:83; and b) a second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:86 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO:90.

[0321] In one embodiment, the PD-L1 inhibitor is a multispecific antibody, such as a bispecific antibody.

[0322] In one embodiment, the PD-L1 inhibitor is in the form of a full length antibody or an antibody fragment.

[0323] 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: i) a polypeptide comprising the first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). Includes; and the second binding arm is iii) a polypeptide comprising the second heavy chain variable region (VH) and a second heavy chain constant region (CH); and iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). Includes.

[0324] In one embodiment, the PD-L1 inhibitor is i) a first heavy chain and a light chain comprising said antigen-binding region having the ability to bind to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a light chain comprising the antigen-binding region having the ability to bind to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region, and the second light chain comprises a second light chain constant region. Includes.

[0325] In one embodiment, (i) the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said first heavy chain and the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said second heavy chain.

[0326] In one embodiment, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E in said first and second heavy chains, respectively.

[0327] In one embodiment, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said first and second heavy chain constant regions (HC).

[0328] In one embodiment, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering of both the first heavy chain constant region and the second heavy chain constant region in the PD-L1 inhibitor are F and E, respectively, and (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is R and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0329] In one embodiment, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first heavy chain constant region and the second heavy chain constant region in the PD-L1 inhibitor are F, E, and A, respectively, and (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0330] 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: a) the sequence depicted 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) a sequence which has at most 6 substitutions, such as at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0331] In one embodiment, the constant region of the first and / or second heavy chain, e.g., the first heavy chain, in the PD-L1 inhibitor comprises: a) the sequence depicted 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) a sequence which has at most 6 substitutions, such as at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0332] In one embodiment, the PD-L1 inhibitor comprises a kappa (κ) light chain constant region.

[0333] In one embodiment, the PD-L1 inhibitor comprises a lambda (λ) light chain constant region.

[0334] In one embodiment, said 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.

[0335] In one embodiment, said 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.

[0336] In one embodiment, said first light chain constant region of the PD-L1 inhibitor is a kappa (κ) light chain constant region and said second light chain constant region is a lambda (λ) light chain constant region, or said first light chain constant region is a lambda (λ) light chain constant region and said second light chain constant region is a kappa (κ) light chain constant region.

[0337] In one embodiment, the kappa (κ) light chain of the PD-L1 inhibitor is a) the sequence shown in SEQ ID NO:16; b) a subsequence of the sequence in a), e.g. a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence which has at most 10 substitutions, such as at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence is selected from the group consisting of:

[0338] In one embodiment, the lambda (λ) light chain of the PD-L1 inhibitor is a) the sequence shown 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) a sequence which has at most 10 substitutions, such as at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence is selected from the group consisting of:

[0339] In one embodiment, the PD-L1 inhibitor is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0340] In one embodiment, the PD-L1 inhibitor is a full length IgG1 antibody.

[0341] In one embodiment, the PD-L1 inhibitor is an antibody of the IgG1m(f) allotype.

[0342] In one embodiment, the PD-L1 inhibitor is a bispecific antibody that binds to CD137 and PD-L1, wherein the bispecific antibody has 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.

[0343] In one embodiment, the PD-L1 inhibitor is akasunlimab or a biosimilar thereof.

[0344] Subjects and Tumors or Cancers to be Treated The subjects treated in accordance with the present disclosure are preferably human subjects.

[0345] In one embodiment, the tumor or cancer is a solid tumor.

[0346] In one embodiment, the tumor is a PD-L1 positive tumor.

[0347] In one embodiment, the tumor or cancer is a head and neck squamous cell carcinoma (HNSCC), such as an HNSCC of the oral cavity, pharynx or larynx.

[0348] In one embodiment, the HNSCC is recurrent, unresectable or metastatic.

[0349] In one embodiment, the tumor or cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

[0350] In one embodiment, the NSCLC is recurrent, unresectable or metastatic.

[0351] In one embodiment, the NSCLC does not have an epidermal growth factor (EGFR) sensitizing mutation and / or an anaplastic lymphoma (ALK) translocation and / or a ROS1 rearrangement.

[0352] In one embodiment, the NSCLC is NTRK1 / 2 / 3 (neurotrophin 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 received prior treatment with the respective targeted therapy.

[0353] In one embodiment, the subject has undergone prior treatment with a PD-1 or PD-L1 inhibitor, e.g., an anti-PD-1 or anti-PD-L1 antibody, preferably at least two doses of a PD-1 or PD-L1 inhibitor.

[0354] In one embodiment, the subject has undergone prior treatment with a platinum-based therapy, or an alternative chemotherapy if platinum is ineligible, such as a gemcitabine-containing regimen.

[0355] In one embodiment, the tumor or cancer has recurred and / or progressed following treatment, such as systemic treatment with a checkpoint inhibitor.

[0356] 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.

[0357] In one embodiment, the cancer or tumor is recurrent and / or refractory, or the subject has progressed following treatment with a PD-1 or PD-L1 inhibitor, e.g., an anti-PD-1 or anti-PD-L1 antibody, administered as a monotherapy or as part of a combination therapy.

[0358] In one embodiment, the last previous treatment was treatment with a PD1 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.

[0359] In one embodiment, the time from progression on last treatment with a PD-1 inhibitor or PD-L1 inhibitor, eg, an anti-PD-1 antibody or an anti-PD-L1 antibody, is 6 months or less.

[0360] In one embodiment, the time since 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 previous treatment is 6 months or less.

[0361] In one embodiment, the cancer or tumor is recurrent and / or refractory, or the subject is progressing during or after i) treatment with an anti-PD-1 or anti-PD-L1 antibody followed by platinum doublet chemotherapy, or ii) treatment with an anti-PD-1 or anti-PD-L1 antibody followed by platinum doublet chemotherapy.

[0362] In a second aspect, the disclosure provides a kit comprising i) a binding agent comprising at least one binding region that binds to CD27 and ii) a PD1 / PD-L1 inhibitor.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] In a third aspect, the present disclosure provides a kit for use in a method for reducing or preventing the progression of a tumor or treating cancer in a subject, the kit comprising i) a binding agent comprising at least one binding region that binds to CD27 and ii) a PD1 / PD-L1 inhibitor.

[0367] 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 present disclosure.

[0368] 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 present disclosure.

[0369] 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 present disclosure.

[0370] 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 present disclosure.

[0371] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising: i) a binding agent comprising at least one binding region that binds to CD27; ii) a PD1 / PD-L1 inhibitor; and iii) optionally, a pharma- ceutically acceptable carrier.

[0372] 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.

[0373] 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.

[0374] In a fifth aspect, the present disclosure provides a pharmaceutical composition for use in a method for reducing or preventing the progression of a tumor or treating cancer in a subject, the pharmaceutical composition comprising i) a binding agent comprising at least one binding region that binds to CD27 and ii) a PD1 / PD-L1 inhibitor.

[0375] 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.

[0376] 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.

[0377] In one embodiment of the pharmaceutical composition for use according to the fifth aspect, the subject is as defined in any aspect or embodiment of the present disclosure.

[0378] In one embodiment of the pharmaceutical composition for use according to the fifth aspect, the method is as defined in any aspect or embodiment of the present disclosure.

[0379] In a sixth aspect, the present disclosure provides a binding agent for use in a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising: i) a binding agent comprising at least one binding region that binds to CD27; and ii) administering to the subject a PD1 / PD-L1 inhibitor.

[0380] 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.

[0381] 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.

[0382] 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 disclosure.

[0383] In a seventh aspect, the present disclosure provides a PD1 / PD-L1 inhibitor for use in a method for reducing or preventing the progression of a tumor or treating cancer in a subject, the method comprising the steps of: i) a binding agent comprising at least one binding region that binds to CD27; and ii) administering to the subject the PD1 / PD-L1 inhibitor.

[0384] 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 disclosure.

[0385] 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.

[0386] 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 disclosure.

[0387] Reference to the documents and works referred to herein is not intended as an admission that any of the same is pertinent 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 regarding the accuracy of the contents of these documents.

[0388] The description (including the following examples) is presented to enable those skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be construed to the fullest extent consistent with the claims.

[0389] Items of this disclosure 1. (i) a binding agent comprising at least one binding region that binds to CD27; and (ii) PD1 / PD-L1 inhibitor 23. A method for reducing or preventing the progression of a tumor or treating cancer in a subject, comprising administering to the subject a

[0390] 2. The method of item 1, wherein the binding agent comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively.

[0391] 3. The method of item 1 or 2, wherein the binding agent comprises two binding regions capable of binding to human CD27, and the antibody comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively.

[0392] 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.

[0393] 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.

[0394] 6. The method of any of the preceding items, wherein the binding agent comprises a VH region and a VL region comprising the sequences set forth in SEQ ID NO:4 and SEQ ID NO:8, respectively.

[0395] 7. The method of any of the preceding items, wherein the binding agent is an antibody, preferably a human or humanized antibody.

[0396] 8. The method of any of the preceding items, wherein the antibody is a full-length antibody further comprising a light chain constant region (CL) and a heavy chain constant region (CH).

[0397] 9. The method of item 8, wherein the light chain constant region is human kappa.

[0398] 10. The method of item 8, wherein the light chain constant region is human lambda.

[0399] 11. The method of any of the preceding items, wherein the binding agent further comprises a heavy chain constant region, which is a heavy chain constant region of a human IgG isotype, and optionally a modified human IgG heavy chain constant region.

[0400] 12. The method of item 11, wherein the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4, such as human IgG1.

[0401] 13. The method of item 11 or 12, wherein the IgG is a modified human IgG containing one or more amino acid substitutions.

[0402] 14. The method of any 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.

[0403] 15. The method of any of items 11 to 14, wherein said 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.

[0404] 16. The method of any of items 11 to 15, wherein the substitution in the heavy chain constant region induces increased CD27 agonism compared to an identical antibody except that it comprises a wild-type IgG1 antibody heavy chain constant region.

[0405] 17. The method of any of items 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 comprising A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

[0406] 18. The method of any 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.

[0407] 19. The method of any of items 11 to 18, wherein the amino acid residue at the position corresponding to position E430 in a human IgG1 heavy chain according to Eu numbering is G.

[0408] 20. The method of any 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.

[0409] 21. The method of any of items 11 to 20, wherein the amino acid residues at positions corresponding to positions E345 and P329 in a human IgG1 heavy chain according to Eu numbering are both R.

[0410] 22. The method of any of items 11 to 21, wherein the binding agent has a pharmacokinetic profile similar to that of a parent antibody comprising a wild-type IgG1 heavy chain constant region.

[0411] 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 NOs:12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and 36.

[0412] 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.

[0413] 25. The method of any of the preceding items, wherein the binding agent comprises a heavy chain constant region which has been modified such that the binding agent induces one or more Fc-mediated effector functions to a lesser extent relative to the parent antibody.

[0414] 26. The method of item 25, wherein the one or more Fc-mediated effector functions are reduced by at least 20%, such as at least 30% or at least 40%, or at least 50% or at least 60% or at least 70%, or at least 80% or at least 90%.

[0415] 27. The method of item 25 or 26, wherein the binding agent does not induce one or more Fc-mediated effector functions.

[0416] 28. The one or more Fc-mediated effector functions are selected from the following group: Complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding 28. The method according to any one of items 25 to 27, selected from the above.

[0417] 29. The method of any of items 25 to 28, wherein the binding agent does not induce C1q binding as measured by the method of example 8.

[0418] 30. The method of any of the preceding items, wherein the binding agent is a monovalent antibody.

[0419] 31. The method of any of the preceding items, wherein the binding agent is a bivalent antibody.

[0420] 32. The method of any of the preceding items, wherein the binding agent is a monospecific antibody.

[0421] 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 of 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.

[0422] 34. The method of any of the preceding items, wherein the CD27 is human CD27, in particular, the human CD27 comprises the sequence shown in SEQ ID NO:1 or the human CD27 variant shown in SEQ ID NO:2.

[0423] 35. The binder is e. a VH region comprising the amino acid sequence set forth in SEQ ID NO:4; f. a VL region comprising the amino acid sequence set forth in SEQ ID NO:8; g. a CH region comprising the amino acid sequence set forth in SEQ ID NO:15; and h. A CL region comprising the amino acid sequence set forth in SEQ ID NO:17. 2. The method of any of the preceding items, comprising:

[0424] 36. The method of any of the preceding items, wherein 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.

[0425] 37. The method of any of the preceding items, wherein the PD-L1 is human PD-L1, particularly a human PD-L1 comprising the sequence shown in SEQ ID NO:98.

[0426] 38. The method of any of the preceding items, wherein the PD1 is human PD1, preferably wherein 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 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 an immunogenic fragment thereof.

[0427] 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 is a PD1 or PD-L1 blocking antibody.

[0428] 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, e.g., an antibody of the IgG1 isotype.

[0429] 41. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is a full-length antibody or an antibody fragment, e.g., a full-length IgG1 antibody.

[0430] 42. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is a monospecific antibody.

[0431] 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 set forth in SEQ ID NOs:99, 100, and 101, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:102, LAS, and SEQ ID NO:103, respectively.

[0432] 44. The method of any of the preceding items, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising 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.

[0433] 45. 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 comprising the amino acid sequence of SEQ ID NO:106 and a light chain comprising the amino acid sequence of SEQ ID NO:107.

[0434] 46. (a) the binding agent is an antibody comprising 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; (b) the PD1 / PD-L1 inhibitor is pembrolizumab or a biosimilar thereof; 2. The method of any of the preceding items.

[0435] 47. (a) the binding agent is an antibody comprising 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; (b) the PD1 / PD-L1 inhibitor is nivolumab or a biosimilar thereof; Any of the methods listed in items 1 to 42.

[0436] 48. (a) the binding agent is an antibody comprising 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; (b) the PD1 / PD-L1 inhibitor is atezolizumab or a biosimilar thereof; Any of the methods listed in items 1 to 42.

[0437] 49. The method of any of items 1 to 42, wherein the PD1 / PD-L1 inhibitor is an antibody, or an antigen-binding fragment thereof, that binds to PD1, and the antibody that binds to PD1 comprises VH regions CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:49, 46, and 45, respectively, and VL regions CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs:52, QAS, and SEQ ID NO:50, respectively.

[0438] 50. The method of item 49, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VH sequence shown in SEQ ID NO:56.

[0439] 51. The method of item 50, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH), and the VH comprises the sequence shown in SEQ ID NO:56.

[0440] 52. The method of any of items 49 to 51, wherein the antibody that binds to PD1 comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence shown in SEQ ID NO:57.

[0441] 53. The method of item 52, wherein the antibody that binds to PD1 comprises a light chain variable region (VL), and the VL comprises the sequence shown in SEQ ID NO:57.

[0442] 54. The method of any of items 49 to 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 set forth in SEQ ID NO:56, and the VL comprises or has the sequence set forth in SEQ ID NO:57.

[0443] 55. The antibody that binds to PD1 comprises a heavy chain constant region, The heavy chain constant region is an aromatic or non-polar amino acid at a position corresponding to position 234 in the human IgG1 heavy chain according to EU numbering, and Amino acids other than glycine at positions corresponding to position 236 in the human IgG1 heavy chain according to EU numbering Including, Any of the methods listed in items 49 to 54.

[0444] 56. The method of item 55, wherein the amino acid at the position corresponding to position 236 is a basic amino acid.

[0445] 57. The method of claim 56, wherein the basic amino acid is selected from the group consisting of lysine, arginine and histidine.

[0446] 58. The method of item 56 or 57, wherein the basic amino acid is arginine (G236R).

[0447] 59. The method according to any of items 55 to 58, wherein the amino acid at the position corresponding to position 234 is an aromatic amino acid.

[0448] 60. The method of item 59, wherein the aromatic amino acid is selected from the group consisting of phenylalanine, tryptophan and tyrosine.

[0449] 61. The method according to any of items 55 to 58, wherein the amino acid at the position corresponding to position 234 is a nonpolar amino acid.

[0450] 62. The method of claim 61, wherein the nonpolar amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan.

[0451] 63. The method of item 61 or 62, wherein the nonpolar amino acid is selected from the group consisting of isoleucine, proline, phenylalanine, methionine and tryptophan.

[0452] 64. The method according to any one of items 55 to 63, wherein the amino acid corresponding to position 234 is phenylalanine (L234F).

[0453] 65. The method of any of items 55 to 64, wherein the amino acid at a position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering in the heavy chain constant region of the antibody that binds to PD1 is an acidic amino acid.

[0454] 66. The method of item 65, wherein the acidic amino acid is aspartic acid or glutamic acid.

[0455] 67. The method of any of items 55 to 66, wherein the amino acid at a position corresponding to position 235 in a human IgG1 heavy chain according to EU numbering in the heavy chain constant region of the antibody that binds to PD1 is glutamic acid (L235E).

[0456] 68. The method of any of items 55 to 67, wherein the amino acids at positions corresponding to 234, 235, and 236 in the heavy chain constant region of the antibody that binds to PD1 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.

[0457] 69. The method of any of items 55 to 68, wherein the amino acid corresponding to position 234 in the heavy chain constant region of the antibody that binds to PD1 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).

[0458] 70. The method of any of items 49 to 69, wherein the heavy chain constant region of the antibody that binds to PD1 comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the HC sequence shown in SEQ ID NO:38.

[0459] 71. The method of any of items 49 to 70, wherein the heavy chain constant region of the antibody that binds to PD1 comprises the sequence shown in SEQ ID NO:38.

[0460] 72. The method according to any one of items 49 to 71, wherein the heavy chain constant region of the antibody that binds to PD1 has an isotype of IgG1.

[0461] 73. The method of any of items 49 to 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.

[0462] 74. The method of any of items 49 to 73, wherein the antibody that binds to PD1 is a monoclonal antibody, a chimeric antibody, or a humanized antibody, or a fragment of such an antibody.

[0463] 75. The method of any of items 49 to 74, wherein the antibody that binds to PD1 has reduced or depleted Fc-mediated effector function.

[0464] 76. The method of any of items 49 to 75, wherein binding of complement protein C1q to the constant region of the PD1-binding 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.

[0465] 77. The method of any of items 49 to 76, wherein binding of the PD1-binding antibody to one or more IgG Fc gamma receptors is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, compared to a wild-type antibody.

[0466] 78. The method of item 77, wherein the one or more IgG Fc gamma receptors are selected from at least one of Fc gamma RI, Fc gamma RII, and Fc gamma RIII.

[0467] 79. The method of item 77 or 78, wherein the IgG Fc gamma receptor is Fc gamma RI.

[0468] 80. The method of any of items 49 to 79, wherein the antibody that binds to PD1 does not have the ability to induce Fc gamma RI-mediated effector function, or the induced Fc gamma RI-mediated effector function is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, compared to a wild-type antibody.

[0469] 81. The method of any of items 49 to 80, wherein the antibody that binds to PD1 does not have the ability to induce at least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis, or at least one of complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis is induced to a reduced extent, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0470] 82. The method of any of items 49 to 81, wherein binding of neonatal Fc receptor (FcRn) to the antibody that binds to PD1 is not affected compared to a wild-type antibody.

[0471] 83. The method of any of items 49 to 82, wherein the antibody that binds to PD1 binds to a native epitope of PD1 present on the surface of a living cell.

[0472] 84. The method of any of items 49 to 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.

[0473] 85. The method of item 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.

[0474] 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) shown in any one of items 50 to 54.

[0475] 87. (a) the binding agent comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:4, a VL region comprising the amino acid sequence set forth in SEQ ID NO:8; (b) the antibody that binds PD1 comprises a VH domain and a VL domain, wherein the VH domain comprises or has a sequence set forth in SEQ ID NO:56, and the VL domain comprises or has a sequence set forth in SEQ ID NO:57. Any of the methods listed in items 49 to 86.

[0476] 88. (a) the binding agent is an antibody comprising a VH region comprising the amino acid sequence set forth in SEQ ID NO:4, a VL region comprising the amino acid sequence set forth in SEQ ID NO:8, a CH region comprising the amino acid sequence set forth in SEQ ID NO:15, and a CL region comprising the amino acid sequence set forth in SEQ ID NO:17; (b) the antibody that binds to PD1 comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:56, a VL region comprising the amino acid sequence set forth in SEQ ID NO:57, a CH region comprising the amino acid sequence set forth in SEQ ID NO:38, and a CL region comprising the amino acid sequence set forth in SEQ ID NO:42; Any of the methods listed in items 49 to 87.

[0477] 89. The method of any of items 1 to 41, wherein the PD1 / PD-L1 inhibitor is a multispecific antibody, e.g. a bispecific antibody.

[0478] 90. The method of item 89, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor comprising a first binding region that binds to CD137 and a second binding region that binds to PD-L1.

[0479] 91. The method of item 90, wherein the CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO:97.

[0480] 92. (a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:79, and a light chain variable region (VL) comprising 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) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:86, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:90; Method of item 90 or 91.

[0481] 93. The method of any of items 90 to 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 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 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 NO:92, respectively.

[0482] 94. (a) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:79 and a light chain variable region (VL) comprising the amino acid sequence set forth in 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 set forth in SEQ ID NO:86 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:90; Any of the methods listed in items 90 to 93.

[0483] 95. The PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm; The first binding arm comprises: (i) a polypeptide comprising the first heavy chain variable region (VH) and a first heavy chain constant region (CH); and (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). Includes; and the second binding arm is (iii) a polypeptide comprising the second heavy chain variable region (VH) and a second heavy chain constant region (CH); and (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). Including, Any of the methods listed in items 90 to 94.

[0484] 96. The PD-L1 inhibitor, (i) a first heavy chain and a light chain comprising the antigen-binding region having the ability to bind to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and (ii) a second heavy chain and a light chain comprising the antigen-binding region having the ability to bind to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region, and the second light chain comprises a second light chain constant region. Including, Any of the methods listed in items 90 to 95.

[0485] 97. The method of item 95 or 96, wherein (i) the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said first heavy chain and the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said second heavy chain.

[0486] 98. The method of any of items 95 to 97, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E in the first and second heavy chains, respectively.

[0487] 99. The method of any of items 95 to 98, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in the first and second heavy chain constant regions (HC).

[0488] 100. The method of any of items 95 to 99, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0489] 101. The method of any of items 95 to 100, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, respectively, and (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0490] 102. The first and / or second heavy chain, e.g., the constant region of the second heavy chain, (a) the sequence depicted 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) a sequence having at most six substitutions, such as at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions or at most one substitution, compared to the amino acid sequence defined in (a) or (b); The amino acid sequence of the present invention is selected from the group consisting of: Any of the methods listed in items 95 to 101.

[0491] 103. The constant region of the first and / or second heavy chain, e.g., the first heavy chain, comprises: (a) the sequence depicted 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) a sequence having at most six substitutions, such as at most five substitutions, at most four substitutions, at most three substitutions, at most two substitutions or at most one substitution, compared to the amino acid sequence defined in (a) or (b). The amino acid sequence of the present invention is selected from the group consisting of: Any of the methods listed in items 95 to 102.

[0492] 104. The method of any of items 95 to 103, wherein the PD-L1 inhibitor comprises a kappa (κ) light chain constant region.

[0493] 105. The method of any of items 95 to 104, wherein the PD-L1 inhibitor comprises a lambda (λ) light chain constant region.

[0494] 106. The method of any of items 95 to 105, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0495] 107. The method of any of items 95 to 106, wherein the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0496] 108. The method of any of items 95 to 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.

[0497] 109. The kappa (κ) light chain is (a) the sequence shown in SEQ ID NO:16; (b) a subsequence of the sequence in (a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) a sequence 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). The amino acid sequence is selected from the group consisting of Any of the methods described in items 104 to 108.

[0498] 110. The lambda (λ) light chain: (a) the sequence shown 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) a sequence 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). The amino acid sequence is selected from the group consisting of Any of the methods listed in items 105 to 109.

[0499] 111. The method of any of items 90 to 110, wherein the PD-L1 inhibitor is an antibody of the IgG1m(f) allotype.

[0500] 112. The method of any of items 90 to 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 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.

[0501] 113. The method of any of items 90 to 112, wherein the PD-L1 inhibitor is akasunlimab or a biosimilar thereof.

[0502] 114. (a) the binding agent comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising 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) 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 (c) 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; Any of the methods listed in items 90 to 113.

[0503] 115. (a) the binding agent comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:4, a VL region comprising the amino acid sequence set forth in SEQ ID NO:8; (b) the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:79 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:83; and (c) the second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:86 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:90; Any of the methods listed in items 90 to 114.

[0504] 116. (a) the binding agent is an antibody comprising a VH region comprising the amino acid sequence set forth in SEQ ID NO:4, a VL region comprising the amino acid sequence set forth in SEQ ID NO:8, a CH region comprising the amino acid sequence set forth in SEQ ID NO:15, and a CL region comprising the amino acid sequence set forth in SEQ ID NO:17; (b) the PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises the first binding region, and the second binding arm comprises the second binding region; (c) the first binding arm of the PD-L1 inhibitor comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO:79, a VL region comprising the amino acid sequence set forth in SEQ ID NO:83; a CH region comprising the amino acid sequence set forth in SEQ ID NO:95, and a CL region comprising the amino acid sequence set forth in SEQ ID NO:16; and (d) the second binding arm of the PD-L1 inhibitor comprises a VH region comprising an amino acid sequence set forth in SEQ ID NO:86, a VL region comprising an amino acid sequence set forth in SEQ ID NO:90, a CH region comprising an amino acid sequence set forth in SEQ ID NO:96, and a CL region comprising an amino acid sequence set forth in SEQ ID NO:17; Any of the methods listed in items 90 to 115.

[0505] 117. (a) 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; (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 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. Any of the methods listed in items 90 to 116.

[0506] 118. (a) 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; (b) the PD-L1 inhibitor is akasunlimab or a biosimilar thereof; Any of the methods listed in items 90 to 117.

[0507] 119. The method of any of items 1 to 38, wherein the PD1 / PD-L1 inhibitor is a PD1 inhibitor selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-224, AMP-514, or their respective biosimilars.

[0508] 120. The method of any of items 1 to 38, wherein the PD1 inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-514, or their respective biosimilars.

[0509] 121. The method of any of items 1 to 38, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, akasunlimab, AUNP12, CA-170, BMS-986189, or their respective biosimilars.

[0510] 122. The method of any of items 1 to 38, wherein the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, akasunlimab, or a biosimilar thereof.

[0511] 123. The method of any of the preceding items, wherein the subject is a human subject.

[0512] 124. The method of any of the preceding items, wherein the tumor or cancer is a solid tumor.

[0513] 125. Any of the methods described above, wherein the tumor is a PD-L1 positive tumor.

[0514] 126. The method of any of the preceding items, wherein the tumor or cancer is head and neck squamous cell carcinoma (HNSCC), e.g., HNSCC of the oral cavity, pharynx, or larynx.

[0515] 127. The method of item 126, wherein the HNSCC is recurrent, unresectable, or metastatic.

[0516] 128. The method of any of items 1 to 125, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

[0517] 129. The method of item 128, wherein the NSCLC is recurrent, unresectable or metastatic.

[0518] 130. The method of items 128 or 129, wherein the NSCLC does not have an epidermal growth factor (EGFR) sensitizing mutation and / or an anaplastic lymphoma (ALK) translocation and / or a ROS1 rearrangement.

[0519] 131. Any of the methods of items 128 to 130, wherein the NSCLC is NTRK1 / 2 / 3 (neurotrophin 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 received prior treatment with the respective targeted therapy.

[0520] 132. The method of any of the preceding items, wherein the subject has undergone prior treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD1 antibody or anti-PD-L1 antibody, preferably at least two doses of a PD1 inhibitor or PD-L1 inhibitor.

[0521] 133. The method of any of the preceding items, wherein the subject has undergone prior treatment with a platinum-based therapy, or an alternative chemotherapy if platinum is ineligible, such as a gemcitabine-containing regimen.

[0522] 134. The method of any of the preceding items, wherein the tumor or cancer is a tumor or cancer that has recurred and / or progressed after treatment, e.g., systemic treatment with a checkpoint inhibitor.

[0523] 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.

[0524] 136. The method of any of the preceding items, wherein the cancer or tumor is recurrent and / or 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.

[0525] 137. The method of any of the preceding items, wherein the last previous treatment was 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.

[0526] 138. The method of any of the preceding items, wherein the time from progression on last treatment with a PD1 inhibitor or PD-L1 inhibitor, e.g., an anti-PD1 antibody or an anti-PD-L1 antibody, is 6 months or less.

[0527] 139. The method of any of the preceding items, wherein the time since the last dose 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 previous treatment is 6 months or less.

[0528] 140. The cancer or tumor is recurrent and / or refractory, or the subject is (i) treatment with an anti-PD1 antibody or an anti-PD-L1 antibody followed by platinum doublet chemotherapy; or (ii) Treatment with anti-PD1 antibody or anti-PD-L1 antibody after platinum doublet chemotherapy The method of any of the preceding items, proceeding during or after

[0529] 141. (i) a binding agent comprising at least one binding domain that binds to CD27; and (ii) PD1 / PD-L1 inhibitor Including the kit.

[0530] 142. The kit of item 141, wherein the binding agent is as defined in any one of items 1 to 140 and / or the PD1 / PD-L1 inhibitor is as defined in any one of items 1 to 140.

[0531] 143. The kit of 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, e.g. for intravenous injection or infusion.

[0532] 144. The kit of any of items 141 to 143 for use in a method for reducing or preventing the progression of a tumor or for treating cancer in a subject.

[0533] 145. A kit for use according to item 144, wherein the tumor or cancer is as defined in any one of items 1 to 140, and / or the subject is as defined in any one of items 1 to 140, and / or the method is as defined in any one of items 1 to 140.

[0534] 146. (i) a binding agent comprising at least one binding domain that binds to CD27; (ii) a PD1 / PD-L1 inhibitor; and (iii) optionally, a pharma- ceutically acceptable carrier; 23. A pharmaceutical composition comprising:

[0535] 147. The pharmaceutical composition of item 146, wherein the binding agent is as defined in any one of items 1 to 140 and / or the PD1 / PD-L1 inhibitor is as defined in any one of items 1 to 140.

[0536] 148. The pharmaceutical composition of item 146 or 147 for use in a method for reducing or preventing the progression of a tumor or for treating cancer in a subject.

[0537] 149. The pharmaceutical composition for use of item 148, wherein the tumor or cancer is as defined in any one of items 1 to 140, and / or the subject is as defined in any one of items 1 to 140, and / or the method is as defined in any one of items 1 to 140.

[0538] 150. A binding agent for use in a method for reducing or preventing the progression of a tumor or treating cancer in a subject, the method comprising the steps of administering to the subject: (i) the binding agent comprising at least one binding region that binds to CD27; and (ii) a PD1 / PD-L1 inhibitor.

[0539] 151. The binder for use of item 150, wherein the method is as defined in any one of items 1 to 140, and / or the binder is as defined in any one of items 1 to 140, and / or the PD1 / PD-L1 inhibitor is as defined in any one of items 1 to 140.

[0540] 152. A PD1 / PD-L1 inhibitor for use in a method for reducing or preventing the progression of a tumor or for treating cancer in a subject, the method comprising the steps of: (i) a binding agent comprising at least one binding region that binds to CD27; and (ii) administering the PD1 / PD-L1 inhibitor to the subject.

[0541] 153. The PD1 / PD-L1 inhibitor for use of item 152, wherein the method is as defined in any one of items 1 to 140, and / or the binding agent is as defined in any one of items 1 to 140, and / or the PD1 / PD-L1 inhibitor is as defined in any one of items 1 to 140.

[0542] Further aspects of the present disclosure are disclosed herein. EXAMPLES

[0543] Example 1: Generation of DuoBody-PD-L1x4-1BB and anti-human CD27 antibodies and their Fc variants The generation of anti-human CD27 antibodies through immunization and hybridoma generation was performed at Aldevron GmbH (Freiburg, Germany). cDNA encoding human CD27 (full length and ECD) was cloned into Aldevron's proprietary expression plasmids. Anti-CD27 antibodies were generated by immunization of OmniRat animals (transgenic rats expressing a diversified repertoire of antibodies with fully human idiotypes; Ligand Pharmaceuticals Inc.) using intradermal application of human CD27 cDNA-coated gold particles using a handheld device for particle bombardment ("gene gun"). Serum samples were collected after the immunization series and tested by flow cytometry in HEK cells transiently transfected with the above-mentioned expression plasmid for full-length human CD27 expression. Antibody-producing cells were isolated from rat spleens and fused with mouse myeloma cells (Ag8) according to standard procedures. RNA from hybridomas producing CD27-specific antibodies was extracted for sequencing.

[0544] From the panel of 71 CD27 antibodies, six antibodies were selected for further characterization based on diversity in binding to primary T cells and in in vitro CD27 binding competition assays. 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.

[0545] The variable regions, which may have single point mutations to remove amino acid residues that were deemed disadvantageous for production of the heavy and light chains of interest (e.g., free cysteines or glycosylation sites), were gene synthesized and cloned into expression vectors containing framework sequences for a human antibody light chain and a human IgG1 heavy chain.

[0546] Six different antibody Fc variants were generated by introduction of one or more of the following amino acid mutations according to Eu numbering: E345R, E430G, P329R, G237A, K326A, E333A (see 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) appeared to have the most optimal biological properties. The sequences of prior art CD27 targeting antibodies used herein as benchmarks were obtained as follows: IgG1-CD27-15 (WO2012004367; SEQ ID 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).

[0547] DuoBody-PD-L1x4-1BB is a bispecific antibody based on the DuoBody technology platform (WO2011131746A2) that binds to 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:92). As a control antibody, the anti-HIV gp120 antibody IgG1-b12 was used in the present application (Barbas et al., J Mol Biol 1993 230:812-823; VH: SEQ ID NO 68 of the present application, VL: SEQ ID NO 72).

[0548] Table 1: List of amino acid sequences TIFF2025516631000010.tif49166TIFF2025516631000011.tif226166TIFF2025516631000012.tif221166TIFF2025516631000013.ti f225166TIFF2025516631000014.tif225166TIFF2025516631000015.tif225166TIFF2025516631000016.tif227166TIFF20255166310 00017.tif220166TIFF2025516631000018.tif224166TIFF2025516631000019.tif227166TIFF2025516631000020.tif226166TIFF202 5516631000021.tif225166TIFF2025516631000022.tif227166TIFF2025516631000023.tif229166TIFF2025516631000024.tif229166

[0549] Example 2: Agonistic activity of anti-CD27 antibodies in a CD27 activation reporter cell assay The CD27 agonist activity of different anti-CD27 antibodies with and without the E345R or E430G hexamerization-enhancing Fc mutations was measured using the CD27 Thaw and Use Bioassay kit (Promega, Custom Assay Services, CAS#CS1979A25). The kit contains NF-κB reporter-Jurkat recombinant cells expressing the firefly luciferase gene under the control of NF-κB response elements along with constitutive expression of human CD27, which was used essentially according to the manufacturer's instructions. Briefly, Thaw-and-Use GloResponse NFκB-luc2 / CD27 cells were thawed and incubated with a dilution series of antibodies (final concentration range 0.04–20 μg / mL) in Bio-Glo Luciferase Assay Buffer in 96-well flat-bottom culture plates (PerkinElmer, catalog #6005680) for 6 h at 37 °C, 5% CO2. The anti-CD27 antibody was wild-type (WT *) IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E, IgG1-CD27-F, and their respective variants harboring E430G or E345R mutations. The anti-CD27 benchmark antibodies were IgG1-CD27-131A (WT and E430G variants) and non-hexamerizing IgG1-CD27-15 (IgG1-CD27-15-P329R-E345R-K439E; harboring a combination of Fc mutations that prevent hexamerization, so the mutations are not functionally relevant in the context of this experiment and are therefore referred to as WT in the figures) as well as the hexamerizing variant of IgG1-CD27-15 containing the E345R mutation. Anti-HIV gp120 human antibody, IgG1-b12-E345R, was used as a non-binding negative control antibody (control). After antibody incubation, Bio-Glo Luciferase Assay Reagent (equilibrated to RT) was added to each well and incubated at RT for 5-10 min. 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.

[0550] Introduction of hexamerization-enhancing Fc mutations (E345R or E430G) resulted in enhanced CD27 agonism for antibody clones IgG1-CD27-A to -E as well as the benchmark antibodies IgG1-CD27-131A (tested with E430G) and IgG1-CD27-15 (tested with E345R) compared to the corresponding WT antibodies (Figure 1).

[0551] IgG-CD27-A, B and C demonstrated enhanced CD27 agonist activity after introduction of E430G or E345R at all concentrations tested, whereas IgG1-CD27-D and E variants containing hexamerization enhancing mutations showed no increase in agonism at the lowest antibody concentrations. IgG1-CD27-F variants with E430G or E345R mutations showed enhanced CD27 agonism only at the highest antibody concentrations tested. For variants IgG1-CD27-A to -E, introduction of the E345R mutation resulted in stronger CD27 activation than the E430G mutation. Antibodies IgG1-CD27-A to -E with the E345R mutation showed higher or similar CD27 activation levels compared to IgG1-CD27-131A with the E430G mutation or CD27-15 with the E345R mutation, respectively. * The WT antibodies for IgG1-CD27-B and IgG1-CD27-F had the F405L mutation in the IgG Fc domain, which is not functionally relevant in the context of this experiment.

[0552] Example 3: Binding affinity of anti-human CD27 antibodies for recombinant human, mouse and cynomolgus CD27 The binding affinity of five anti-human CD27 IgG1 antibodies (IgG1-CD27-A, -B, -C, -D and -E) for recombinant human, cynomolgus and mouse CD27 proteins was determined using label-free biolayer interferometry on an Octet HTX instrument (ForteBio, Portsmouth, UK). Experiments were performed using bispecific antibodies that contain 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 and non-binding antibodies (as described in Labrijn AF et al., Nat Protoc. 2014 Oct;9(10):2450-63).

[0553] To determine the affinity of CD27 antibodies for human and mouse CD27, 100 nM of recombinant His-tagged mouse or human CD27 protein (Sino Biological, Catalog #10039-H08B1 [human], Catalog #50110-M08H [mouse]) was loaded onto a preconditioned anti-Penta-HIS (HIS1K) biosensor (ForteBio, Catalog #18-5120) for 600 seconds.

[0554] To evaluate the affinity of CD27 antibodies for cynomolgus CD27, 5 μg / mL of recombinant cynomolgus CD27-Fc fusion protein (R&D systems, catalog #9904-CD-100) was loaded onto an activated Amine Reactive 2nd Generation (AR2G) biosensor (ForteBio, catalog #18-5092).

[0555] After a 300 second baseline measurement in Sample Diluent (ForteBio, Cat #18-1104), CD27 antibody association (200 seconds) and dissociation (1,000 seconds) were determined for an antibody concentration series from 0.78 to 800 nM with 2-fold dilution steps in Sample Diluent. An antibody molecular mass of 150 kDa was used for calculations. The reference sensor was incubated with Sample Diluent.

[0556] Data were acquired using Data Acquisition Software v11.1.1.19 (ForteBio) and analyzed using Data Analysis Software v9.0.0.14 (ForteBio). Data traces were corrected for each antibody by subtraction of the reference sensor. The Y-axis was aligned to the last 10 seconds of baseline, and Interstep Correction alignment for dissociation and Savitzky-Golay filtering were applied. Data traces were excluded from analysis if the response was <0.05 nM and the calculated equilibrium was close to saturation (Req / Rmax>95% using a dissociation time of 50 seconds). Data were fitted with a 1:1 model with the window of interest for the association set at 200 seconds and the dissociation time set at 50 seconds. Dissociation times were calculated using the coefficient of determination (R 2 ) (preferentially >0.98), were selected based on visual inspection of the curves and a signal decay of at least 5% during the association step.

[0557] The affinity for human CD27 was determined using a K in the nanomolar range. D The K values could be accurately determined for three CD27 antibodies (IgG1-CD27-A, -B, -C) along with their K values (Table 2). For IgG1-CD27-D and -E, biolayer interferometry experiments confirmed binding to human CD27 with similar ranges of affinities, although the exact K values could not be determined due to suboptimal curve fitting. D It was not possible to calculate values (shown in Table 2).

[0558] IgG1-CD27-A and -B also have the same K range as human CD27. D Results with IgG1-CD27-C, -D and -E also confirmed binding to cynomolgus CD27 with a similar range of affinities, although the exact K values were unclear due to suboptimal curve fitting. D It was not possible to calculate values (shown in Table 2).

[0559] Binding to recombinant mouse CD27 was only observed for the antibody IgG1-CD27-C.

[0560] Table 2. Binding affinities of IgG1-CD27-A to -E antibodies to CD27 from the indicated species TIFF2025516631000025.tif73166 * : Binding was observed, but KD, k on and k dis is a low confidence value due to suboptimal curve fitting resulting in unreliable interpretation using the 1:1 model. nb: no binding was observed.

[0561] Example 4: Binding of anti-CD27 antibodies to cell surface expressed human and cynomolgus CD27 Anti-CD27 antibodies IgG1-CD27-A to -E against human and cynomolgus monkey CD27 expressed on the cell surface * and IgG1-CD27-131A of the prior art * Binding of was analyzed by flow cytometry using transiently transfected HEK293F cells and primary T cells that endogenously express CD27. The non-binding control antibody IgG1-b12-FEAR was used as a negative control antibody.

[0562] FreeStyle 293-F suspension cells (HEK293F; ThermoFisher, catalog #R79007) were transiently transfected with the mammalian expression vector pSB encoding full-length human or cynomolgus CD27 using 293fectin Transfection Reagent (ThermoFisher, catalog #12347019) according to the manufacturer's instructions.

[0563] 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, catalog #S-1135) by low-density gradient centrifugation using Lymphocyte Separation Medium (LSM; Corning, catalog #25-072CV) according to the manufacturer's instructions.

[0564] Cells were seeded in 96-well plates (100,000 cells / well; Greiner Bio-one, catalog #650180) for sequential incubations with intervening washing steps using FACS buffer consisting of PBS (Lonza, catalog #BE17-517Q) + 1% BSA (Roche, catalog #10735086001) + 0.02% sodium azide (Bio-World, catalog #41920044-3). The following incubations were applied: antibody concentration series (final concentrations of 0.0001-10 μg / mL) for 30 min at 4 °C; live / dead marker FVS510 (BD, Cat #564406, dilution of 1:1,000 in PBS) for 20 min at RT; PE-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, Cat #109-116-098, dilution of 1:500) for 30 min at 4 °C; and anti-CD3 antibodies for T cell identification (anti-human CD3: BD, Cat #555335, dilution of 1:10; anti-cynomolgus CD3: Miltenyi, Cat #130-091-998, dilution of 1:10) for 30 min at 4 °C. All samples were analyzed on a FACSCelesta flow cytometer (BD) and FlowJo software. Data were processed and visualized using GraphPad Prism.

[0565] All tested antibodies showed dose-dependent binding to human CD27 in both human T cells and transfected HEK293F cells (Figure 2A, Figure 2B). The highest maximal binding was observed for IgG1-CD27-B and IgG1-CD27-C, compared to intermediate binding for IgG1-CD27-A and IgG1-CD27-131A, and low binding for IgG1-CD27-D and IgG1-CD27-E, with the difference being most pronounced using human T cells. For binding to cynomolgus monkey CD27 T cells, the highest binding was observed for IgG1-CD27-B, followed by Ig1-CD27-131A and IgG1-CD27-A. Lower binding was observed for IgG1-CD27-D and -E, while IgG1-CD27-C showed the least binding to cynomolgus monkey T cells. All CD27 antibodies showed dose-dependent binding to cynomolgus CD27-transfected HEK cells. The highest maximal binding was observed for IgG1-CD27-B and IgG1-CD27-131-A, and somewhat lower binding was observed for IgG1-CD27-A, -D, and -E. IgG1-CD27-C showed the lowest binding to cynomolgus CD27-transfected HEK cells (Figure 2C, Figure 2D).

[0566] In conclusion, IgG1-CD27-A and IgG1-CD27-B showed dose-dependent binding to human and cynomolgus CD27 expressed endogenously on human or cynomolgus T cells and transiently expressed in transfected HEK cells. IgG1-CD27-A and IgG-CD27-131A showed comparable binding to human T cells, while IgG1-CD27-B showed higher maximal binding. *Note: IgG1-CD27-A, -B, -C, -D and -E had the mutations F405L-L234F-L235E-D265A in the IgG Fc domain that are not functionally relevant in the context of this experiment. IgG1-CD27-131A had the functionally irrelevant F405L mutation in the IgG1 Fc domain.

[0567] Example 5: Binding of anti-CD27 antibodies to the native human CD27-A59T variant Approximately 19% of the human population expresses a naturally occurring CD27 variant harboring the A59T mutation in the extracellular domain (SEQ ID NO. 2). Binding to human CD27-A59T was confirmed by the anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, and IgG1-CD27-D. * and benchmark IgG1-CD27-131A were tested by flow cytometry. The non-binding antibody IgG1-b12-FEAL was used as a negative control antibody. Transiently transfected HEK293F cells (15,000 cells / well) expressing human CD27-A59T were incubated with the primary test antibodies IgG1-CD27-A to -C, the non-binding control antibody IgG1-b12 (control), and a concentration series (0.0001 to 10 μg / mL using 10-fold dilution steps) of the prior art benchmark IgG-CD27-131A, which has previously been described (WO2018 / 058022) to bind to CD27-A59T. After incubation, the antibodies were PE-labeled using a 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.

[0568] 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 between the different antibodies (Figure 3). *Note: IgG1-CD27-A, -B and -C had the mutations F405L-L234F-L235E-D265A in the IgG Fc domain that are not functionally relevant in the context of this experiment. IgG1-CD27-131A had the functionally irrelevant F405L mutation in the IgG1 Fc domain.

[0569] Example 6: Induction of human T cell proliferation by anti-CD27 antibodies Because enhancing IgG hexamerization through Fc-Fc interactions with the introduction of the E345R or E430G mutation enhanced 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 mutation to increase proliferation of TCR-activated T cells was tested in vitro.

[0570] Additionally, Fc mutations reported to reduce binding to C1q and FcγR (G237A or P329R) or enhance binding to C1q (K326A / E333A double mutation) were introduced to test their potential effect on the CD27 agonist activity of CD27 antibodies with E345R or E430G mutations. The K326A / E333A double mutation was previously shown to enhance C1q binding and contribute to the enhanced agonist activity of a DR5-specific humanized IgG1 antibody containing Fc-Fc interaction enhancing mutations (WO2018 / 146317A1). In addition to E430G or E345R, the mutations G237A, P329R, or K326A / E333A were introduced into IgG1-CD27-A, IgG1-CD27-B and IgG1-C (Table 3) and their effects on T cell proliferation were determined using human PBMCs obtained from healthy donors (Sanquin Blood Bank, the Netherlands).

[0571] Table 3. Mutations in the Fc domain of antibodies IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C and their biological effects TIFF2025516631000026.tif93166 * X in IgG1-CD27-X refers to the IgG1-CD27 clones IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C.

[0572] PBMCs, 5 x 10 in PBS 6 The PBMCs were resuspended at a density of 100,000 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 in 96-well round-bottom plates (Greiner Bio-one, Cat#650180) in T-cell Activation Medium (ATCC, Cat#80528190) supplemented with 5% Normal Human Serum (NHS; Sanquin, Product#B0625) with 0.1 μg / mL of anti-CD3 antibody clone UCHT1 (Stemcell Technologies, Cat#60011) to activate T cells, and CD27 antibody (final concentration of 1 μg / mL) for 96 h at 37°C / 5% CO2. CD4 by flow cytometry was analyzed. + and CD8 +For identification of viable cells in T cell subsets, cells were sequentially incubated with the live / dead marker FVS510 (1:1,000) for 20 min at RT and with a staining mix for lymphocyte markers containing 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) for 30 min in the dark at 4 °C. Samples were measured on a FACSCelesta (BD Biosciences) flow cytometer and viable CD4 + and CD8 + T cell subsets (FVS510 - CD14 - CD19 - CD4 + and FVS510 - CD14 - CD19 - CD8 + ) were analyzed as a readout for T cell proliferation using FlowJo 10 software. T cell proliferation was expressed as a percentage of cell proliferation or mitotic index, both calculated by using FlowJo software (version 10). The percentage of cell proliferation (division) was calculated based on the percentage of cells that underwent CFSE dilution (CFSE low peaks ). Mitotic index is the average number of divisions a cell has undergone. Heat maps were generated using GraphPad Prism version 8. Proliferation assays were performed using PBMCs from four different healthy donors.

[0573] IgG1-CD27-A, -B and -C variants carrying the E430G or E345R mutations upregulated CD8 +Introduction of additional mutations (P329R, G237A or K326A / E333A) into the IgG1-CD27-A, -B or -C variants carrying the E430G mutation induced a small increase in CD8 T cell proliferation across four PBMC donors. + In contrast, introduction of the P329R mutation into IgG1-CD27-A and IgG1-CD27-C variants carrying the E345R mutation suppressed activation of CD8 + Consistently, they increased the ability of IgG1-CD27-A to enhance T cell proliferation, which was particularly true for IgG1-CD27-A, which increased the measured CD8 + T cell proliferation was comparable for IgG-CD27-A-E345R, IgG1-CD27-B-E345R and IgG1-CD27-C-E345R in each of the donors, while introduction of the additional P329R mutation significantly increased CD8 proliferation for clone IgG1-CD27-A-E345R compared to IgG1-CD27-B-E345R or IgG1-CD27-C-E345R. + This consistently led to higher increases in T cell proliferation. + The effect of the E345R mutation in combination with the P329R mutation on T cell proliferation was consistently greater for clone IgG1-CD27-A than for IgG1-CD27-B and IgG1-CD27-C. Across all antibody variants tested, IgG1-CD27-A-E345R-P329R significantly increased CD8 T cell proliferation in all donors. + induced the greatest increase in T cell proliferation (Figure 4A).

[0574] The addition of the mutations G237A or K326A-E333A to CD27 antibody variants with the E345R mutation significantly increased CD8 expression in any of the clones tested compared to antibodies containing the single mutation E345R. + It did not increase or only minimally increased T cell proliferation (Figure 4A).

[0575] CD4 +Also in T cells, the highest and most consistent increase in T cell proliferation was observed in the presence of IgG1-CD27-A-E345R-P329R (Figure 4B). + T cell proliferation was generally comparable between IgG1-CD27-A, -B and -C variants carrying only the E430G or E345R mutations, whereas introduction of the additional P329R mutation significantly increased CD4 T cell proliferation for IgG1-CD27-A variants carrying the E345R variant compared to IgG1-CD27-A-E430G or IgG1-CD27-B or -C variants carrying either the E430G or E345R mutations. + This led to a greater increase in T cell proliferation. This effect was observed in three of the four donors tested. In donor 1, CD4 + The effects of additional mutations besides E430G or E345R on T cell proliferation were generally small, and the effects observed in this donor were not reproduced in the other three donors.

[0576] The combination of E345R with the P329R mutation also inhibited CD4 + Although it consistently increased T cell proliferation, 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. + A modest increase in T cell proliferation was observed for IgG1-CD27-B-E345R-P329R compared to IgG1-CD27-B-E345R in two of four donors.

[0577] Introduction of P329R, G327A or K326A / E333A mutations into IgG1-CD27-A, -B or -C variants carrying the E430G mutation enhances CD4 +Similarly, no or inconsistent effects were observed following introduction of G327A or K326A / E333A in IgG1-CD27-A, -B or -C variants carrying the E345R mutation.

[0578] In summary, IgG1-CD27-A-E345R-P329R inhibits activated CD8 + and CD4 + We demonstrated that IgG1-CD27-A-E345R-P329R consistently induced the highest increase in T cell proliferation and induced the most efficient CD27 agonism. DR5-specific, hexamerization-enhanced antibodies with the P329R mutation have previously shown a reduced ability to induce DR5 agonism compared to DR5-specific, hexamerization-enhanced antibodies without the P329R mutation (Overdijk et al, Mol Canc Ther 2020). Therefore, it was considered surprising that introducing the P329R mutation in addition to the E345R mutation in IgG1-CD27-A would enhance CD27 agonist activity. Furthermore, it is not known why the combined effect of the E345R+P329R mutations was consistently greater for IgG1-CD27-A than for IgG1-CD27-B or IgG1-CD27-C.

[0579] Example 7: Induction of human T cell proliferation by anti-CD27 antibody IgG1-CD27-A-P329R-E345R TCR stimulated human CD4 + and CD8 + The ability of IgG1-CD27-A-P329R-E345R to increase T cell proliferation was analyzed in a CSFE dilution assay using human healthy donor PBMCs and compared with the prior art anti-CD27 clone 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 using T cells without anti-CD3 stimulation were included to test potential CD27 agonist activity of the antibodies in the absence of T cell receptor activation (Figure 5A and Figure 5B). Such activity is undesirable as it poses a safety risk if the antibody is able to induce proliferation of resting T cells.

[0580] Using FlowJo software, the percentage of proliferated T cells (Figure 5A, Figure 5B, Figure 5C, Figure 5D) was calculated as the percentage of cells with a reduction in CFSE fluorescence, indicative of cell division. The expansion proliferation index (Figure 5E and Figure 5F) identified the fold increase in cells in a well and was calculated using the Proliferation Modeling tool in FlowJo version 10. Manual adjustments to the peaks were made when necessary to more consistently define the number of peaks present.

[0581] Neither the CD27 antibodies of the invention nor the prior art antibodies tested here induced proliferation of unstimulated T cells, i.e., in the absence of CD3 cross-linking (FIGS. 5A and 5B).

[0582] Most CD27 antibodies inhibited activating CD4 at the highest antibody concentrations tested. + and CD8 + The antibody IgG1-CD27-A-P329R-E345R of the present invention induced a certain degree of proliferation of T cells (FIGS. 5C and 5D). Based on this, the expansion proliferation index was calculated (FIGS. 5E and 5F). The antibody IgG1-CD27-A-P329R-E345R of the present invention showed a significantly higher in vitro CD4 T cell proliferation rate than the prior art anti-CD27 clones IgG1-CD27-131A, IgG1-CD27-CDX1127 and IgG1-CD27-BMS986215. + and CD8 + It significantly enhanced T cell proliferation. *For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants with the F405L mutation, which are not functionally relevant in the context of this experiment, were used.

[0583] Example 8: Binding of C1q to membrane-bound CD27 antibodies The P329R mutation was 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 an in vitro cellular C1q binding assay using human healthy donor T cells. The anti-HIV gp120 antibody IgG1-b12-F405L was used as a non-binding isotype control antibody (control). T cells were enriched from human healthy donor PBMCs using RosetteSep Human T cell Enrichment cocktail (Stemcell, catalog #15061) and resuspended in culture medium (RPMI 1640 [Gibco, catalog #A10491-01] supplemented with 0.1% BSA and 1% Pen / Strep [Lonza, catalog #DE17-603E]). T cells (2 × 10 6 T cells (1000 cells / well) were pre-incubated for 15 min at 37 °C in polystyrene 96-well round-bottom plates containing an antibody dilution series (eight 5-fold dilutions starting at a final assay concentration of 15 μg / mL) to allow antibody binding to T cells. Cells were then chilled on ice, supplemented with NHS as a source of human C1q (final assay concentration of 20% NHS) and incubated on ice for 45 min. Cells were subsequently incubated with FITC-labeled rabbit anti-human C1q antibody (DAKO, catalog #F0254; 20 μg / mL) for 30 min on ice and resuspended in FACS buffer containing TO-PRO-3 (ThermoFisher, catalog #T3605; 1:5,000 dilution). C1q binding was determined by flow cytometry measuring the FITC signal in live cells.

[0584] Membrane-bound WT IgG1-CD27-A antibody did not exhibit 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 CD27 antibodies on the T cell surface (Figure 6), consistent with increased binding avidity of hexameric C1q protein to hexameric antibody ring structures on the cell surface. Introduction of the P329R mutation in IgG1-CD27-A-E345R (IgG1-CD27-A-P329R-E345R) resulted in loss of C1q binding (Figure 6), demonstrating that IgG1-CD27-A-P329R-E345R cannot bind C1q.

[0585] These data indicate that IgG1-CD27-A-P329R-E345R is unable to bind C1q upon binding to CD27 on the cell surface of T cells. This indicates that C1q binding does not contribute to the antibody-induced CD27 agonist activity of IgG1-CD27-A-P329R-E345R. This is in contrast to what has been previously described for other hexamerization-enhancing agonist antibodies. Furthermore, the lack of C1q binding indicates that IgG1-CD27-A-P329R-E345R is unable to activate the classical pathway of complement activation. Therefore, it is expected that IgG1-CD27-A-P329R-E345R will not induce complement activation and CDC in T cells, where these activities are undesirable.

[0586] Example 9: Binding of anti-CD27 antibodies to human Fc receptors Binding of IgG1-CD27-A-P329R-E345R to human FcγR variants was analyzed using a Biacore surface plasmon resonance (SPR) system and compared to anti-HIV gp120 antibody IgG1-b12 (control). Biacore Series S Sensor Chips CM5 (Cytiva, Cat#29104988) were covalently coated with anti-His antibody using an Amine-Coupling and His Capture Kit (Cytiva, Cat#BR100050 and Cat#29234602) according to the manufacturer's instructions. Next, 125 nM of Fcγ receptors FcγRIa, FcγRIIa (167-His[H] and 167-Arg[R]), FcγRIIb, or FcγRIIIa (176-Phe[F] and 176-Val[V]) (Sino Biological, Catalog #10256-H08S-B, Catalog #10374-H27H, Catalog #10374-H27H1-B, Catalog #10259-H27H-B, Catalog #10389-H27H-B, and Catalog #10389-H27H1-B) in HBS-P+ (Cytiva, Catalog #BR100827) were captured onto the surface. After three cycles of buffer, antibody samples were injected over 36 cycles to generate binding curves using an antibody range of 0-3,000 nM for FcγRI and 0-10,000 nM for other FcγRs. Each sample analyzed on the FcR-coated surface (active surface) was also analyzed on a parallel flow cell without FcR (reference surface) that was used for background correction. Dissociation from the anti-His-coated surface was performed by regeneration of the surface using 10 mM glycine-HCl (pH 1.5) (Cytiva, catalog #BR100354). Sensorgrams were generated using Biacore Insight Evaluation software (Cytiva) and a four-parameter logistic (4PL) fit was applied to calculate the relative binding of IgG1-CD27-A-P329R-E345R to the reference sample (control).

[0587] Binding of IgG1-CD27-A-P329R-E345R to the high affinity receptor FcγRIa was strongly reduced compared to the control antibody, although some binding was observed at higher antibody concentrations (Figure 7A). IgG1-CD27-A-P329R-E345R did not bind to the human low affinity receptors FcγRIIa (Figures 7B and 7C), FcγRIIb (Figure 7D) and FcγRIIIa (Figures 7E and 7F).

[0588] In conclusion, IgG1-CD27A-P329R-E345R shows minimal or no binding to human IgG Fc receptors (FcγRIa) or (FcγRIIa, FcγRIIb, and FcγRIIIa).

[0589] Example 10: Binding of anti-CD27 antibody IgG1-CD27-A-E345R-P329R to human T cells 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 (control). Human PBMCs were isolated from buffy coats obtained from human healthy donors. PBMCs (1 × 10 5Cells were pelleted by centrifugation at 300 x g for 3 min at 4 °C. Cells were resuspended in 50 μL / well of serial antibody dilutions in FACS buffer (ranging from 0.0015 to 10 μg / mL in 3-fold dilution steps) and incubated for 30 min at 4 °C. Cells were pelleted, washed twice with FACS buffer, and incubated with FITC-conjugated secondary antibody (FITC AffiniPure F(ab')2 fragment goat anti-human IgG, F(ab')2 fragment specific, Jackson ImmunoResearch, catalog #109-096-097, diluted 1:100) in 50 μL / well for 30 min in the dark at 4 °C. Cells were pelleted again, washed twice with FACS buffer, and stained with BV711-conjugated anti-human CD19 antibody (BioLegend, catalog #302246, 1:50), AlexaFluor700-conjugated anti-human CD8a antibody (BioLegend, catalog #301028, 1:100), APC-eFluor780-conjugated anti-human CD4 antibody (Invitrogen, catalog #47-0048-42, 1:50), PE-CF594-conjugated mouse anti-human CD56 antibody (BD Biosciences, catalog #564849, 1:100), and PE-Cy7-conjugated mouse anti-human CD14 antibody (BD The cells were incubated in 50 μL / well of staining mix for lymphocyte markers containing 50 μL / well of 50-μL ...Binding curves were analyzed using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism 8 software.

[0590] The anti-CD27 antibody IgG1-CD27-A-P329R-E345R inhibits CD4 + and CD8 + It showed dose-dependent binding to healthy donor T cells with similar binding characteristics for T cells (Figure 8).

[0591] Example 11: FcγR-independent induction of CD27 cell signaling by anti-CD27 antibody IgG1-CD27-A-P329R-E345R CD27-specific monoclonal antibodies that can induce CD27 signaling independently of secondary FcγR-mediated cross-linking may be immunostimulatory in the absence of FcγR-positive cells, which is an advantage in tumors with a low frequency of FcγR-bearing cells.

[0592] The CD27 agonist activity of IgG1-CD27-A-P329R-E345R was tested in the presence or absence of FcγR-bearing cells and compared with the corresponding WT antibody IgG1-CD27-A as well as the prior art antibody IgG1-CD27-131A. * , IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 * The non-binding antibody IgG1-b12-P329R-E345R was used as a negative control (control). CD27 reporter assays were performed essentially as described in Example 2, with the exception that in this example, Thaw-and-Use GloResponse NFκB-luc2 / CD27 Jurkat cells were cultured in the presence of human FcyRIIb expressing cells that can facilitate FcγR-mediated cross-linking of membrane-bound antibodies.

[0593] Thaw-and-Use effector FcγRIIb CHO-K1 cells (Promega, Cat#JA2251) were plated undiluted or in three increasing dilutions (1 / 3, 1 / 9, 1 / 27) in 96-well flat-bottom culture plates (PerkinElmer, Cat#0815) and incubated overnight at 37°C / 5% CO2. Supernatants of adherent FcyRIIb-expressing cells were replaced by Thaw-and-Use NFκB-luc2 / CD27 Jurkat cell suspensions at fixed cell concentrations (starting with a 1:1 NFκB-luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratio for undiluted FcγRIIb CHO-K1 cells) in Bio-Glo Luciferase Assay Buffer containing serial dilutions of antibody (final concentration range 0.0002-10 μg / mL). After 6 h incubation at 37° C. / 5% CO 2 , plates were equilibrated to RT and bioluminescence was measured and presented as RLU as described in Example 2.

[0594] IgG1-CD27-A-P329R-E345R induced dose-dependent CD27 activation independent of FcγRIIb-expressing cells (Figure 9A). In contrast, the corresponding WT antibody IgG1-CD27-A, which does not have the E345R hexamerization-enhancing mutation and the P329R mutation, showed CD27 agonism only in the presence of FcγRIIb-expressing cells (Figures 9A-E). Similarly, CD27 activation by the prior art antibodies IgG1-CD27-131A, IgG1-CD27-CDX1127 and IgG1-CD27-BMS986215 was also dependent on the presence of FcγRIIb-expressing cells and gradually decreased with decreasing NFκB-luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratio (Figures 9F-J).

[0595] In conclusion, these data indicate 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 were dependent on the presence of FcγR-bearing cells to induce CD27 agonism. * For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants with the F405L mutation, which are not functionally relevant in the context of this experiment, were used.

[0596] Example 12: Pharmacokinetic (PK) analysis of anti-CD27 antibody IgG1-CD27-A-P329R-E345R in the absence of target binding studied in mice Anti-CD27 antibody IgG1-CD27-A-P329R-E345R in the absence of target binding * The pharmacokinetic characteristics of the corresponding WT antibody IgG1-CD27-A were analyzed in mice. *IgG1-CD27-A does not bind to mouse CD27 (Example 3, Table 2), therefore, experiments were designed to test the pharmacokinetic behavior of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R in vivo in the absence of target binding. The study was carried out by Crown Bioscience (China) by qualified personnel in accordance with the approved IACUC protocol and Crown Bioscience, Inc. Standard Operating Procedures. Eleven to 12 week old female SCID mice (CB-17, Vital River Laboratory Animal Technology Co., Ltd. (VR, Beijing, China; 3 mice / group) were intravenously injected with 500 μg of antibody (25 mg / kg) in an injection volume of 200 μL. 40 μL blood samples were collected at 10 min, 4 h, 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 determination of total human IgG concentrations by ELISA. 96-well ELISA plates (Greiner, Cat #655092) were filled with 2 μg / mL of anti-human IgG (Sanquin, The The anti-human IgG-coated plates were then coated overnight at 4° C. with ELISA kit (Roche, Netherlands, product #M9105, lot #8000260395) and subsequently blocked for 1 h with PBSA (PBS supplemented with 0.2% bovine serum albumin [BSA, Roche, catalog #10735086001]). The anti-human IgG-coated plates were then incubated at RT for 1 h on a plate shaker with an intermediate washing step for 1 h. Plasma samples serially diluted in Buffer (PBSA supplemented with 0.05% Tween 20 [Sigma-Aldrich, Cat. #P1379]) were sequentially incubated with a polyclonal peroxidase-conjugated goat anti-human IgG secondary antibody (Jackson, Cat. #109-035-098) for 1 h at RT, and finally with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Cat. #11112422001). The reaction was stopped by adding 2% oxalic acid (Riedel de Haen, Cat. #33506).A reference curve was generated using a dilution series of each material used for injection. The absorbance was measured at 405 nm in an EL808 microtiter plate reader (BioSPX) and the total human IgG concentration (μg / mL) was plotted.

[0597] There were no substantial differences between the PK profiles of IgG1-CD27-A-P329R-E345R and the counterpart WT antibody IgG1-CD27-A, as determined by measuring plasma IgG levels at different time points after intravenous injection in mice (Figure 10).

[0598] A steeper decline in the early (distribution) phase was observed for IgG1-CD27-A-P329R-E345R and its WT counterpart (IgG1-CD27-A) compared to that predicted for human IgG1 in mice, but the terminal clearance of both antibodies was consistent with the predicted kinetics for human wild-type IgG1 based on a two-compartment model (Bleeker WK, Teeling JL, Hack CE. Blood. 2001 Nov 15;98(10):3136-42).

[0599] Together, this demonstrates that the introduction of the P329R and E345R mutations did not affect the pharmacokinetic properties of IgG1-CD27-A in the absence of target binding. Note: The experiments described in this example used variants of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R with the F405L mutation, which is not functionally relevant in the context of this experiment.

[0600] Example 13: Induction of antibody-dependent cellular phagocytosis by anti-CD27 antibody IgG1-CD27-A-P329R-E345R Antibody-dependent cellular cytotoxicity (ADCC) is mediated primarily through FcγRIIIa expressed on NK cells, whereas 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). 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 FcγRIa-expressing immune cells, we used CTV-labeled CD27 + The ability of IgG1-CD27-A-P329R-E345R to induce ADCP was analyzed in vitro using Burkitt's lymphoma Daudi cells as target cells and human monocyte-derived macrophages (hMDMs) as effector cells (E:T=2:1).

[0601] hMDMs were isolated from PBMCs by positive selection using CD14 microbeads (Miltenyi Biotec, Cat. No. 130-050-201) according to the manufacturer's instructions. PBMCs were centrifuged (1,200 RPM, 5 min, RT) and diluted to 1.25 × 10 in ice-cold monocyte isolation buffer (PBS, 0.5% BSA, 2 mM EDTA). 7 PBMCs were resuspended at a density of 1000 ng / mL. 20 μL of CD14 microbeads were added per 80 μL of PBMC suspension and incubated with agitation for 15 min at 4°C on a roller bank. 30 mL of ice-cold monocyte isolation buffer was added and the PBMC / CD14 microbead mixture was centrifuged (300×g, 10 min, 4°C) and resuspended in 6 mL of ice-cold monocyte isolation buffer. LS columns (Miltenyi Biotec, Cat. No. 130-042-401) were rinsed with 3 mL of ice-cold monocyte isolation buffer and 3 mL of the PBMC / CD14 microbead mixture was loaded onto each column. CD14 - After draining the cells and washing the column three times with ice-cold monocyte isolation buffer, CD14 +Monocytes were harvested in 3 mL of ice-cold monocyte isolation buffer. CD14 cells were detected using ViaStain™ Viability Dye Acridine Orange / Propidium Iodide (AOPI; Nexcelom Bioscience, Cat. No. CS2-0106) on a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience). + 100 mm 200 µm plate with UpCell™ Surface to count cells and allow cell harvesting by placing plates at room temperature 2 Macrophage colony-stimulating factor (M-CSF; Gibco, catalog no. PH9501; final concentration of 50 ng / mL) and 3 mL of monocyte suspension (i.e., 2.4 × 10 6 monocytes) in Celgene® GMP DC medium (CellGenix, catalog no. 20801-0500) supplemented with 0.8 × 10 6 Macrophages were resuspended at a density of 1 × 10 cells / mL. After 3 days of incubation, 2 mL of fresh medium containing 5× M-CSF was added to the plate. After 7 days of incubation (37 °C, 5% CO2), macrophages were detached from the surface by leaving the plate at RT for 1–1.5 h. Detached macrophages were pelleted by centrifugation, counted using an AOPI, and resuspended in culture medium (RPMI 1640 with 10% DBSI) at a density of 1 × 10 6 The cells were resuspended at a density of 1000 cells / mL.

[0602] Human Burkitt's lymphoma Daudi cells (ATCC® CCL-213™) were labeled using the CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific, Cat. No. C34557) according to the manufacturer's instructions. Briefly, Cell Trace Violet (CTV) was added at 1 × 10 per mL in PBS to a final concentration of 0.2 μM. 6Daudi cells and incubated for 20 min at 37°C in the dark (15 mL incubation volume). 10 mL of DBSI was added to inactivate unbound dye. Cells were pelleted by centrifugation (300 x g, 5 min), washed in PBS, and counted in an AOPI. CTV-labeled Daudi cells were added to culture medium at 0.5 x 10 6 The cells were resuspended at a density of 1000 cells / mL.

[0603] For the ADCP assay, hMDMs (50,000 cells / well) and CTV-labeled Daudi cells (25,000 cells / well) were seeded together (E:T=2:1) in a final volume of 150 μL of culture medium in 96-well plates on ice and incubated for 4 h (37 °C, 5% CO2) with anti-CD27 antibody IgG1-CD27-A-P329R-E345R or anti-CD20 antibody IgG1-CD20 (concentration range of 0.000001-10 μg / mL in 10-fold dilutions). After incubation, 100 μL of Human BD Fc Block™ (BD Biosciences, catalog no. 564220; 1:100 in FACS buffer) was added and incubated for 10 min at 4 °C. Cells were pelleted by centrifugation (300×g, 5 min), 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 min. Cells were washed, resuspended in FACS buffer, collected and analyzed on a FACSymphony™ A3 Cell Analyzer (BD Biosciences). Data was analyzed using FlowJo software to measure viable target cell numbers and phagocytic hMDMs, and processed and visualized using GraphPad Prism software.

[0604] The percentage of viable Daudi cells for each condition was calculated by the following formula: TIFF2025516631000027.tif15128

[0605] The amount of phagocytic hMDM for each condition was determined by TO-PRO-3 - CD11b + CTV + Determined as % of cells.

[0606] IgG1-CD27-A-P329R-E345R did not increase the percentage of phagocytic hMDMs or reduce the percentage of viable Daudi cells in phagocytosis assays using hMDMs from four different human healthy donors, demonstrating that residual FcγRIa binding did not result in FcγRIa-mediated effector function for IgG1-CD27-A-P329R-E345R (data from a representative human healthy donor is shown in FIG. 11). The positive control antibody IgG1-CD20 efficiently 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.

[0607] In conclusion, residual binding to FcγRIa is consistent with CD27 + was not sufficient to induce IgG1-CD27-A-P329R-E345R-dependent ADCP of cells.

[0608] Example 14: Target-independent fluid-phase complement activation by anti-CD27 antibody IgG1-CD27-A-P329R-E345R as determined by measuring C4d deposition Antibodies with enhanced Fc-Fc interactions generally exist as monomeric IgG1 molecules in solution, which upon target binding hexamerize on the cell surface to form a C1q docking site in the case of the active Fc region (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 the introduction of the P329R mutation, which results in the lack of C1q binding for membrane-bound IgG1-CD27-A-P329R-E345R (Figure 6). To confirm that IgG1-CD27-A-P329R-E345R is unable to activate complement in solution in the absence of target binding, target-independent complement activation was examined by the determination of C4d deposition, which is considered an indicator for activation of the classical complement pathway. Fluid-phase C4d fragment deposition by IgG1-CD27-A-P329R-E345R was analyzed by enzyme-linked immunosorbent assay (ELISA) using MicroVue™ C4d Enzyme Immunoassay (EIA; Quidel, Cat. No. A008) and was performed according to the manufacturer's protocol. Heat Aggregated Gamma Globulin (HAGG; 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. Introduction of E345R / E430G / S440Y (RGY) Fc mutations in IgG1 antibodies has been described to induce the formation of hexamers in solution, resulting in fluid-phase complement activation (Diebolder, CA 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.

[0609] 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. 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 (monoclonal IgG) or 1,000 μg / mL (HAGG) and incubated for 1 h at 37 °C. In parallel, a "No Antibody" sample (no antibody, 90% NHS) and a "PBS Alone" sample (no antibody, no NHS) were included as negative controls. Samples were then diluted 1:250 in chilled Complement Specimen Diluent provided in the kit. Meanwhile, mouse anti-human C4d antibody-coated strips were placed into a 96-well plate and the assay wells were washed three times with 250–300 μL of wash buffer with a 1 min waiting step after the first wash. Test samples were added to wells (100 μL / well) and, as a negative control, only complement sample diluent (blank) was used in the ELISA. In parallel, 100 μL of standard (standard AE) and internal control provided by the kit were added to separate wells. The plate was incubated for 30 min at RT. Then, the plate was washed 5 times with the washing buffer described above. 50 μL of C4d conjugate (peroxidase-conjugated goat anti-human C4d) was added to the wells and the plate was incubated for 30 min at RT. After 5 washing steps with the washing buffer described above, 100 μL of C4d substrate [0.7% 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid diammonium salt] was added and the plate was again incubated for 30 min at RT. Finally, 50 μL of stop solution provided by the kit was added and within 1 h, the optical density was measured at 405 nm using an ELISA plate reader (EL808 BioSPX, BioTek).

[0610] IgG1-CD27-A-P329R-E345R and the control antibody IgG1-b12-P329R-E345R (with 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 (FIG. 12). In contrast, the positive control antibody IgG1-b12-RGY, known to form hexamers in solution, induced C4d deposition to the same level as HAGG.

[0611] These data indicate that IgG1-CD27-A-P329R-E345R did not induce target-independent, fluid-phase complement activation in vitro.

[0612] Example 15: Ability of anti-CD27 antibody IgG1-CD27-A-P329R-E345R to compete for ligand binding with CD70 To determine whether the anti-CD27 antibody IgG1-CD27-A-P329R-E345R interferes with the interaction of CD27 with its natural ligand CD70, binding of saturating concentrations of biotinylated recombinant human CD70 extracellular domain (ECD) to CD27 endogenously expressed on the human Burkitt's lymphoma cell line Daudi was studied in the presence and absence of excess IgG1-CD27-A-P329R-E345R.

[0613] Daudi cells (ATCC® CCL-213™) cultured in RPMI 1640 medium (Gibco, Cat. No. A10491-01) supplemented with 10% donor bovine serum with iron (DBSI; Gibco, Cat. No. 20731-030) were seeded at 50,000 cells / well in round-bottom 96-well plates (Greiner Bio One, Cat. No. 650261). Cells were pelleted by centrifugation (300×g, 3 min, 4° C.) and resuspended in FACS buffer (PBS, 1% BSA [Roche, Cat. No. 1073508600]) containing anti-CD27 or control antibodies (final concentration of 50 μg / mL). Biotinylated recombinant human CD70 ECD (Abcam, Cat. No. ab271443) was added at saturating concentration (6 μg / mL) and cells were incubated at 4° C. for 30 min.

[0614] Cells were washed twice and resuspended in FACS buffer containing Brilliant Violet (BV) 421™-labeled streptavidin (BioLegend, Cat. No. 405225; 0.0025 μg / mL final concentration) and R-Phycoerythrin (PE)-labeled polyclonal AffiniPure F(ab')2 fragment goat-anti-human IgG Fc (Jackson ImmunoResearch, Cat. No. 109 116098; 0.0025 μg / mL final concentration) for 30 minutes at 4°C. 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. Data were collected on a BD FACSymphony™ A3 flow cytometer (BD Biosciences) and analyzed using FlowJo software. For compensation, one drop of UltraComp eBeads™ Compensation Beads (Life Technologies, Cat. No. 01-2222-42) was added to each well. 2 μL of each antibody was added and the mix was incubated for 20 minutes. The plates were spun down 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 viable cells. Cells were spun down and resuspended in TO-PRO-3 diluted in FACS buffer. Data was processed and visualized using GraphPad Prism.

[0615] IgG1-CD27-A-P329R-E345R or IgG1-CD27-A inhibits CD27 +It did not block binding of CD70 ECD to Daudi cells, and CD70 binding levels were comparable to Daudi cells incubated with non-binding isotype control antibodies IgG1-b12-P329R-E345R or IgG1-b12, or cells without antibody (Figure 13). Prior art anti-CD27 antibodies IgG1-CD27-BMS986215 and IgG1-CD27-131A also showed a weak blocking effect on CD27 binding to CD70 ECD. In contrast, CD70 was unable to bind to surface CD27 on Daudi cells in the presence of prior art anti-CD27 antibody IgG1-CD27-CDX1127, previously reported to block ligand binding (Vitale et al., 2012) (Figure 13).

[0616] In conclusion, binding of IgG1-CD27-A-P329R-E345R does not block binding of CD27 by its natural ligand CD70 on Daudi cells.

[0617] Example 16: Expression of T cell activation markers upon incubation of polyclonally stimulated human PBMCs with anti-CD27 antibodies The effect of IgG1-CD27-A-P329R-E345R on the expression of T cell activation markers in polyclonally activated T cells was studied using 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 a prior art anti-CD27 antibody.

[0618] 75,000 freshly isolated PBMCs were seeded per well in cell culture medium in 96-well U-bottom plates (Greiner Bio-One). Duplicate wells were incubated simultaneously with anti-CD3 antibodies (UCHT1 clone; Stemcell; 0.1 μg / mL); and IgG1-CD27-A-P329R-E345R (0.0005 to 30 μg / mL in 3-fold dilutions); 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, duplicate control wells containing untreated (no anti-CD3 or anti-CD27 antibodies) cells were supplemented with culture medium alone. A fluorescence minus one (FMO) control was used to set the gates to identify activation marker-positive cells. For the FMO control, all antibodies used in the experiment except those corresponding to the activation markers in duplicate wells were added to 75,000 PBMCs per well from one donor activated with anti-CD3 antibodies. Untreated cells from each donor in a single well without staining antibody were included as negative controls. To detect viable cells, untreated cells from each donor were stained with 4',6-diamidino-2-phenylindole (DAPI) alone in a single well.

[0619] After 2 or 5 days of incubation (37°C, 5% CO2), plates were washed once with FACS buffer and analyzed by flow cytometry with antibodies for T cell activation markers 4-1BB, CD25, CD107a, human leukocyte antigen (HLA)-DR; as well as CD4 + and CD8 +After 30 min incubation at 4°C, all plates were washed twice with FACS buffer and cells were resuspended in FACS buffer. Samples were analyzed on a BD LSRFortessa Cell Analyzer using FlowJo software to identify CD4 + and CD8 + The median fluorescence intensity (MFI) and percentage of positive cells for each T cell activation marker on T cells were determined. Anti-CD27 antibody-induced changes in the expression levels of T cell activation markers were presented as the fold change in MFI of anti-CD27 antibody samples compared to the non-binding control antibody IgG1-b12-P329R-E345R. Samples were analyzed on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) using FlowJo software.

[0620] IgG1-CD27-A-P329R-E345R inhibits activated CD4 + Increased 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. CD8 + On T cells, incubation with IgG1-CD27-A-P329R-E345R resulted in increased expression of HLA-DR, CD107a and 4-1BB after both 2 and 5 days of incubation (Figure 14B).

[0621] The expression of T cell activation markers was also evaluated after 2 and 5 days of incubation with the three prior art antibodies. IgG1-CD27-131A and IgG1-CD27-BMS986215 inhibited CD4 + and CD8 +Although it induced comparable increases in HLA-DR, 4-1BB, CD25, and CD107a expression on T cells, the effect of incubation with IgG1-CD27-CDX1127 for 2 or 5 days on T cell activation marker expression was weaker.

[0622] In conclusion, incubation of polyclonally activated PBMCs with IgG1-CD27-A-P329R-E345R significantly inhibited CD4 + and CD8 + This resulted in increased expression of activation markers HLA-DR, CD25, CD107a and 4-1BB on T cells.

[0623] Example 17: OVA-specific CD8 in OVA protein-immunized mice after injection of anti-CD27 antibody in human CD27-KI mouse model + T cell percentage The effect of IgG1-CD27-A-P329R-E345R treatment on the expansion of antigen-specific T cells in the hCD27 KI OVA model in splenocytes was analyzed by flow cytometry.

[0624] 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 was developed in collaboration with Crown Bioscience's HuGEMM™ platform and featured a humanized drug target (CD27 in this case) in mice with a functional immune system. In the hCD27 KI mice, exons 1-5 of the mouse CD27 gene, which encodes the extracellular domain, were replaced by human CD27 exons 1-5. OVA-specific T cells were induced in vivo in hCD27-KI mice by subcutaneous (sc) injection of the immunogen ovalbumin (OVA) and the agonistic effect of IgG1-CD27-A-P329R-E345R was tested by simultaneously treating the mice with the antibody intravenously (iv).

[0625] On day 0, mice were injected sc with 5 mg OVA (InvivoGen, Cat. No. vac-pova-100, Lot No. EFP-42-04) and treated by iv tail vein injection with IgG1-CD27-A-P329R-E345R (30 mg / kg), IgG1-CD27-CDX1127 (30 mg / kg) or IgG1-b12-P329R-E345R (30 mg / kg). On days 12 and 21, mice were boosted with OVA and treated with the same antibodies as on day 0. On days 10, 19 and 24, blood was collected via the cheek pouch or saphenous vein into BD Microtainer® blood collection tubes containing dipotassium ethylenediaminetetraacetate (K2-EDTA; BD, Cat. No. 365974) and used immediately in further analysis. On day 28, mice were euthanized and spleens were removed under aseptic conditions.

[0626] Excised splenic tissue in RPMI1640 medium (Thermo Fisher Scientific, Cat. No. C22400500BT) was transferred to gentleMACs™ C Tubes (Miltenyi Biotec, Cat. No. 130-093-237) and mechanically dissociated into a single cell suspension using a gentleMACS™ Dissociator (Miltenyi, Cat. No. 130-093-235) according to the manufacturer's instructions. After dissociation, the cell suspension was filtered through a 70 μm cell strainer (Falcon, Cat. No. 352350). Samples were then washed twice by resuspension in 3 mL of wash buffer (sterile PBS [Hyclone, SH0256.01B] supplemented with 4% FBS [Gibco, Cat. No. 10099 141]). Cells were counted with a Cellometer Auto T4 (Nexcelom Bioscience) and the number of cells was determined to be 2 × 10 per tube. 6 The cells were adjusted to 100 spleen cells.

[0627] 2×10 6Spleen cells 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. 10099 141]) supplemented with 1 μg / mL purified rat anti-mouse CD16 / CD32 (Mouse BD Fc Block™, BD Biosciences, Cat. No. 553141). After 10 min preincubation in the dark at 2-8 °C, 10 μL of PE-labeled OVA tetramer (MBL Life science, Cat. No. TS 5001 1C) was added and the samples were gently vortexed before further incubation for 30-60 min in the dark at 2-8 °C. Without washing, labeled antibodies and compounds used for flow cytometry gating of T cell subsets were added. Samples were gently vortexed and incubated for an additional 30 min in the dark at 2–8 °C. Samples were then washed twice by resuspension in 2 mL of wash buffer and centrifuged at 300 × g for 5 min. Finally, cells were resuspended in 250 μL of wash buffer and analyzed on a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences). Data were processed using Kaluza Analysis Software (Beckman Coulter).

[0628] IgG1-CD27-A-P329R-E345R increased OVA-specific CD8 expression in the spleens of mice co-injected with OVA protein vaccination. + Increased the percentage of OVA-specific CD8 T cells in mice treated with 30 mg / kg IgG1-CD27-CDX1127 + The percentage of T cells was lower in the IgG1-CD27-A-P329R-E345R-treated group and similar to the IgG1-b12-P329R-E345R-treated group (Figure 15). Similar observations were made in peripheral blood samples.

[0629] Example 18: OVA-specific CD8 from spleens of OVA-immunized mice injected with anti-CD27 antibody+ IFNγ secretion by T cells Excised spleen tissue (see Example 17) in RPMI1640 medium was gently mashed through a 70 μm cell strainer (Falcon, Cat. No. 352350), pelleted by centrifugation (1,500 rpm, 5 min), and resuspended in 10 mL of Ammonium-Chloride-Potassium (ACK) Lysing Buffer (Invitrogen, Cat. No. A1049201). After 3-5 min of incubation at RT, samples were washed twice with 10-20 mL of PBS and resuspended in 5 mL of Cellular Technology Limited (CTL) Test™ Medium (ImmunoSpot, Cat. No. CTLT-005) supplemented with 50 U / mL penicillin and 50 μg / mL streptomycin (pen / strep, Gibco, Cat. No. 15070-063). The harvested spleen cells were again filtered through a 70 μm cell strainer and counted on a Vi-CELL™ XR Cell Viability Analyzer (Beckman Coulter) to obtain a total of 3.125 × 10 6 The concentration was adjusted to cells / mL.

[0630] 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 MultiScreenHTS IP Filter (MSIP) white plates (mAb AN18) were washed 4 times with 200 μL of sterile PBS per well and conditioned with 200 μL of CTL-Test Medium containing pen / strep (RT, 30 min). The medium was removed and 5 × 10 cells were plated per well. 5 10 spleen cells were inoculated with 2 μg / mL OVA in duplicate. 257-264Plates were incubated with peptide SIINFEKL (Invivogen, Cat. No. vac-sin), or scrambled control peptide FILKSINE (SB-PEPTIDE, Cat. No. SB073-1MG) in a total volume of 180 μL / well for 20 h in a humidified incubator (37 °C, 5% CO2). As a positive control for IFNγ production, spleen cells were incubated in parallel with a cell stimulation cocktail consisting of 500 ng / mL phorbol myristate acetate (PMA) and 10 μg / mL ionomycin (PMA + ionomycin, Dakewe Biotech, Cat. No. DKW ST PI). Cultures of spleen cells without peptide were included as negative controls. After incubation, cells were removed and plates were washed 5 times with PBS. Plates were then sequentially incubated with biotinylated detection mAb (R4-6A2; RT, 2 h), streptavidin-horseradish peroxidase (HRP; RT, 1 h), and finally 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (all provided by the kit), with five washing steps with PBS in between. When distinct spots appeared, the reaction was stopped by extensive washing in deionized water. Spots were counted on an AID iSpot ELISpot Reader (Autoimmun Diagnostika [AID] GMBH, ELR08IFL) using spotAID V8 software (AID). ELISpot data were analyzed using GraphPad Prism software and presented in bar graphs and as the mean number of spots per well ± SEM from all mice (n = 5) per treatment group.

[0631] As demonstrated by ELISpot analysis, spleen cells from all IgG1-CD27-A-P329R-E345R-treated animal groups showed increased IFNγ production in response to treatment with OVA peptide (Figure 16). Stimulation of spleen cells with a scrambled control peptide did not induce IFNγ production or induced minimal IFNγ production, suggesting that IFNγ was produced by OVA-specific T cells. In contrast, IFNγ production was not observed in spleen cells from mice treated with 30 mg / kg IgG1-CD27-CDX1127.

[0632] Example 19: Effect of IgG1-CD27-A-P329R-E345R treatment on T cell activation in OVA-immunized mice in vivo CD8 + The effect of IgG1-CD27-A-P329R-E345R treatment on T cell activation was measured using CD8 + In vivo studies were performed by analyzing the expression of PD-1 on T cells. Mice were treated as described in Example 17. Methods for obtaining splenocytes and analyzing them by FACS are also described in Example 17.

[0633] IgG1-CD27-A-P329R-E345R inhibited CD8 + Induced an increase in the percentage of CD8 T cells. + PD-1 + The percentage of T cells was lower in animals treated with IgG1-CD27-CDX1127 or the control antibody IgG1-b12-P329R-E345R (Figure 17).

[0634] Example 20: Effect of IgG1-CD27-A-P329R-E345R treatment on in vivo induction of T cell subsets in OVA-immunized mice The effect of IgG1-CD27-A-P329R-E345R on the expansion of T cell subsets was studied by analyzing the expression of CD44 and CD62L in splenocyte samples from OVA-treated hCD27-KI mice. Memory CD8 T cells derived from the spleens of IgG1-CD27-A-P329R-E345R-treated, OVA-immunized, hCD27-KI mice were + T cells were quantified by flow cytometry. Memory T cells were classified as effector memory (CD44 + CD62L - ) and pre-effector T cells (CD44 - CD62L - ;Nakajima,Y.,K et al 2018). Mice were treated as described in Example 17. Methods for obtaining spleen cells and analyzing them by FACS are also described in Example 17.

[0635] IgG1-CD27-A-P329R-E345R (30 mg / kg) significantly increased pre-effector T cells and effector memory CD8 T cells in the spleen at day 28 compared to spleen cells from mice treated with IgG1-b12-P329R-E345R. + Induced an increase in the percentage of CD45 T cells (Figure 18). + Within the population, IgG1-CD27-A-P329R-E345R induced a higher percentage of pre-effector and effector memory T cells than IgG1-CD27-CDX1127 (30 mg / kg), but similar mean percentages of these T cell populations were downregulated by CD8 + In the fractions, both anti-CD27 antibodies induced

[0636] Example 21: Effect of IgG1-CD27-A-P329R-E345R treatment on in vivo expansion of T cells in OVA-immunized mice The effect of IgG1-CD27-A-P329R-E345R on T cell expansion was studied by analyzing the expression of CD3 in spleen cells and blood samples from OVA-treated hCD27-KI mice. Mice were treated as described in Example 17. Methods for obtaining spleen cells and blood samples and analyzing them by flow cytometry are also described in Example 17.

[0637] Treatment of OVA-immunized hCD27-KI mice with 30 mg / kg IgG1-CD27-A-P329R-E345R significantly increased CD3+ expression in the spleen compared to treatment with the non-binding control antibody IgG1-b12-P329R-E345R. + In contrast, treatment with the benchmark antibody IgG1-CD27-CDX1127 (30 mg / kg) did not increase the percentage of CD3 T cells in the spleen. + This resulted in a depletion of T cells. Similar observations were made in peripheral blood samples.

[0638] Example 22: Effect of IgG1-CD27-A-P329R-E345R on T cell cytokine production in antigen-specific studies The ability of IgG1-CD27-A-P329R-E345R to increase cytokine production was studied using T cells stimulated with cognate antigen.PBMCs were isolated from buffy coats obtained from healthy human donors by Ficoll-Paque density gradient separation (GE Healthcare, Cat. No. 17 1440 03) according to the manufacturer's instructions.

[0639] Human magnetic CD14 and CD8 microbeads (Miltenyi Biotec, catalog numbers 130 050 201 and 130 045 201, respectively) were used to isolate CD14 from human PBMCs. + Positive selection of monocytes and CD14 - Negative selection of PBLs and CD8 from frozen PBLs +The cell suspension was centrifuged and sorted into magnetic activated cell sorting (MACS) buffer (Dulbecco's phosphate-buffered saline [DPBS] containing 5 mM EDTA and 1% human albumin) at 1 × 10 per 80 μL of MACS buffer. 7 Viable cells were resuspended at 1 x 10 7 Twelve μL of CD14 or CD8 microbeads were added per cell. Subsequent MACS separation was performed using an automated magnetic cell separation instrument or by manual separation. Automated MACS separation was performed using an autoMACS® Pro Separator (Miltenyi Biotec) according to the manufacturer's instructions. Eluted CD14 + Monocytes and CD8 + T cells were centrifuged (8 min, 300 × g, RT), resuspended in X-VIVO 15 medium (Lonza) and used for further use with Erythrosin B solution; i.e., monocyte differentiation into iDCs or CD8+ cells with PD-1 and / or CLDN6 specific T cell receptor (TCR) mRNA. + T cells were counted for electroporation.

[0640] For generation of monocyte-derived iDCs, up to 40 x 10 6 PBMC-derived CD14 +Monocytes were cultured in T175 flasks for 5 days (37°C, 5% CO2) in DC medium (RPMI 1640, 5% pooled human serum [PHS; One Lambda, catalog number A25761], 1x minimum essential medium non-essential amino acid solution [MEM NEAA, Life Technologies, catalog number 11140 035], 1 mM sodium pyruvate [Life Technologies, catalog number 11360 039]) supplemented with 100 ng / mL human granulocyte / macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec, catalog number 130-093-868) and 50 ng / mL human IL-4 (Miltenyi Biotec, catalog number 130093 924). After 3 days of culture, half of the medium per flask was replaced. The medium obtained from the flask contained non-adherent monocytes, so it was centrifuged (8 min, 300×g, RT), the supernatant was discarded, and the cell pellet was resuspended in fresh DC medium and then transferred back to the original flask with 200 ng / mL GM-CSF and 200 ng / mL IL-4 (final concentrations). After 5 days of incubation, i...

Claims

1. (i) A binder comprising at least one binding region that binds to CD27; and (ii) PD1 / PD-L1 inhibitors A pharmaceutical product for use in a method for reducing or preventing the progression of tumors or treating cancer in a subject, which includes a step of administering the product to the subject, (a) comprising the binder, (b) comprising the PD1 / PD-L1 inhibitor, or (c) A combination of the binder and the PD1 / PD-L1 inhibitor, Pharmaceuticals.

2. The pharmaceutical product according to claim 1, wherein the binder comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 containing sequences shown in SEQ ID NO: 5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 containing sequences shown in SEQ ID NO: 9, 10, and 11, respectively.

3. The pharmaceutical product according to claim 1, wherein the binder comprises two binding regions having the ability to bind to human CD27, and the binder comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising sequences shown in SEQ ID NO: 5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising sequences shown in SEQ ID NO: 9, 10, and 11, respectively.

4. The pharmaceutical product according to claim 1, wherein the binder comprises a VH region including the sequence shown in SEQ ID NO:

4.

5. The pharmaceutical product according to claim 1, wherein the binder comprises a VL region containing the sequence shown in SEQ ID NO:

8.

6. The pharmaceutical product according to claim 1, wherein the binder comprises a VH region and a VL region, respectively, which include the sequences shown in SEQ ID NO:4 and SEQ ID NO:

8.

7. The pharmaceutical product according to claim 1, wherein the binder is an antibody, preferably a human antibody or a humanized antibody.

8. The pharmaceutical product according to claim 7, 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 pharmaceutical product according to claim 8, wherein the constant region of the light chain is human kappa.

10. The pharmaceutical product according to claim 8, wherein the constant region of the light chain is human lambda.

11. The pharmaceutical product according to claim 1, wherein the binder further comprises a heavy chain constant region, the heavy chain constant region being a heavy chain constant region of a human IgG isotype, and optionally a modified human IgG heavy chain constant region.

12. The pharmaceutical product according to claim 11, wherein the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3, or IgG4, for example, human IgG1.

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

14. The pharmaceutical product according to claim 11, wherein the modified human IgG is a modified human IgG1 comprising one or more amino acid substitutions, for example, two or more amino acid substitutions.

15. The pharmaceutical product according to claim 11, wherein the modified human IgG heavy chain constant region comprises up to 10 amino acid substitutions, for example, up to 9, for example, up to 8, for example, up to 7, for example, up to 6, for example, up to 5, for example, up to 4, for example, up to 3, for example, up to 2 amino acid substitutions.

16. The pharmaceutical product according to claim 11, wherein the substitution in the heavy chain constant region induces an increase in CD27 agonism compared to the same antibody except that it includes the heavy chain constant region of a wild-type IgG1 antibody.

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

18. The pharmaceutical product according to claim 11, wherein the amino acid residue at the position corresponding to position E345 in the human IgG monohelic chain according to EU numbering is R.

19. The pharmaceutical product according to claim 11, wherein the amino acid residue at the position corresponding to position E430 in the human IgG single chain according to EU numbering is G.

20. The pharmaceutical product according to claim 11, wherein the amino acid residue at the position corresponding to position P329 in the human IgG mono heavy chain according to EU numbering is R.

21. The pharmaceutical product according to claim 11, wherein both amino acid residues at positions E345 and P329 in the human IgG monohelic acid according to EU numbering are R.

22. The pharmaceutical product according to claim 11, wherein the binder has a pharmacokinetic profile similar to that of a parent antibody containing a wild-type IgG1 heavy chain constant region.

23. The pharmaceutical product according to claim 1, wherein the binder comprises a heavy chain constant region containing a sequence selected from the group including 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 pharmaceutical product according to claim 1, wherein the binder comprises a heavy chain constant region including the sequence shown in SEQ ID NO:

15.

25. The pharmaceutical product according to claim 1, wherein the binder includes a heavy chain constant region, and the heavy chain constant region is modified such that the binder induces one or more Fc-mediated effector functions to a lower degree than that of the parent antibody.

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

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

28. The one or more Fc-mediated effector functions are grouped as follows: 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 A pharmaceutical product according to claim 25, which is more selected.

29. The pharmaceutical product according to claim 25, wherein the binder does not induce C1q bonding when measured by the method of Example 8.

30. The pharmaceutical product according to claim 1, wherein the binder is a monovalent antibody.

31. The pharmaceutical product according to claim 1, wherein the binder is a bivalent antibody.

32. The pharmaceutical product according to claim 1, wherein the binder is a monospecific antibody.

33. The pharmaceutical product according to claim 1, wherein the binder is a bispecific antibody comprising a first antigen-binding region having the ability to bind to human CD27 as described in claim 1, and a second antigen-binding region having the ability to bind to a different epitope on human CD27 or to a different target.

34. The pharmaceutical product according to claim 1, wherein CD27 is human CD27, and in particular, the human CD27 comprises the sequence shown in SEQ ID NO:1 or the human CD27 variant shown in SEQ ID NO:

2.

35. The aforementioned binder is e. The VH region containing the amino acid sequence shown in SEQ ID NO:4; f. The VL region containing the amino acid sequence shown in SEQ ID NO:8; g. CH region containing the amino acid sequence shown in SEQ ID NO:15; and h. CL region containing the amino acid sequence shown in SEQ ID NO:17 The pharmaceutical product according to claim 1, including the above.

36. The pharmaceutical product according to claim 1, wherein the binder comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO:35 and a light chain containing the amino acid sequence shown in SEQ ID NO:

25.

37. The pharmaceutical product according to claim 1, wherein PD-L1 is human PD-L1, particularly human PD-L1 comprising the sequence shown in SEQ ID NO:

98.

38. The pharmaceutical product according to claim 1, wherein PD1 is human PD1, preferably having or containing the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 59, or the amino acid sequence of 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 shown in SEQ ID NO: 58 or SEQ ID NO: 59, or is an immunogenic fragment thereof.

39. The pharmaceutical product according to claim 1, 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 pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is an isotype antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, for example, an IgG1 isotype antibody.

41. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is a full-length antibody or antibody fragment, for example, a full-length IgG1 antibody.

42. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is a monospecific antibody.

43. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 99, 100, and 101, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 102, LAS, and SEQ ID NO: 103, respectively.

44. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising a VH region containing the amino acid sequence of SEQ ID NO:104 and a VL region containing the amino acid sequence of SEQ ID NO:

105.

45. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO:106 and a light chain containing the amino acid sequence of SEQ ID NO:

107.

46. (a) The binder is an antibody comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO:35 and a light chain containing the amino acid sequence shown in SEQ ID NO:25; (b) The PD1 / PD-L1 inhibitor is pembrolizumab or a biosimilar thereof. The pharmaceutical product according to claim 1.

47. (a) The binder is an antibody comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO:35 and a light chain containing the amino acid sequence shown in SEQ ID NO:25; (b) The PD1 / PD-L1 inhibitor is nivolumab or a biosimilar thereof. The pharmaceutical product according to claim 1.

48. (a) The binder is an antibody comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO:35 and a light chain containing the amino acid sequence shown in SEQ ID NO:25; (b) The PD1 / PD-L1 inhibitor is atezolizumab or a biosimilar thereof. The pharmaceutical product according to claim 1.

49. The pharmacopoeia according to claim 1, wherein the PD1 / PD-L1 inhibitor is an antibody that binds to PD1, or an antigen-binding fragment thereof, and the antibody that binds to PD1 comprises VH regions CDR1, CDR2, and CDR3 containing sequences shown in SEQ ID NO: 49, 46, and 45, respectively, and VL regions CDR1, CDR2, and CDR3 containing sequences shown in SEQ ID NO: 52, QAS, and SEQ ID NO: 50, respectively.

50. The pharmaceutical product according to claim 49, wherein the antibody that binds to PD1 comprises a VH having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the heavy chain variable region (VH) sequence shown in SEQ ID NO:

56.

51. The pharmaceutical product according to claim 50, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH), and the VH comprises the sequence shown in SEQ ID NO:

56.

52. The pharmaceutical product according to claim 49, wherein the antibody that binds to PD1 comprises a VL having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the light chain variable region (VL) sequence shown in SEQ ID NO:

57.

53. The pharmaceutical product according to claim 52, wherein the antibody that binds to PD1 includes a light chain variable region (VL), and the VL includes the sequence shown in SEQ ID NO:

57.

54. The pharmaceutical product according to claim 49, wherein the antibody that binds to PD1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising or having the sequence shown in SEQ ID NO: 56, and the VL comprising or having the sequence shown in SEQ ID NO:

57.

55. The antibody that binds to PD1 includes a heavy chain constant region, The heavy chain steady region is Aromatic or nonpolar amino acids at the position corresponding to position 234 in the human IgG monochain according to EU numbering, and Non-glycine amino acids at the position corresponding to position 236 in the human IgG1 heavy chain according to EU numbering. including, The pharmaceutical product according to claim 49.

56. The pharmaceutical product according to claim 55, wherein the amino acid at the position corresponding to the 236th position is a basic amino acid.

57. The pharmaceutical product according to claim 56, wherein the basic amino acid is selected from the group consisting of lysine, arginine, and histidine.

58. The pharmaceutical product according to claim 56, wherein the basic amino acid is arginine (G236R).

59. The pharmaceutical product according to claim 55, wherein the amino acid at the position corresponding to the 234th position is an aromatic amino acid.

60. The pharmaceutical product according to claim 59, wherein the aromatic amino acid is selected from the group consisting of phenylalanine, tryptophan, and tyrosine.

61. The pharmaceutical product according to claim 55, wherein the amino acid at the position corresponding to the 234th position is a nonpolar amino acid.

62. The pharmaceutical product according to claim 61, wherein the nonpolar amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

63. The pharmaceutical product according to claim 61, wherein the nonpolar amino acid is selected from the group consisting of isoleucine, proline, phenylalanine, methionine, and tryptophan.

64. The pharmaceutical product according to claim 55, wherein the amino acid at the position corresponding to the 234th position is phenylalanine (L234F).

65. The pharmaceutical product according to claim 55, wherein the amino acid at the position corresponding to position 235 in the human IgG1 heavy chain according to EU numbering in the heavy chain constant region of the antibody that binds to PD1 is an acidic amino acid.

66. The pharmaceutical product according to claim 65, wherein the acidic amino acid is aspartic acid or glutamic acid.

67. The pharmaceutical product according to claim 55, wherein the amino acid at the position corresponding to position 235 in the human IgG1 heavy chain according to the EU numbering in the heavy chain constant region of the antibody that binds to PD1 is glutamic acid (L235E).

68. The pharmaceutical product according to claim 55, wherein the amino acids at positions 234, 235, and 236 in the heavy chain constant region of the antibody bound to PD1 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 pharmaceutical product according to claim 55, wherein the amino acid corresponding to position 234 in the heavy chain constant region of the antibody that binds to PD1 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 pharmaceutical product according to claim 49, wherein the heavy chain constant region of the antibody that binds to PD1 comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the HC sequence shown in SEQ ID NO:

38.

71. The pharmaceutical product according to claim 49, wherein the heavy chain constant region of the antibody that binds to PD1 includes the sequence shown in SEQ ID NO:

38.

72. The pharmaceutical product according to claim 49, wherein the isotype of the heavy chain constant region of the antibody that binds to PD1 is IgG1.

73. The pharmaceutical product according to claim 49, 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 pharmaceutical product according to claim 49, wherein the antibody that binds to PD1 is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fragment of such an antibody.

75. The pharmaceutical product according to claim 49, wherein the antibody that binds to PD1 has reduced or depleted Fc-mediated effector function.

76. The pharmaceutical product according to claim 49, wherein the binding of complement protein C1q to the constant region of the antibody that binds to PD1 is preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody.

77. The pharmaceutical product according to claim 49, wherein the binding of one or more IgG Fc gamma receptors to the antibody that binds to PD1 is preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody.

78. The pharmaceutical product according to claim 77, wherein the one or more IgG Fc gamma receptors are selected from at least one of Fc gamma RI, Fc gamma RII, and Fc gamma RIII.

79. The pharmaceutical product according to claim 77, wherein the IgG Fc gamma receptor is an Fc gamma radioisotope.

80. The pharmacopoeia according to claim 49, wherein the antibody that binds to PD1 does not have the ability to induce Fc gamma RI-mediated effector function, or the induced Fc gamma RI-mediated effector function is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody.

81. The pharmacopoeia according to claim 49, wherein the antibody that binds to PD1 does not have the ability to induce at least one of complement-dependent cell-mediated lysis, antibody-dependent cell-mediated lysis, apoptosis, homotyped adhesion, and / or phagocytosis, or at least one of complement-dependent cell-mediated lysis, antibody-dependent cell-mediated lysis, apoptosis, homotyped adhesion, and / or phagocytosis is induced to a reduced degree, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%.

82. The pharmaceutical product according to claim 49, wherein the binding of neonatal Fc receptor (FcRn) to the antibody that binds to PD1 is unaffected compared to that of a wild-type antibody.

83. The pharmaceutical product according to claim 49, wherein the antibody that binds to PD1 binds to a native epitope of PD1 present on the surface of a living cell.

84. The pharmaceutical product according to claim 49, wherein the antibody that binds to PD1 is a polyspecific antibody comprising a first antigen-binding region that binds to PD1 and at least one further antigen-binding region that binds to another antigen.

85. The pharmaceutical product according to 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 pharmaceutical product according to claim 84, 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 claim 50.

87. (a) The binder comprises a VH region containing the amino acid sequence shown in SEQ ID NO:4 and a VL region containing the amino acid sequence shown in SEQ ID NO:8; (b) The antibody that binds to PD1 comprises a VH region and a VL region, wherein the VH region contains or has the sequence shown in SEQ ID NO: 56, and the VL region contains or has the sequence shown in SEQ ID NO: 57, The pharmaceutical product according to claim 49.

88. (a) The binder is an antibody comprising a VH region containing the amino acid sequence shown in SEQ ID NO:4, a VL region containing the amino acid sequence shown in SEQ ID NO:8, a CH region containing the amino acid sequence shown in SEQ ID NO:15, and a CL region containing the amino acid sequence shown in SEQ ID NO:17; (b) The antibody that binds to PD1 includes a VH region containing the amino acid sequence shown in SEQ ID NO: 56, a VL region containing the amino acid sequence shown in SEQ ID NO: 57, a CH region containing the amino acid sequence shown in SEQ ID NO: 38, and a CL region containing the amino acid sequence shown in SEQ ID NO:

42. The pharmaceutical product according to claim 49.

89. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is a polyspecific antibody, for example, a bispecific antibody.

90. The pharmaceutical product according to claim 89, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor comprising a first binding region that binds to CD137 and a second binding region that binds to PD-L1.

91. The pharmaceutical product according to claim 90, wherein CD137 is human CD137, in particular human CD137 comprising the sequence shown in SEQ ID NO:

97.

92. (a) The first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:79, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:83; and (b) The second binding region of the PD-L1 inhibitor includes a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:86, and a light chain variable region (VL) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:90, The pharmaceutical product according to claim 90.

93. (a) The pharmacopoeia according to claim 90, wherein the first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 80, 81, and 82, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 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) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 87, 88, and 89, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 91, DDN, and SEQ ID NO: 92, respectively.

94. (a) The first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO:79 and a light chain variable region (VL) containing 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) containing the amino acid sequence shown in SEQ ID NO:86 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:90, The pharmaceutical product according to claim 90.

95. The PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm, The first connecting arm, (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and (ii) A polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). Includes; Furthermore, the second connecting arm (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and (iv) A polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). including, The pharmaceutical product according to claim 90.

96. The aforementioned PD-L1 inhibitor, (i) a first heavy chain and a light chain comprising an antigen-binding region having the ability to bind to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and (ii) A second heavy chain and light chain comprising the antigen-binding region having the ability to bind to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region. including, The pharmaceutical product according to claim 90.

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

98. The pharmaceutical product according to claim 95, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E in the first and second heavy chains, respectively.

99. The pharmaceutical product according to claim 95, wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A in the first and second heavy chain constant regions (HC), respectively.

100. The pharmaceutical product according to claim 95, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU numbering of both the first heavy chain steady region and the second heavy chain steady region are F and E, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain steady region is L and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain steady region is R and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L.

101. The pharmaceutical product according to claim 95, wherein the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L.

102. The steady region of the first and / or second heavy chain, for example, the second heavy chain, (a) Sequence [IgG1-Fc_FEAL] shown in SEQ ID NO: 94 or 96; (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having up to six substitutions, for example, up to five substitutions, up to four substitutions, up to three substitutions, up to two substitutions, or up to one substitution, compared to the amino acid sequence defined in (a) or (b). It comprises an amino acid sequence selected from the group consisting of, or is essentially composed of, or consists of, The pharmaceutical product according to claim 95.

103. The first and / or second heavy chain, for example, the steady region of the first heavy chain, (a) Sequence shown in SEQ ID NO: 93 or 95 [IgG1-Fc_FEAR]; (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having up to six substitutions, for example, up to five substitutions, up to four, up to three, up to two substitutions, or up to one substitution, compared to the amino acid sequence defined in (a) or (b). It comprises an amino acid sequence selected from the group consisting of, or is essentially composed of, or consists of, The pharmaceutical product according to claim 95.

104. The pharmaceutical product according to claim 95, wherein the PD-L1 inhibitor comprises a kappa (κ) light chain constant region.

105. The pharmaceutical product according to claim 95, wherein the PD-L1 inhibitor comprises a lambda (λ) light chain constant region.

106. The pharmaceutical product according to claim 95, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

107. The pharmaceutical product according to claim 95, 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 pharmaceutical product according to claim 95, wherein the first light chain steady region is a kappa (κ) light chain steady region and the second light chain steady region is a lambda (λ) light chain steady region, or the first light chain steady region is a lambda (λ) light chain steady region and the second light chain steady region is a kappa (κ) light chain steady region.

109. The kappa (κ) light chain is (a) The sequence shown in SEQ ID NO:16, (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having up to 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions, or up to 1 substitution. The amino acid sequence includes an amino acid sequence selected from the group consisting of the following: The pharmaceutical product according to claim 104.

110. The lambda (λ) light chain is (a) The sequence shown in SEQ ID NO:17, (b) subsequences of the sequence in (a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in (a); and (c) A sequence having up to 10 substitutions compared to the amino acid sequence defined in (a) or (b), for example, up to 9 substitutions, up to 8, up to 7, up to 6, up to 5, up to 4 substitutions, up to 3, up to 2 substitutions, or up to 1 substitution. The amino acid sequence includes an amino acid sequence selected from the group consisting of the following: The pharmaceutical product according to claim 105.

111. The pharmaceutical product according to claim 90, wherein the PD-L1 inhibitor is an IgG1m(f) allotype antibody.

112. The pharmacopoeia according to claim 90, wherein the PD-L1 inhibitor is a bispecific antibody that binds to CD137 and PD-L1, and the bispecific antibody comprises (i) a first heavy chain containing the amino acid sequence shown in SEQ ID NO:75 and a first light chain containing the amino acid sequence shown in SEQ ID NO:76, and (ii) a second heavy chain containing the amino acid sequence shown in SEQ ID NO:77 and a second light chain containing the amino acid sequence shown in SEQ ID NO:

78.

113. The pharmaceutical product according to claim 90, wherein the PD-L1 inhibitor is acasunlimab or a biosimilar thereof.

114. (a) The binder comprises heavy chain variable (VH) regions CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NO: 5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NO: 9, 10, and 11, respectively; (b) The first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 80, 81, and 82, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 84, GAS, and SEQ ID NO: 85, respectively; and (c) The second binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 87, 88, and 89, respectively, and a light chain variable region (VL) containing CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 91, DDN, and SEQ ID NO: 92, respectively. The pharmaceutical product according to claim 90.

115. (a) The binder comprises a VH region containing the amino acid sequence shown in SEQ ID NO:4 and a VL region containing the amino acid sequence shown in SEQ ID NO:8; (b) The first binding region of the PD-L1 inhibitor comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO:79 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:83; and (c) The second binding region of the PD-L1 inhibitor includes a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO:86 and a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:

90. The pharmaceutical product according to claim 90.

116. (a) The binder is an antibody comprising a VH region containing the amino acid sequence shown in SEQ ID NO:4, a VL region containing the amino acid sequence shown in SEQ ID NO:8, a CH region containing the amino acid sequence shown in SEQ ID NO:15, and a CL region containing the amino acid sequence shown in SEQ ID NO:17; (b) The PD-L1 inhibitor is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises a first binding region and the second binding arm comprises a second binding region; (c) The first binding arm of the PD-L1 inhibitor comprises a VH region containing the amino acid sequence shown in SEQ ID NO:79, a VL region containing the amino acid sequence shown in SEQ ID NO:83, a CH region containing the amino acid sequence shown in SEQ ID NO:95, and a CL region containing the amino acid sequence shown in SEQ ID NO:16; and (d) The second binding arm of the PD-L1 inhibitor includes a VH region containing the amino acid sequence shown in SEQ ID NO:86, a VL region containing the amino acid sequence shown in SEQ ID NO:90, a CH region containing the amino acid sequence shown in SEQ ID NO:96, and a CL region containing the amino acid sequence shown in SEQ ID NO:

17. The pharmaceutical product according to claim 90.

117. (c) The binder comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO:35 and a light chain containing 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, and the bispecific antibody has (i) a first heavy chain containing the amino acid sequence shown in SEQ ID NO:75 and a first light chain containing the amino acid sequence shown in SEQ ID NO:76, and (ii) a second heavy chain containing the amino acid sequence shown in SEQ ID NO:77 and a second light chain containing the amino acid sequence shown in SEQ ID NO:

78. The pharmaceutical product according to claim 90.

118. (c) The binder comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO:35 and a light chain containing the amino acid sequence shown in SEQ ID NO:25; (d) The PD-L1 inhibitor is acasunlimab or a biosimilar thereof. The pharmaceutical product according to claim 90.

119. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is a PD1 inhibitor selected from pembrolizumab, nivolumab, semiprimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, INCMGA00012 (MGA012), AMP-224, AMP-514, or their respective biosimilars.

120. The pharmaceutical product according to claim 1, wherein the PD1 inhibitor is selected from pembrolizumab, nivolumab, semiprimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, INCMGA00012 (MGA012), AMP-514, or their respective biosimilars.

121. The pharmaceutical product according to claim 1, wherein the PD1 / PD-L1 inhibitor is a PD-L1 inhibitor selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, akasunlimab, AUNP12, CA-170, BMS-986189, or their respective biosimilars.

122. The pharmaceutical product according to claim 1, wherein the PD-L1 inhibitor is selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, akasunlimab, or their respective biosimilars.

123. The pharmaceutical product according to claim 1, wherein the subject is a human subject.

124. The pharmaceutical product according to claim 1, wherein the tumor or cancer is a solid tumor.

125. The pharmaceutical product according to claim 1, wherein the tumor is a PD-L1-positive tumor.

126. The pharmaceutical product according to claim 1, wherein the tumor or cancer is head and neck squamous cell carcinoma (HNSCC), for example, HNSCC of the oral cavity, pharynx, or larynx.

127. The pharmaceutical product according to claim 126, wherein the HNSCC is recurrent, unresectable, or metastatic.

128. The pharmaceutical product according to claim 1, wherein the tumor or cancer is non-small cell lung cancer (NSCLC), for example, squamous or non-squamous NSCLC.

129. The pharmacopoeia according to claim 128, wherein the NSCLC is recurrent, unresectable, or metastatic.

130. The pharmacopoeia according to claim 128, wherein the NSCLC does not have epidermal growth factor (EGFR) sensitizing mutations and / or anaplastic lymphoma (ALK) translocations and / or ROS1 rearrangements.

131. The pharmaceutical product according to claim 128, wherein the NSCLC is NTRK1 / 2 / 3 (neurotrophic factor receptor tyrosine kinase 1 / 2 / 3) fusion positive and / or has a mutation in the KRAS (KRAS oncogene, GTPase), BRAF (B-Raf oncogene, serine / threonine kinase), or MET (MET oncogene, receptor tyrosine kinase) gene and / or has a RET (ret oncogene) gene rearrangement, and the subject has received prior treatment with the respective targeted therapy.

132. The pharmaceutical product according to claim 1, wherein the subject has previously been treated with a PD1 inhibitor or a PD-L1 inhibitor, for example, an anti-PD1 antibody or an anti-PD-L1 antibody, preferably at least two doses of a PD1 inhibitor or a PD-L1 inhibitor.

133. The pharmacopoeia according to claim 1, wherein the subject has previously received platinum-based therapy, or, if platinum is unsuitable, alternative chemotherapy, such as a gemcitabine-containing regimen.

134. The pharmacopoeia according to claim 1, wherein the tumor or cancer is a tumor or cancer that has recurred and / or progressed after treatment, for example, systemic treatment with a checkpoint inhibitor.

135. The pharmacopoeia according to claim 1, wherein the subject has received at least one prior line of systemic therapy, such as a systemic therapy comprising a PD1 inhibitor or a PD-L1 inhibitor, such as an anti-PD1 antibody or an anti-PD-L1 antibody.

136. The pharmacopoeia according to claim 1, wherein the cancer or tumor is recurrent and / or refractory, or the subject has progressed after treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD1 antibody or anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

137. The pharmacopoeia according to claim 1, wherein the previous last treatment was treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD1 antibody or an anti-PD-L1 antibody, administered as monotherapy or as part of a combination therapy.

138. The pharmacopoeia according to claim 1, wherein the time since progression to the last treatment with a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD1 antibody or anti-PD-L1 antibody, is 6 months or less.

139. The pharmacopoeia according to claim 1, wherein the time since the last dose of a PD1 inhibitor or PD-L1 inhibitor, such as an anti-PD1 antibody or anti-PD-L1 antibody, as part of a previous last treatment is six months or less.

140. The aforementioned cancer or tumor is recurrent and / or refractory, or the subject is (i) Platinum doublet chemotherapy after treatment with an anti-PD1 antibody or anti-PD-L1 antibody, (ii) Treatment with an anti-PD1 antibody or anti-PD-L1 antibody after platinum doublet chemotherapy The pharmacopoeia according to claim 1, which is progressing during or after the period described above.

141. (i) A binder comprising at least one binding region that binds to CD27, and (ii) PD1 / PD-L1 inhibitors A kit that includes this.

142. The kit according to claim 141, wherein the binder 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.

143. The kit according to claim 141, wherein the binder, the PD1 / PD-L1 inhibitor, and, if present, one or more additional therapeutic agents are for systemic administration, particularly for injection or infusion, for example, for intravenous injection or infusion.

144. The kit according to claim 141 for use in a method for reducing or preventing the progression of tumors or for treating cancer in a subject.

145. The kit according to claim 144, 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.

146. (i) A binder comprising at least one binding region that binds to CD27; (ii) PD1 / PD-L1 inhibitors; and (iii) Optional, a pharmaceutically acceptable carrier A pharmaceutical composition containing [the specified substance].

147. The pharmaceutical composition according to claim 146, wherein the binder 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.

148. The pharmaceutical composition according to claim 146 for use in a method for reducing or preventing the progression of a tumor or for treating cancer in a subject.

149. The pharmaceutical composition 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.

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