Combination therapy

Abstract

The present invention relates to novel combination therapies or pharmaceutical compositions, and their use in the treatment of cancer, particularly cancers expressing carcinoembryonic antigen (CEA).

Description

[Technical Field]

[0001] The present invention relates to combination therapies and their use in pharmaceuticals, including the treatment of cancer, particularly cancer expressing carcinoembryonic antigen (CEA). The combination therapy or pharmaceutical composition comprises (a) a bispecific polypeptide having a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), and (b) a PD-1 inhibitor. The present invention also relates to pharmaceutical compositions, the use of the combination therapy of the present invention, and methods of using the same. Cancer can be a solid tumor. [Background technology]

[0002] Cancer immunotherapy Cancer is a leading cause of premature death in developed countries. The goal of cancer immunotherapy is to initiate an effective immune response against tumor cells. This can be achieved, for example, by disrupting tolerance to tumor antigens, enhancing the anti-tumor immune response, and stimulating a local cytokine response at the tumor site. The main effector cells of a long-lasting anti-tumor immune response are activated tumor-specific effector T cells. A potent expansion of activated tumor-specific effector T cells can redirect the immune response against the tumor. In this context, various immunosuppressive mechanisms induced by the tumor microenvironment suppress the activity of effector T cells. Several immunosuppressive mediators are expressed by tumor cells. Such mediators inhibit T cell activation, either directly or indirectly, by inducing regulatory T cells (Tregs) or myeloid-derived suppressor cells, for example. Thus, by depleting, inhibiting, reversing, or inactivating such regulatory cells, an anti-tumor effect can be provided, and immunosuppression in the tumor microenvironment can be reversed. Furthermore, incomplete activation of effector T cells by dendritic cells (DCs), for example, can result in suboptimal activated or inactive T cells, leading to an inefficient antitumor response. In contrast, proper induction by DCs can result in a potent expansion of activated effector T cells, redirecting and enhancing the immune response against the tumor. In addition, natural killer (NK) cells play a crucial role in tumor immunology by attacking tumor cells through the expression of downregulated human leukocyte antigens (HLA) and by inducing antibody-dependent cytotoxicity (ADCC). Therefore, stimulating NK cells can also reduce tumor growth.

[0003] CD40 CD40, a 48kDa transmembrane cell surface glycoprotein, is a costimulatory receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily (Banchereau J, Bazan F, Blanchard D, et al. The CD40 antigen and its ligand. Annu Rev Immunol. 1994;12:881-922; Elgueta R, Benson MJ, de Vries VC, et al. Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunol Rev. 2009 May;229(1):152-72). CD40 is expressed in a variety of cell types and can be detected in antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages. In addition, CD40 is expressed in granulocytes, endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells (Banchereau J, Bazan F, Blanchard D, et al. The CD40 antigen and its ligand. Annu Rev Immunol. 1994;12:881-922; Elgueta R, Benson MJ, de Vries VC, et al. Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunol Rev. 2009 May;229(1):152-72; Korniluk A, Kemona H, Dymicka-Piekarska V. Multifunctional CD40L: pro- and anti-neoplastic activity. Tumour Biol. 2014 Oct;35(10):9447-57; Peters AL, Stunz LL, Bishop GA. CD40 and autoimmunity: the dark side of a great activator.Semin Immunol.2009 Oct;21(5):293-300.).Consistent with its widespread expression in normal cells, CD40 is also present on the membranes of a wide range of malignant cells, including non-Hodgkin and Hodgkin lymphomas, myeloma, and certain types of cancers, including those of the nasopharynx, bladder, cervix, kidney, and ovary (Elgueta R, Benson MJ, de Vries VC, et al. Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunol Rev. 2009 May;229(1):152-72.; Eliopoulos AG, Young LS. The role of the CD40 pathway in the pathogenesis and treatment of cancer. Curr Opin Pharmacol. 2004 Aug;4(4):360-7).

[0004] CD40 is a transmembrane protein expressed by activated T cells, but also by B cells, platelets, mast cells, macrophages, basophils, natural killer (NK) cells, and non-hematopoietic cells (smooth muscle cells, endothelial cells, and epithelial cells). It interacts with a single ligand, CD40L (CD154) (Elgueta R, Benson MJ, de Vries VC, et al. Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunol Rev. 2009 May;229(1):152-72., Korniluk A, Kemona H, Dymicka-Piekarska V. Multifunctional CD40L: pro-and anti-neoplastic activity. Tumour Biol. 2014 Oct;35(10):9447-57). The binding of CD40 to CD40L as part of cell-cell interactions activates an intracellular signaling pathway involving a series of adapter molecules known as TNFR activators (TRAFs). To initiate this intracellular signaling, multiple CD40 receptor trimers must form a higher-order cluster on the cell membrane (Peters AL, Stunz LL, Bishop GA. CD40 and autoimmunity: the dark side of a great activator. Semin Immunol. 2009 Oct;21(5):293-300; Werneburg BG, Zoog SJ, Dang TT, et al. Molecular characterization of CD40 signaling intermediates. J Biol Chem. 2001 Nov 16;276(46):43334-42).CD40 clustering forms a signaling complex that enables the assembly of multiple TRAFs, which in turn leads to the activation of downstream transcription factors, including NFκB (Elgueta R, Benson MJ, de Vries VC, et al. Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunol Rev. 2009 May;229(1):152-72; Kornbluth RS, Stempniak M, Stone GW. Design of CD40 agonists and their use in growing B cells for cancer immunotherapy. Int Rev Immunol. 2012 Aug;31(4):279-88).

[0005] The molecular outcomes of CD40 signaling depend on the cell type expressing CD40 and their microenvironment (Vonderheide RH, Glennie MJ. Agonistic CD40 antibodies and cancer therapy. Clin Cancer Res. 2013 Mar 01;19(5):1035-43). APC, particularly "licensing" of DCs, leads to the upregulation of membrane costimulatory molecules and MHC, as well as the production of pro-inflammatory cytokines (Caux C, Massacrier C, Vanbervliet B, et al. Activation of human dendritic cells through CD40 cross-linking. J Exp Med. 1994 Oct 1;180(4):1263-72; van Kooten C, Banchereau J. Functions of CD40 on B cells, dendritic cells and other cells. Curr Opin Immunol. 1997 Jun;9(3):330-7). Therefore, CD40 is involved in the functional maturation of APCs and, consequently, the activation of antigen-specific T cells (Ma DY, Clark EA. The role of CD40 and CD154 / CD40L in dendritic cells. Semin Immunol. 2009 Oct;21(5):265-72; Moran AE, Kovacsovics-Bankowski M, Weinberg AD. The TNFRs OX40, 4-1BB, and CD40 as targets for cancer immunotherapy. Curr Opin Immunol. 2013 Apr;25(2):230-7).CD40 also plays a role in humoral immunity by activating resting B cells and increasing their antigen-presenting function (Vonderheide RH, Glennie MJ. Agonistic CD40 antibodies and cancer therapy. Clin Cancer Res. 2013 Mar 01;19(5):1035-43; Zarnegar B, He JQ, Oganesyan G, et al. Unique CD40-mediated biological program in B cell activation requires both type 1 and type 2 NF-kappa B activation pathways. Proc Natl Acad Sci US A. 2004 May 25;101(21):8108-13). Furthermore, CD40 is involved in innate immunity induction through stimulation of cells such as macrophages, granulocytes, and NK cells (Rakhmilevich AL, Alderson KL, Sondel PM. T-cell-independent antitumor effects of CD40 ligation. Int Rev Immunol. 2012 Aug;31(4):267-78).

[0006] Monoclonal CD40 agonist antibodies are thought to induce antitumor effects through two distinct mechanisms: (i) tumor-specific immune activation, and (ii) direct tumor-killing effects via, for example, apoptosis, antibody-dependent cytotoxicity (ADCC), and / or complement-dependent cytotoxicity (CDC) (Khong A, Nelson DJ, Nowak AK, et al. The use of agonistic anti-CD40 therapy in treatments for cancer. Int Rev Immunol. 2012 Aug;31(4):246-66). Treatment with CD40 agonists induces the activation of several different immune cells that contribute to the antitumor immune response.T cells, particularly cytotoxic T lymphocytes (CTLs), are essential for the antitumor effect induced by CD40 agonists, as demonstrated in preclinical models (Byrne KT, Vonderheide RH. CD40 Stimulation Obviates Innate Sensors and Drives T Cell Immunity in Cancer. Cell Rep. 2016 Jun 21;15(12):2719-32; Mangsbo SM, Broos S, Fletcher E, et al. The human agonistic CD40 antibody ADC-1013 eradicates bladder tumors and generates T-cell-dependent tumor immunity. Clin Cancer Res. 2015 Mar 01;21(5):1115-26; Tutt AL, O'Brien L, Hussain A, et al. T Cell Immunity to Lymphoma Following Treatment with Anti-CD40 Monoclonal Antibody. The Journal of Immunology.2002;168(6):2720-2728, van Mierlo GJ, den Boer AT, Medema JP, et al.CD40 stimulation leads to effective therapy of CD40(-) tumors through induction of strong systemic cytotoxic T lymphocyte immunity.Proc Natl Acad Sci US A.2002 Apr 16;99(8):5561-6).DC activation and subsequent T cell priming may play a central role when the presence of antigen-cross-presenting DCs is required for the antitumor effect of CD40 agonist therapy in T cell-dependent models (Beatty GL, Chiorean EG, Fishman MP, et al. CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science. 2011 Mar 25;331(6024):1612-6; Beatty GL, Li Y, Long KB. Cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists. Expert Rev Anticancer Ther. 2017 Feb;17(2):175-186; Long KB, Gladney WL, Toooker GM, et al. IFNgamma and CCL2 Cooperate to Redirect Tumor-Infiltrating Monocytes to Degrade Fibrosis and Enhance Chemotherapy Efficacy in Pancreatic). Carcinoma.Cancer Discov.2016 Apr;6(4):400-413, Lum HD, Buhtoiarov IN, Schmidt BE, et al.In vivo CD40 ligation can induce T-cell-independent antitumor effects that involve macrophages.J Leukoc Biol.2006 Jun;79(6):1181-92).NK cells are also capable of cytotoxic killing of tumor cells and have been shown to contribute to a reduction in tumor growth in response to CD40 agonists (Turner JG, Rakhmilevich AL, Burdelya L, et al. Anti-CD40 Antibody Induces Antitumor and Antimetastatic Effects: The Role of NK Cells. The Journal of Immunology. 2001;166(1):89). CD40-activated B cells can further enhance the anti-tumor immune response by presenting antigens to T cells and producing tumor-targeting antibodies (Jackaman C, Cornwall S, Graham PT, et al. CD40-activated B cells contribute to mesothelioma tumor regression. Immunol Cell Biol. 2011 Feb;89(2):255-67; ​​Liu M, Sun Q, Wang J, et al. A New Perspective: Exploring Future Therapeutic Strategies For Cancer By Understanding The Dual Role Of B Lymphocytes In Tumor Immunity. Int J Cancer. 2018 Sep 5).In addition, CD40 agonists have been found to convert tumor-associated macrophages (TAMs) into activated macrophages with antitumor properties that can promote tumor reduction independently of T cells (Beatty GL, Chiorean EG, Fishman MP, et al. CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science. 2011 Mar 25;331(6024):1612-6; Beatty GL, Li Y, Long KB. Cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists. Expert Rev Anticancer Ther. 2017 Feb;17(2):175-186; Long KB, Gladney WL, Toooker GM, et al. IFNgamma and CCL2 Cooperate to Redirect Tumor-Infiltrating Monocytes to Degrade Fibrosis and Enhance Chemotherapy Efficacy in Pancreatic). Carcinoma.Cancer Discov.2016 Apr;6(4):400-413, Lum HD, Buhtoiarov IN, Schmidt BE, et al.In vivo CD40 ligation can induce T-cell-independent antitumor effects that involve macrophages.J Leukoc Biol.2006 Jun;79(6):1181-92).

[0007] DCs are the most important APCs for generating antigen-specific T cell responses (Flamar AL, Xue Y, Zurawski SM, et al. Targeting concatenated HIV antigens to human CD40 expands a broad repertoire of multifunctional CD4+ and CD8+ T cells. AIDS. 2013 Aug 24;27(13):2041-51). Their central role in inducing an antitumor immune response has been demonstrated in preclinical models lacking Batf3, which in turn leads to a deficiency in cross-presenting DCs (cDC1), resulting in impaired rejection of immunogenic tumors and unresponsiveness to immunotherapy due to impaired priming of tumor-targeted CTLs (Hildner K, Edelson BT, Purtha WE, et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science. 2008 Nov 14;322(5904):1097-100; Sanchez-Paulete AR, Cueto FJ, Martinez-Lopez M, et al. Cancer Immunotherapy with Immunomodulatory Anti-CD137 and Anti-PD-1 Monoclonal Antibodies Requires BATF3-Dependent Dendritic Cells. Cancer Discov. 2016). Jan;6(1):71-9).According to these data, the presence of cross-presenting DCs in human tumors correlates with CD8+ T cell infiltration and is associated with a better prognosis and a superior response to immunotherapy (Broz ML, Binnewies M, Boldajipour B, et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell. 2014 Nov 10;26(5):638-52; Sanchez-Paulete AR, Teijeira A, Cueto FJ, et al. Antigen Cross-Presentation and T-Cell Cross-Priming In Cancer Immunology And Immunotherapy. Ann Oncol. 2017 Sep 01). CD40-mediated signaling on DCs induces activation of antigen presentation mechanisms and upregulation of costimulatory molecules such as CD80 and CD86, thereby enabling DCs to present antigens to T cells and activate them (Beatty GL, Li Y, Long KB. Cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists. Expert Rev Anticancer Ther. 2017 Feb;17(2):175-186; Gladue RP, Paradis T, Cole SH, et al. The CD40 agonist antibody CP-870,893 enhances dendritic cell and B-cell activity and promotes anti-tumor efficacy in SCID-hu mice. Cancer Immunol Immunother. 2011 Jul;60(7):1009-17), improving the ability to produce cytokines, particularly IL-12, which are helpful in shaping the T cell response.

[0008] CD40 expression can be detected in all blood dendritic cells (DCs), with the highest expression observed in a subpopulation called cDC1 (Carenza C, Calcaterra F, Oriolo F, et al. Costimulatory Molecules and Immune Checkpoints Are Differentially Expressed on Different Subsets of Dendritic Cells [Original Research]. Frontiers in Immunology. 2019 2019-June-11;10(1325), MacDonald KP, Munster DJ, Clark GJ, et al. Characterization of human blood dendritic cell subsets. Blood. 2002 Dec 15;100(13):4512-20). Recent studies have focused on the role of cDC1 in driving T cell responses against tumors, demonstrating the potential of CD40 agonists alone or in combination with other therapies in enhancing cDC1 priming of tumor-targeted T cells (Hegde S, Krisnawan VE, Herzog BH, et al. Dendritic Cell Paucity Leads to Dysfunctional Immune Surveillance in Pancreatic Cancer. Cancer Cell. 2020 Mar 16;37(3):289-307 e9; Morrison AH, Diamond MS, Hay CA, et al. Sufficiency of CD40 activation and immune checkpoint blockade for T cell priming and tumor immunity. Proc Natl Acad Sci US A. 2020 Mar 25; Zhang L, Li Z, Skrzypczynska KM, et al. Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.Cell.2020;181(2):442-459.e29).Single-cell RNA sequencing studies confirm the presence of cDC1 cells in primary tumor tissue that may respond to CD40 agonists (Chevrier S, Levine JH, Zanotelli VRT, et al. An Immune Atlas of Clear Cell Renal Cell Carcinoma. Cell. 2017;169(4):736-749.e18; Zhang L, Li Z, Skrzypczynska KM, et al. Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer. Cell. 2020;181(2):442-459.e29; Zhang Q, He Y, Luo N, et al. Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma. Cell. 2019;179(4):829-845.e20). Therefore, targeting CD40 on DCs has the ability to expand the tumor-specific T cell pool and potentially represents a way to treat immunologically "cold" tumors.

[0009] Carcinoembryonic antigen (CEA) Carcinoembryonic antigens (CEAs) describe a family of highly related glycoproteins (some of which are cell surface-immobilized glycosylphosphatidylinositol (GPIs)) involved in cellular functions such as cell adhesion, phagocytosis, proliferation, and signaling. CEAs are generally characterized by being members of the CD66 family of molecules (with CEAs including examples of CD66a, CD66b, CD66c, CD66d, CD66e, and CD66f molecules). Currently, 29 CEA family genes have been identified, which are generally referred to as carcinoembryonic antigen-associated cell adhesion molecules (CEACAMs). Examples of CEACAM genes are CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21.

[0010] CEA (particularly CEACAM5) is normally produced during fetal development and is present only at very low levels in the blood of healthy adult humans. However, the levels of CEA found in cancer are increased, and in that context, it is characterized as a tumor-associated antigen (TAA). CEA is associated with many types of cancer and tumors, including gastric cancer, pancreatic cancer, lung cancer, breast cancer, and medullary thyroid cancer. CEACAM1, CEACAM6, CEACAM7, and CEACAM5 are particularly associated with cancer and tumors (Zi-Wen Han, Zhi-Wu Lyv, Bin Cui, et al. The old CEACAMs find their new role in tumor immunotherapy. Invest New Drugs volume. 2020 38:1888-1898; Chaogu Zheng, Jing Feng1, Di Lu1, et al. A Novel Anti-CEACAM5 Monoclonal Antibody, CC4, Suppresses Colorectal Tumor Growth and Enhances NK Cells-Mediated Tumor Immunity. PLoS One. 2011;6(6):e21146).

[0011] Despite the progress made in the development of immunotherapies for the treatment of various cancers over the past decade, there remains a need for new and effective drugs to treat cancer, particularly cancers that express CEA.

[0012] Therefore, the present invention seeks to provide improved polypeptide-based therapies for the treatment of cancer, particularly cancers that express CEA.

[0013] PD-1 The programmed death 1 (PD-1) receptor is a negative regulator of antitumor T cell effector function when engaged with its ligand, PD-L1, which is expressed on the cell surface within tumors (Ribas and Wolchok 2018). PD-1 is an immune checkpoint, and its inhibitory function is mediated by the tyrosine phosphatase SHP-2, which dephosphorylates downstream signaling molecules of the T cell receptor (TCR) signaling molecule. PD-1 has two ligands: programmed death ligand 1 (PD-L1, also known as CD274 or B7-H1), which is widely expressed by many somatic cells, primarily upon exposure to pro-inflammatory cytokines, and programmed death ligand 2 (PD-L2, also known as CD273 or B7-DC), which has more limited expression in antigen-presenting cells. Inflammation-inducible PD-L1 expression in the tumor microenvironment leads to PD-1-mediated T cell exhaustion and inhibits the antitumor cytotoxic T cell response. PD-L1 is expressed in both tumor cells and bone marrow cells. PD-1 resistance can be broadly subdivided into primary resistance or secondary (acquired) resistance (Kluger et al. 2020). [Overview of the project]

[0014] Therefore, the present invention seeks to provide improved polypeptide-based therapies for the treatment of cancer, particularly cancers that express CEA.

[0015] The inventors have surprisingly found that combination therapy comprising a CD40-CEA bispecific antibody or its antigen-binding fragment and a PD-1 inhibitor (such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or their antigen-binding fragments) is remarkably effective in treating cancer. As shown in the examples herein, the combination of a CD40×CEA bispecific antibody and a PD-1 inhibitor (such as an anti-PD-1 antibody or an anti-PD-L1 antibody) remarkably produced synergistic effects both in vitro and in vivo compared to the use of CD40×CEA bispecific or a PD-1 inhibitor alone. The inventors also hereby show that this effect is supported by the finding that the CD40×CEA bispecific antibody induces upregulation of PD-1 and PD-L1 expression. Such an effect could not have been predicted prior to the invention.

[0016] In a first aspect, the present invention provides a combination therapy comprising (a) a bispecific polypeptide comprising a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to CEA, and (b) a PD-1 inhibitor which is formulated for parenteral delivery.

[0017] A second aspect of the present invention provides a pharmaceutical composition comprising: (a) a bispecific polypeptide having a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA); and (b) a PD-1 inhibitor formulated for parenteral delivery.

[0018] Using such combination therapies and pharmaceutical compositions, highly effective and safe cancer immunotherapy can be established. A third aspect of the present invention provides combination therapies or pharmaceutical compositions for pharmaceutical use, particularly for use in the treatment of cancer and / or tumors in a subject. Preferably, the tumor is a solid tumor.

[0019] A fourth aspect of the present invention provides the use of combination therapy or pharmaceutical composition in the preparation of a pharmaceutical product. Preferably, the pharmaceutical product is for the treatment of cancer and / or tumors as described herein.

[0020] A fifth aspect of the present invention provides a method for treating cancer and / or tumors in a subject, comprising: (a) administering to the subject an effective amount of a bispecific polypeptide comprising a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA); and (b) administering to the subject an effective amount of a PD-1 inhibitor, wherein the PD-1 inhibitor is administered parenterally.

[0021] A sixth aspect of the present invention provides a bispecific polypeptide for pharmaceutical use, comprising a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), wherein the bispecific polypeptide is for use in combination with a PD-1 inhibitor, and the PD-1 inhibitor is formulated for parenteral administration.

[0022] A seventh aspect of the present invention provides a bispecific polypeptide for use in the treatment of cancer and / or tumors in a subject, comprising a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), wherein the bispecific polypeptide is for use in combination with a PD-1 inhibitor, and the PD-1 inhibitor is formulated for parenteral administration.

[0023] An eighth aspect of the present invention comprises combination therapies, pharmaceutical compositions, bispecific polypeptides, methods, or uses, substantially described herein with reference to the specification and drawings.

[0024] Clinical advances with immune cytokines have so far been unimpressive, as tumor-binding entities only confer limited tumor localization, and most immune cytokines terminate in other compartments, leaving side effects lingering. Bispecific antibodies that limit activity to tumors, as described in the combination therapies and pharmaceutical compositions of the present invention, would offer a clear advantage over immune cytokines, as they are inactive in the absence of cancer and / or tumors, particularly cancer and / or tumors expressing CEA.

[0025] To avoid affecting parts of the immune system not involved in inducing tumor immunity, to avoid systemic toxicity from CD40 activators, and to achieve high efficacy in the tumor region, the molecular format design of CD40 agonists can be optimized. For example, a favorable efficacy / safety profile can be obtained with a CD40-CEA bispecific antibody that requires crosslinking by binding to CEA for CD40 activation to occur. Thus, CD40-expressing cells, such as dendritic cells present in tumor tissue, are preferentially activated, while CD40-expressing cells in other tissues with low or absent CEA expression are not activated. This would allow for concentrated activation of CD40-expressing cells specifically in tumor tissue while limiting the toxicity induced by general CD40 activation. [Modes for carrying out the invention]

[0026] Structure of a bispecific polypeptide in a combination therapy or pharmaceutical composition In its broadest sense, “polypeptide” is used herein to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptide mimetic compounds. Therefore, the term “polypeptide” also includes short peptide sequences, as well as longer polypeptides and proteins. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including D or L optical isomers, as well as both amino acid analogs and peptide mimetic compounds.

[0027] As used herein, the term “dual specificity” means that a polypeptide can specifically bind to at least two target entities. Therefore, as used herein, dual specificity can describe a polypeptide that can specifically bind to more than two target entities, such as at least three, at least four, or at least five target entities. In a preferred embodiment, a dual specific polypeptide can specifically bind to two target entities.

[0028] Therefore, the first and / or second binding domains may be selected from the group consisting of antibodies and their antigen-binding fragments, as well as CD40 ligands.

[0029] "Antibodies or their antigen-binding fragments" include not only substantially intact antibody molecules, but also chimeric antibodies, humanized antibodies, isolated human antibodies, single-chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and their antigen-binding fragments and derivatives. Suitable antigen-binding fragments and derivatives include Fv fragments (e.g., single-chain Fv and disulfide-bonded Fv), Fab-like fragments (e.g., Fab fragments, Fab' fragments and F(ab)2 fragments), single variable domains (e.g., VH and VL domains), and single-domain antibodies (dAb [i.e., dAb-linker-dAb], including single and dual formats, as well as nanobodies). The potential advantages of using antibody fragments rather than whole antibodies are several times greater. Smaller fragments can result in improved pharmacological properties, such as better penetration into solid tissues. Furthermore, antigen-binding fragments such as Fab, Fv, ScFv, and dAb antibody fragments can be expressed in E. coli and secreted from there, making it possible to easily mass-produce these fragments.

[0030] In one preferred embodiment, the polypeptide is a bispecific antibody (many examples of which are described in detail below).

[0031] In one embodiment, the antigen-binding fragment is selected from the group consisting of Fv fragments (such as single-chain Fv fragments or disulfide-linked Fv fragments), Fab-like fragments (such as Fab fragments, Fab' fragments, or F(ab)2 fragments), and single-domain antibodies.

[0032] Furthermore, the expression “antibody or antigen-binding fragment” is intended to encompass antibody mimics (e.g., non-antibody scaffold structures that allow for the introduction of variability at specific locations while maintaining high stability). Those skilled in the art of biochemistry will be familiar with many such molecules, such as those discussed in Gebauer & Skerra, 2009 (whose disclosure is incorporated herein by reference). Exemplary antibody mimetic compounds include afibody (also known as trinectin, Nygren, 2008, FEBS J, 275, 2668-2676), CTLD (also known as tetranectin, Innovations Pharmac. Technol. (2006), 27-30), adnectin (also known as monobody, Meth. Mol. Biol., 352 (2007), 95-109), anticarin (Drug Discovery Today (2005), 10, 23-33), DARPin (ankyrin; Nat. Biotechnol. (2004), 22, 575-582), avimer (Nat. Biotechnol. (2005), 23, 1556-1561), and microbody (FEBS Examples include J, (2007), 274, 86-95, peptide aptamer (Expert. Opin. Biol. Ther. (2005), 5, 783-797), Kunitz domain (J. Pharmacol. Exp. Ther. (2006) 318, 803-809), affilin (Trends. Biotechnol. (2005), 23, 514-522), and affimer (Avacta Life Sciences, Wetherby, UK).

[0033] Furthermore, chimeric T cell receptors (also known as chimeric immune receptors, and chimeric antigen receptors or CARs) (see Pule et al., 2003, whose disclosure is incorporated herein by reference) are also included within the scope of bispecific polypeptides. These are engineered receptors that transfer arbitrary specificity to immune effector cells. Typically, CARs are used to transfer the specificity of monoclonal antibodies to T cells, and the transfer of their coding sequence is facilitated by retroviral vectors. The most common form of such molecules is a fusion containing a single-stranded variable fragment (scFv) derived from a monoclonal antibody, fused with the transmembrane and endodomains of CD3-zeta. When T cells express this fusion molecule, they recognize and kill target cells that express the specificity of the transferred monoclonal antibody.

[0034] Those skilled in the art will further understand that bispecific polypeptides also include modified versions of antibodies and their antigen-binding fragments, which are modified by covalent bonding of, for example, polyethylene glycol or other suitable polymers (see below), whether currently existing or in the future.

[0035] Methods for generating antibodies and antibody fragments are well known in the art. For example, antibodies may be generated by any one of several methods using the induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi et al., 1989, Winter et al., 1991; these disclosures are incorporated herein by reference), or the generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, hybridoma techniques, human B-cell hybridoma techniques, and Epstein-Barr virus (EBV)-hybridoma techniques (Kohler et al., 1975, Kozbor et al., 1985, Cote et al., 1983, Cole et al., 1984; these disclosures are incorporated herein by reference).

[0036] Preferred methods for the production of monoclonal antibodies are also disclosed in “Monoclonal Antibodies: A manual of techniques”, H. Zola (CRC Press, 1988, the disclosure of which is incorporated herein by reference) and “Monoclonal Hybridoma Antibodies: Techniques and Applications”, J.GR. Hurrell (CRC Press, 1982, the disclosure of which is incorporated herein by reference).

[0037] Similarly, antibody fragments can be obtained using methods well known in the art (e.g., Harlow & Lane, 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, New York, the disclosure of which is incorporated herein by reference). For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression of the DNA encoding the fragment in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems). Alternatively, antibody fragments can be obtained by pepsin or papain digestion of the whole antibody by conventional methods.

[0038] Those skilled in the art will understand that human or humanized antibodies are preferably used for human therapy or diagnosis. The humanized form of a non-human (e.g., mouse) antibody is preferably a genetically engineered chimeric antibody or antibody fragment having a minimal portion derived from the non-human antibody. Humanized antibodies include antibodies in which the complementary determinant region of a human antibody (recipient antibody) is replaced with residues from the complementary determinant region of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired function. In some cases, Fv framework residues of the human antibody are replaced with corresponding non-human residues. Humanized antibodies may also contain residues not found in the recipient antibody or in the transferred complementary determinant region or framework sequence. Generally, humanized antibodies contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the complementary determinant region corresponding to that of the non-human antibody, and all or substantially all of the framework region corresponding to that of the relevant human consensus sequence. Humanized antibodies optimally include at least a portion of the antibody constant region, such as the Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986, Riechmann et al., 1988, and Presta, 1992, the disclosures of which are incorporated herein by reference).

[0039] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often called imported residues and are usually obtained from imported variable domains. Humanization can be carried out as essentially described by substituting human complementarity-determining regions with corresponding rodent complementarity-determining regions (see, e.g., Jones et al., 1986, Reichmann et al., 1988, Verhoeyen et al., 1988, US4,816,567, the disclosures of which are incorporated herein by reference). Thus, such humanized antibodies are chimeric antibodies in which substantially fewer than the intact human variable domains are substituted with corresponding sequences from non-human species. In practice, humanized antibodies can typically be human antibodies in which several complementarity-determining region residues, and possibly several framework residues, are substituted with residues from similar sites in a rodent antibody.

[0040] Human antibodies can also be identified using a variety of techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991; Marks et al., 1991; Cole et al., 1985; and Boerner et al., 1991, the disclosures of which are incorporated herein by reference).

[0041] Those skilled in the art will understand that bispecific polypeptides, such as antibodies, can be in any preferred structural format.

[0042] Therefore, in the exemplary embodiment of the bispecific antibody, (a) Binding domain B1 and / or binding domain B2 are intact IgG antibodies (or together form intact IgG antibodies), (b) The binding domain B1 and / or the binding domain B2 are Fv fragments (e.g., scFv), (c) The binding domain B1 and / or the binding domain B2 are Fab fragments and / or (d) The binding domain B1 and / or binding domain B2 are single-domain antibodies (e.g., domain antibodies and nanobodies).

[0043] Those skilled in the art will understand that the bispecific antibody may contain a human Fc region or a variant of said region, and that the region is an IgG1 region, an IgG2 region, an IgG3 region, or an IgG4 region, preferably an IgG1 region or an IgG4 region.

[0044] By manipulating the Fc region of therapeutic monoclonal antibodies or Fc fusion proteins, it becomes possible to generate molecules more suitable for the required pharmacological activity (Strohl, 2009, the disclosure of which is incorporated herein by reference).

[0045] "CD40 ligand" refers to a non-antibody molecule capable of binding to CD40, such as CD40L (CD154, e.g., GenBank: D31797.2), or a fragment or variant of CD40L that retains their ability to bind to CD40.

[0046] (a) Manipulated Fc region for increased half-life One approach to improving the efficacy of therapeutic antibodies is to increase their serum persistence, thereby enabling higher circulating levels, less frequent administration, and lower doses.

[0047] The half-life of IgG depends on its pH-dependent binding to the neonatal receptor FcRn. FcRn, expressed on the surface of endothelial cells, binds to IgG in a pH-dependent manner, protecting it from degradation.

[0048] Some antibodies that selectively bind to FcRn at pH 6.0 but not at pH 7.4 exhibit longer half-lives in various animal models. In addition, some antibodies that bind to FcRn with higher affinity at pH 6.0 but remain at lower affinity at pH 7.4 also exhibit longer half-lives.

[0049] Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L (Hinton et al., 2004, its disclosure incorporated herein by reference) and M252Y / S254T / T256E+H433K / N434F (Vaccaro et al., 2005, its disclosure incorporated herein by reference), have been shown to increase binding affinity to FcRn and the in vivo half-life of IgG1.

[0050] (b) Fc area manipulated for the modified effect function To ensure the absence of CD40 activation in the absence of CEA, the Fc portion of the bispecific antibody should either not bind to FcγR or bind with very low affinity, because FcγR-mediated crosslinking of the CD40 antibody can induce activation. "Very low affinity" includes the Fc portion exhibiting at least a 10-fold reduced affinity for FcγRI, FcγRII, and III compared to wild-type IgG1, when half of the maximum binding is determined by the concentration achieved by flow cytometry analysis of FcγR-expressing cells (Hezareh et al., 2001) or FcγR ELISA (Shields et al., 2001).

[0051] Another factor to consider is that FcγR engagement can also induce antibody-dependent cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) in antibody-coated cells. In one embodiment, to enhance tumor-dependent CD40 activation and to avoid depletion of CD40-expressing cells, the isotype of the CD40-CEA bispecific antibody should preferably be silent.

[0052] The four human IgG isotypes bind to the activated Fcγ receptor (FcγRI, FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the first component of complement (C1q) with different affinities, resulting in very different effector functions (Bruhns et al., 2009, its disclosure incorporated herein by reference). The IgG1 molecule has the highest affinity and ability to induce effector function, while IgG2, IgG3, and IgG4 are less effective (Bruhns, 2012, Hogarth and Pietersz, 2012, Stewart et al., 2014) (Wang et al. 2015, Vidarson et al. 2014). In addition, certain mutations within the Fc region of IgG1 dramatically reduce FcγR affinity and effector function while preserving neonatal FcR (FcRn) interactions (Ju and Jung, 2014; Leabman et al., 2013; Oganesyan et al., 2008; Sazinsky et al., 2008).

[0053] The most widely used IgG1 variants are N297A alone or in combination with D265A, as well as L234 and L235 mutations, including the so-called "LALA" double mutant L234A / L235A. Another site described for further silencing IgG1 through mutation is P329 (see US2012 / 0251531).

[0054] Therefore, selecting a mutant IgG1 format that has low effector function but retains binding to FcRn may result in a bispecific antibody that exhibits CD40 CEA-dependent activation, a favorable potency / safety profile, and good PK properties.

[0055] Advantageously, the polypeptide is unable to induce antibody-dependent cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). “Unable” means that the polypeptide’s ability to induce ADCC, etc., is at least 10-fold lower compared to wild-type IgG1, as demonstrated, for example, by monocyte-dependent ADCC or CDC assays reported by Hezareh et al. 2001.

[0056] In one embodiment, the Fc region is located at the following position: It may be a variant of the human IgG1 Fc region containing mutations in one or more of L234, L235, P239, D265, N297, and / or P329.

[0057] Advantageously, alanine can be present at the mutated location(s).

[0058] Optionally, IgG1 variants may be variants of the human IgG1 Fc region, including mutants L234A and L235A (i.e., LALA double mutants, see SEQ ID NO: 336).

[0059] Those skilled in the art will understand that bispecific polypeptides can be of several different structural formats (see, for example, Chan & Carter, 2016, the disclosure of which is incorporated herein by reference).

[0060] In an exemplary embodiment, the bispecific antibody is (a) A bivalent bispecific antibody such as an IgG-scFv bispecific antibody (for example, B1 is intact IgG and B2 is scFv bound to B1 at the N-terminus of the light chain and / or the C-terminus of the light chain and / or the N-terminus of the heavy chain and / or the C-terminus of the heavy chain of the IgG, or vice versa), (b) Monovalent bispecific antibodies such as DuoBody® (Genmab AS, Copenhagen, Denmark) or "Nobu-in-Hole" bispecific antibodies (e.g., scFv-KIH, scFv-KIH) r , BiTE-KIH or BiTE-KIH r (See Xu et al., 2015, mAbs 7(1):231-242) (c) scFv2-Fc bispecific antibody (such as ADAPTIR® bispecific antibody from Aptevo Therapeutics), (d) BiTE / scFv2 bispecific antibody, (e) DVD-Ig bispecific antibody, (f) DART-based bispecific antibodies (e.g., DART2-Fc or DART), (g) DNL-Fab3 bispecific antibody, and (h) Selected from the group consisting of scFv-HSA-scFv bispecific antibodies.

[0061] For example, a bispecific antibody may be an IgG-scFv antibody. The IgG-scFv antibody may be either VH-VL oriented or VL-VH oriented. In one embodiment, the scFv may be stabilized by an SS bridge between VH and VL.

[0062] In one embodiment, binding domains B1 and B2 are directly fused with each other.

[0063] In alternative embodiments, binding domains B1 and B2 are linked via a polypeptide linker. For example, the polypeptide linker may be a short linker peptide consisting of about 10 to about 25 amino acids. The linker is typically rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa.

[0064] Therefore, the linker can be selected from the group consisting of amino acid sequences SGGGGSGGGGS (SEQ ID NO: 337), SGGGGSGGGGSAP (SEQ ID NO: 338), NFSQP (SEQ ID NO: 339), KRTVA (SEQ ID NO: 340), GGGSGGGG (SEQ ID NO: 341), GGGGSGGGGS (SEQ ID NO: 342), GGGGSGGGGSGGGGS (SEQ ID NO: 343), GTSTGSGKPGSGEGSTKG (SEQ ID NO: 344) (Whitlow et al. 1993), THTCPPCPEPKSSDK (SEQ ID NO: 345), GGGS (SEQ ID NO: 346), EAAKEAAKGGGGS (SEQ ID NO: 347), EAAKEAAK (SEQ ID NO: 348), or (SG)m, where m = 1 to 7.

[0065] In a preferred embodiment, the linker may be selected from the group consisting of SEQ ID NOs: 341, 342, and 343. In a particularly preferred embodiment, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 343).

[0066] As used herein, the term “amino acid” includes the standard 20 genetically encoded amino acids and their corresponding “D” type (compared to the natural “L” type) stereoisomers, omega-amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derived amino acids (see below).

[0067] When amino acids are specifically listed, such as "alanine," "Ala," or "A," unless explicitly stated otherwise, this term refers to both L-alanine and D-alanine. Other unconventional amino acids can also be suitable components of bispecific polypeptides, as long as the desired functional properties are maintained by the polypeptide. For the peptides shown, where appropriate, each encoded amino acid residue is represented by a single letter corresponding to the common name of the conventional amino acid.

[0068] In one embodiment, the bispecific polypeptide contains or consists of an L-amino acid.

[0069] Those skilled in the art will understand that a bispecific polypeptide may contain, or consist of, one or more modified or derivatized amino acids.

[0070] Chemical derivatives of one or more amino acids can be achieved by reaction with functional side groups. Examples of such derivatized molecules include those in which a free amino group is derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxybenzoxy group, a t-butyloxycarbonyl group, a chloroacetyl group, or a formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters, or other types of esters and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include peptides containing naturally occurring amino acid derivatives of 20 standard amino acids. For example, proline may be substituted with 4-hydroxyproline, lysine with 5-hydroxylysine, histidine with 3-methylhistidine, serine with homoserine, and lysine with ornithine. Derivatives also include peptides containing one or more additions or deletions, as long as the desired activity is maintained. Other modifications include amidation, amino-terminal acylation (e.g., acetylation or thioglycolate amidation), terminal carboxylamilation (e.g., by ammonia or methylamine), and similar terminal modifications.

[0071] Those skilled in the art will further understand that peptide mimetic compounds can also be useful. The term "peptide mimetic" refers to a compound that mimics the three-dimensional structure and desirable characteristics of a particular peptide as a therapeutic agent.

[0072] For example, the polypeptide includes not only molecules in which amino acid residues are linked by peptide (-CO-NH-) bonds, but also molecules in which the peptide bonds are reversed. Such retro-inversopeptide mimetic compounds can be prepared using methods known in the art, such as those described in Meziere et al. (1997), which are incorporated herein by reference. This approach involves preparing pseudopeptides that contain changes including the backbone, rather than the orientation of the side chains. Retroinversopeptides containing NH-CO bonds instead of CO-NH peptide bonds are far more resistant to proteolysis. Alternatively, the polypeptide may be a peptide mimetic compound in which one or more amino acid residues are linked by -y(CH2NH)- bonds instead of conventional amide bonds.

[0073] In further alternatives, the peptide bond may be completely partitioned, provided that a suitable linker moiety is used to maintain the spacing between carbon atoms of the amino acid residues, and it may be advantageous that the linker moiety has substantially the same charge distribution and substantially the same planarity as the peptide bond.

[0074] Furthermore, it will be understood that the polypeptide may be conveniently blocked at its N-terminus or C-terminus to help reduce its sensitivity to exoprotein digestion.

[0075] Various non-coding or modified amino acids, such as D-amino acids and N-methylamino acids, have also been used to modify mammalian peptides. In addition, the proposed bioactive three-dimensional structure may be stabilized by covalent modifications such as cyclization, or by the incorporation of lactams or other types of bridges; see, for example, Veber et al., 1978 and Thursell et al., 1983, incorporated herein by reference.

[0076] In one embodiment, one of binding domains B1 or B2 is an immunoglobulin molecule, and one of binding domains B1 or B2 is a Fab fragment, the Fab fragment being fused to the C-terminus of the heavy chain of the immunoglobulin via the light chain of the Fab fragment.

[0077] For example, a polypeptide may have a format as shown in Figure 23. Such a format is referred to as the "RUBY™ format" (as described in pending UK Patent Application No. 1820556.7 and PCT Application No. WO2020 / 127354). Antibodies of the "RUBY™ format" and "Optimized RUBY™ format" described herein are particularly preferred for bispecific polypeptides.

[0078] A bispecific polypeptide containing one or more mutations may promote the association of an immunoglobulin heavy chain polypeptide with an immunoglobulin light chain polypeptide and / or promote the association of a Fab heavy chain polypeptide with a Fab light chain polypeptide.

[0079] In one embodiment, one or more mutations prevent the formation of aggregates and Fab byproducts.

[0080] Those skilled in the art will understand that in one embodiment, mutations can prevent the formation of aggregates and / or Fab byproducts by generating steric hindrance and / or charge mismatch.

[0081] "Steric hindrance" refers to a delay in a reaction caused by steric bulk, i.e., the size of the amino acid molecule, which otherwise prevents the association of two protein surfaces that could occur in the presence of a smaller amino acid.

[0082] "Charge incompatibility" means that the charges are incompatible, preventing the formation of products and thus avoiding the formation of unwanted products. For example, if two negatively charged parts are present, they may repel each other, preventing the formation of unwanted products.

[0083] As described above, the mutation restricts the formation of Fab byproducts and / or aggregates, for example, by creating a surface that restricts the formation of aggregates or byproduct Fab fragments. In one embodiment, the mutation prevents the formation of Fab byproducts by generating steric hindrance and / or inter-charge mismatch (resulting in improper chain charge mismatch). The mutation can also promote correct inter-chain interactions (i.e., between the first heavy chain polypeptide and the first light chain polypeptide, and / or between the second heavy chain polypeptide and the second light chain polypeptide) by, for example, creating salt or disulfide crosslinks.

[0084] Therefore, the mutation may support the formation of a bispecific polypeptide.

[0085] In one embodiment, the proportion of aggregates formed during production is 25% or less. Optionally, the proportion of aggregates is 20%, 17.5%, 15%, 13.5%, or 10% or less. Preferably, the proportion of aggregates is less than 10%. Optionally, these measurements are performed when the chains of the bispecific polypeptide are transfected in equal proportions, for example, in a 1:1:1 ratio when three chains are used during production.

[0086] Alternatively, the chain transfection ratio can be optimized. Optionally, when the chain transfection ratio is optimized, the percentage of aggregates may be 3.5%, 3%, 2.5%, or 2% or less.

[0087] In one embodiment, the bispecific polypeptide comprises one or more mutant pairs, each containing two functionally compatible mutants.

[0088] "Functionally compatible mutations" mean that the mutations have complementary functions. For example, one mutation in a pair (within one chain) may form a positively charged region, while the other mutation (within another chain) forms a negatively charged region. Together, these mutations act in a functionally compatible manner, promoting the association of each chain.

[0089] In one embodiment, the bispecific polypeptide is a group of regions as follows: (a) CH1 and C-kappa or C-lambda regions of immunoglobulins, and / or (b) CH1 and C-kappa or C-lambda regions of Fab, and / or (c) The VL and VH regions of immunoglobulins, and / or (d) Contains one or more mutation pairs in one or more of the VL and VH regions of Fab.

[0090] Therefore, in one embodiment, the mutation pair is located within the CH1 and C-kappa or C-lambda regions of the Fab and / or immunoglobulin, and the mutation pair is (a) Cavity and protrusion surface variations (i.e., steric variations), and / or (b) Hydrophobic exchange mutations, and / or (c) Charge mutations (i.e., salt mutations), and / or (d) A mutation that results in the formation of a disulfide bridge is selected.

[0091] Mutation pairs may, alternatively or additionally, be located within the VH and VL regions of Fab and / or immunoglobulins, and mutation pairs within the VH and VL regions are (a) Charge mutations (i.e., salt mutations), and / or (b) Double charge variation, and / or (c) A mutation resulting in the formation of a disulfide bridge is selected.

[0092] In one embodiment of a bispecific polypeptide, (a) One or more of the following locations in the CH1 domain: H168, F170, L145, S183, and T187 (according to the EU numbering system), and / or (b) Any position within the C Kappa or C Lambda domain that is selected from one or more of the following positions: 132–138, 173–179, 130–136, 111–117, and 134–140 (according to the EU numbering system), and / or (c) The following position ranges in the VL: positions 41-47, 117-123, and 46-52 (according to the IMGT numbering system), one or more of these positions, and / or (d) The position is selected from the group consisting of the following position ranges in VH: positions 41-47, positions 46-52, and positions 117-123 (according to the IMGT numbering system).

[0093] In one embodiment, the bispecific polypeptide is (a) One or more of the following locations in the CH1 domain: H168, F170, L145, S183, and T187 (according to the EU numbering system), and / or (b) A position in the C Kappa or C Lambda domain that is selected from one or more of the following position ranges: positions 132–138, 173–179, 130–136, 111–117, and 134–140 (according to the Kabat numbering system), and / or (c) The following position ranges in the VL: positions 41-47, 117-123, and 46-52 (according to the IMGT numbering system), one or more of these positions, and / or (d) The position is selected from the group consisting of the following position ranges in VH: positions 41-47, positions 46-52, and positions 117-123 (according to the IMGT numbering system).

[0094] In one embodiment, the bispecific polypeptide is (a) One or more of the following locations in the CH1 domain: H168, F170, L145, S183, and T187 (according to the EU numbering system), and / or (b) Any position within the C Kappa or C Lambda domain that is selected from one or more of the following positions: 132–138, 173–179, 130–136, 111–117, and 134–140 (according to the EU numbering system), and / or (c) The following position ranges in the VL: positions 41-47, 117-123, and 46-52 (according to the IMGT numbering system), one or more of these positions, and / or (d) The position is selected from the group consisting of the following position ranges in VH: positions 41-47, positions 46-52, and positions 117-123 (according to the IMGT numbering system).

[0095] Each mutation within the ranges shown above is a relevant functional mutation, and therefore a relevant interface between chains, as it is located at a position where it comes into contact with an amino acid within the corresponding domain / chain.

[0096] Therefore, it will be understood by those skilled in the art that the preference for mutations in the positional range shown above is that the relevant functional feature is whether the position is in contact with a corresponding position on another chain, i.e., the relevant position is a position in the VH chain that is in contact with a corresponding position in the VL chain, or a position in the C-lambda that is in contact with a position in the CH1 chain.

[0097] In one embodiment, the mutation is VH X44R / E / D / K, X49C, X120K VL X44R / E / D / K, X49D X120C CH1 H168A / G, F170G / A, L145Q, S183V, T187E / D, C Kappa / C Lambda S / T114A, V133T, L135Y / W, N / S137K / R / H, S176W / V / Y It is selected from the group consisting of the following. *Numbering follows the IMGT system for VH / VL domains and the EU numbering system for permanent domains. *X refers to any amino acid.

[0098] The use of " / " in the context of considering mutations illustrates possible alternative amino acids; for example, "X44R / E / D / K" indicates that R, E, D, or K can be substituted for the amino acid "X" at position 44.

[0099] In one embodiment, the mutation is VH X44R / E / D / K, X49C, X120K VL X44R / E / D / K, X49D X120C CH1 H168A / G, F170G / A, L145Q, S183V, T187E / D, C Kappa / C Lambda S / T114A, V133T, L135Y / W, N / S137K / R / H, S176W / V / Y It is selected from the group consisting of the following. *Numbering follows the IMGT system for VH / VL domains and the Kabat numbering system for stationary domains. *X refers to any amino acid.

[0100] In one embodiment, the bispecific polypeptide is (a) One or more of the following locations in the CH1 domain: H168, F170, L145, S183, and T187 (according to the EU numbering system), and / or (b) One or more of the following locations in the C Kappa domain: L135, S176, V133, S114, and N137 (according to the EU numbering system), and / or one or more of the following locations in the C Lambda domain: L135, S176, V133, T114, and S137 (according to the EU numbering system), and / or (c) One or more of the following locations in the VL: Q44, Q120, and A49 (according to the IMGT numbering system), and / or (d) The following locations in VH: one or more of the following locations: Q44, G49, and Q120 (according to the IMGT numbering system), selected from the group.

[0101] In one embodiment, the bispecific polypeptide is (a) One or more of the following locations in the CH1 domain: H168, F170, L145, S183, and T187 (according to the EU numbering system), and / or (b) One or more of the following locations in the C Kappa domain: L135, S176, V133, S114, and N137 (according to the Kabat numbering system), and / or one or more of the following locations in the C Lambda domain: L135, S176, V133, T114, and S137 (according to the Kabat numbering system), and / or (c) One or more of the following locations in the VL: Q44, Q120, and A49 (according to the IMGT numbering system), and / or (d) The following locations in VH: one or more of the following locations: Q44, G49, and Q120 (according to the IMGT numbering system), selected from the group.

[0102] For example, mutations are (a) One or more of the following mutations in the CH1 domain: H168A, F170G, L145Q, S183V, and T187E (according to the EU numbering system), and / or (b) One or more of the following mutations in the C kappa domain: L135Y, S176W, V133T, S176V, S114A, and N137K (according to the EU numbering system), and / or one or more of the following mutations in the C lambda domain: L135Y, S176W, V133T, S176V, T114A, and S137K (according to the EU numbering system), and / or (c) One or more of the following mutations in VL: Q44R, Q44E, Q120C, Q44D, and A49D (according to the IMGT numbering system), and / or (d) The following mutations in VH may be selected from the group consisting of one or more of the following: Q44E, Q44R, G49C, Q44K, and Q120K (according to the IMGT numbering system).

[0103] For example, mutations are (a) One or more of the following mutations in the CH1 domain: H168A, F170G, L145Q, S183V, and T187E (according to the EU numbering system), and / or (b) One or more of the following mutations in the C kappa domain: L135Y, S176W, V133T, S176V, S114A, and N137K (according to the Kabat numbering system), and / or one or more of the following mutations in the C lambda domain: L135Y, S176W, V133T, S176V, T114A, and S137K (according to the Kabat numbering system), and / or (c) One or more of the following mutations in VL: Q44R, Q44E, Q120C, Q44D, and A49D (according to the IMGT numbering system), and / or (d) The following mutations in VH may be selected from the group consisting of one or more of the following: Q44E, Q44R, G49C, Q44K, and Q120K (according to the IMGT numbering system).

[0104] The above mutations are in the "RUBY (trademark) format".

[0105] In further embodiments, the polypeptide may have a format shown in Figure 23 with further optimized variations, referred to as the "Optimized RUBY® Format".

[0106] While bispecific polypeptides in the "RUBY™ format" can be produced with excellent purity and reproducibility, bispecific polypeptides in the "Optimized RUBY™ format" can be produced with even higher purity and reproducibility. Furthermore, bispecific polypeptides in the "Optimized RUBY™ format" are engineered to have a reduced risk of inducing immunogenic responses directed towards the bispecific polypeptide itself.

[0107] The optimized mutations are described below as including “Optimized Mutation Set 1” and “Optimized Mutation Set 2” – “Set 2a” and / or “Set 2b”. Those skilled in the art will understand that various combinations of these optimized mutations can be used in bispecific polypeptides and in combination with any of the “RUBY® Format” mutations described above. Combinations of “RUBY® Format” mutations and “Optimized RUBY® Format” mutations used in the same bispecific antibody are described below. It will also be understood that variations of these mutations described herein are functional. All mutations within the variable domain (VH or VL) are numbered according to the IMGT numbering system, and all mutations within the constant domain are numbered according to the EU numbering system.

[0108] Mutation Set 1 - Mutations in Variable Domain Weight (VH): T65E, T65A, T65I.

[0109] Mutation Set 2 - Any individual and / or any combination of mutations listed in Sets 2a and 2b. Set 2a - Mutations in CH1: Y180A, Y180G, Y180I, Y180N, Y180S, Y180T, Y180V, or Y180W, and / or S183N or S183T, and / or V188G, preferably Y180T. Set 2b - Mutations in the C kappa domain: A111R, A111T, A111W, or A111V, and / or T109P, preferably T109P and / or A111V, and / or mutations in the variable domain light (VL): I126A, I126G, I126H, I126N, I126P, I126Q, I126S, or I126T.

[0110] In one embodiment, the mutation is (a) T65 position in VH (according to the IMGT numbering system), and / or (b) One or more of the following positions in CH1: Y180, S183, and V188, preferably Y180 (according to the EU numbering system), and / or (c) C. The following locations in the Kappa Domain: one or more of A111 and T109 (according to the EU or Kabat numbering system), and / or (d) It is located in a position selected from the group consisting of I126th position in the VL (according to the IMGT numbering system).

[0111] In certain embodiments, the mutation is located at position T65 in the variable domain weight (VH) (according to the IMGT numbering system).

[0112] In certain embodiments, the mutation is one or more of the following positions in CH1: Y180, S183, and V188, preferably Y180 (according to the EU numbering system).

[0113] In certain embodiments, the mutation is one or more of the following positions in the C-kappa domain: A111 and T109 (according to the EU numbering system), and / or position I126 in the VL (according to the IMGT numbering system).

[0114] In one embodiment, the mutation is (a) X65E / A / I in VH (according to the IMGT numbering system), and / or (b) One or more of the following mutations in CH1: X180A / G / I / N / S / T / V / W, X183N / T, and X188G, preferably X180T (according to the EU numbering system), and / or (c) The following mutations in the C kappa domain: X111R / T / W / V and X109P, preferably one or more of X111V and X109P (according to the EU or Kabat numbering system), and / or (d) X126A / G / H / N / P / Q / S / T in VL (according to the IMGT numbering system) It is selected from the group consisting of the following. *X refers to any amino acid.

[0115] In certain embodiments, the mutation is X65E / A / I in VH (according to the IMGT numbering system). *X refers to any amino acid.

[0116] In certain embodiments, the mutation is one or more of the following mutations in CH1: X180A / G / I / N / S / T / V / W, X183N / T, and X188G, preferably X180T (according to the EU numbering system). *X refers to any amino acid.

[0117] In certain embodiments, the mutation is one or more of the following mutations in the C kappa domain: X111R / T / W / V and X109P, preferably X111V and X109P (according to the EU or Kabat numbering system), and / or the mutation is X126A / G / H / N / P / Q / S / T in the VL (according to the IMGT numbering system). *X refers to any amino acid.

[0118] For example, mutations are (a) The following mutations in VH: one or more of T65E, T65A, and T65I (according to the IMGT numbering system), and / or (b) One or more of the following mutations in CH1: Y180A, Y180G, Y180I, Y180N, Y180S, Y180T, Y180V, Y180W, S183N, S183T, V188G, preferably Y180T (according to the EU numbering system), and / or (c) The following mutations in the C kappa domain: A111R, A111T, A111W, A111V, and T109P, preferably one or more of T109P and A111V (according to the EU or Kabat numbering system), and / or (d) The following mutations in the VL may be selected from the group consisting of one or more of I126A, I126G, I126H, I126N, I126P, I126Q, I126S, and I126T (according to the IMGT numbering system).

[0119] In certain cases, the mutation is one or more of the following mutations in VH: T65E, T65A, and T65I (according to the IMGT numbering system).

[0120] In certain cases, the mutation is one or more of the following mutations in CH1: Y180A, Y180G, Y180I, Y180N, Y180S, Y180T, Y180V, Y180W, S183N, S183T, V188G, preferably Y180T (according to the EU numbering system).

[0121] In certain cases, the mutations are one or more of the following mutations in the C kappa domain: A111R, A111T, A111W, A111V, and T109P, preferably T109P and A111V (according to the EU or Kabat numbering system), and / or one or more of the following mutations in the VL: I126A, I126G, I126H, I126N, I126P, I126Q, I126S, and I126T (according to the IMGT numbering system).

[0122] As discussed above, any combination of the "RUBY(trademark) format" mutation and the "optimized RUBY(trademark) format" mutation can be used with the same bispecific polypeptide, such as one or more of the following "RUBY(trademark) format" mutations in (a) to (d), or variations described herein, and can be combined with one or more of the following "optimized RUBY(trademark) format" mutations in (e) to (g), or variations described herein: (a) One or more of the following mutations in the CH1 domain: H168A, F170G, and / or T187E (according to the EU numbering system) (b) One or more of the following mutations in the C kappa domain: L135Y, S176W, S114A, and / or N137K (according to the EU or Kabat numbering system), and / or one or more of the following mutations in the C lambda domain: L135Y, S176W, T114A, and / or S137K (according to the Kabat numbering system), (c)Variant mutations in VL: Q44R or Q44E (according to the IMGT numbering system), (d) Variations in VH: Q44E or Q44R (according to the IMGT numbering system), (e) Variations in VH: T65E, T65A, or T65I (according to the IMGT numbering system), (f) Mutations in CH1: Y180T (according to the EU numbering system), and / or (g) Variations in C kappa: T109P and / or A111V (according to the EU or Kabat numbering system).

[0123] Therefore, in certain embodiments, a bispecific antibody having a combined "RUBY(trademark) format" mutation and an "optimized RUBY(trademark) format" mutation is defined as having the following mutations: ● One or more of the following mutations in the CH1 domain: H168A, F170G, Y180T, and / or T187E (according to the EU numbering system) ●One or more of the following mutations in the C kappa domain: T109P, A111V, L135Y, S176W, S114A, and / or N137K (according to the EU or Kabat numbering system), and / or one or more of the following mutations in the C lambda domain: L135Y, S176W, T114A, and / or S137K (according to the Kabat numbering system), ●Vulnerabilities in VL: Q44R or Q44E (according to the IMGT numbering system), and / or ●The following mutations in VH may include: Q44E or Q44R, and / or one or more of T65E, T65A, or T65I (according to the IMGT numbering system).

[0124] In one embodiment, one or more Fab fragments are linked to the C-terminus of an immunoglobulin via a linker.

[0125] In one embodiment, the bispecific polypeptide is tetravalent and can bind to each of the two antigens in a divalent state.

[0126] In one embodiment, the bispecific polypeptide comprises an immunoglobulin arranged as an antibody having two arms, thus two binding sites for a first antigen, and two of the Fab fragments, each providing a binding site for a second antigen. Thus, there are two binding sites for the first antigen and two binding sites for the second antigen. The bispecific polypeptide of this embodiment may comprise three polypeptide chains: (1) chain H1, which comprises the heavy chain, linker, and light chain of IgG; (2) chain L1, which is the light chain in IgG; and (3) chain H2, which is the heavy chain in the added (bound) Fab. In a preferred embodiment, the bispecific polypeptide may comprise six polypeptide chains: (a) two chains H1, which comprises the heavy chain, linker, and light chain of IgG; (b) two chains L1, which are the light chains in IgG; and (c) two chains H2, which are the heavy chains in the added (bound) Fab. This structure can be used for both "RUBY™ format" antibodies and "Optimized RUBY™ format" antibodies.

[0127] In one embodiment, binding domain B1 is immunoglobulin and binding domain B2 is Fab. In an alternative embodiment, binding domain B1 is Fab and binding domain B2 is immunoglobulin.

[0128] In one embodiment, a bispecific polypeptide may modulate and / or activate the activity of target immune system cells, the modulation being an increase or decrease in the activity of such cells. Such cells include T cells, dendritic cells, and natural killer cells.

[0129] In another embodiment, the bispecific polypeptide may modulate and / or activate the activity of myeloid cells such as macrophages, monocytes, and myeloid-derived suppressor cells.

[0130] Monocytes and macrophages also express CD40 and can promote immune responses against tumors. In fact, the mouse anti-CD40 substitute antibody FGK45 has been shown to mediate antitumor activity, including macrophages, independently of T cell and NK cell function (Lum HD, Buhtoiarov IN, Schmidt BE, et al. In vivo CD40 ligation can induce T-cell-independent antitumor effects that involve macrophages. J Leukoc Biol. 2006 Jun;79(6):1181-92). However, the effects of CD40 agonists on macrophages and other myeloid cell populations also result in increased production of IFN-γ and CCL5, promoting improved T cell influx into tumors (Huffman AP, Lin JH, Kim SI, et al. CCL5 mediates CD40-driven CD4+ T cell tumor infiltration and immunity. JCI Insight. 2020 May 21;5(10)).

[0131] Several studies have shown that CD40 agonist antibodies can convert TAMs into activated macrophages with an antitumor phenotype. FGK45 interacts with TAMs after in vivo treatment, resulting in their increased expression of MHCII and CD86 (Beatty GL, Chiorean EG, Fishman MP, et al. CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans. Science. 2011 Mar 25;331(6024):1612-6). Similar effects have been observed with CD11b + F4 / 80 +This has been observed in macrophages, the spleen (Luheshi NM, Coates-Ulrichsen J, Harper J, et al. Transformation of the tumor microenvironment by a CD40 agonist antibody correlates with improved responses to PD-L1 blockade in a mouse orthotopic pancreatic tumor model. Oncotarget. 2016 Apr 5;7(14):18508-20), and the liver, and treatment can lead to hepatotoxicity due to its strong effect on macrophages (Byrne KT, Vonderheide RH. CD40 Stimulation Obviates Innate Sensors and Drives T Cell Immunity in Cancer. Cell Rep. 2016 Jun 21;15(12):2719-32; Medina-Echeverz J, Ma C, Duffy AG, et al. Systemic Agonistic Anti-CD40 Treatment of Tumor-Bearing Mice Modulates Hepatic Myeloid-Suppressive Cells and Causes Immune-Mediated Liver Damage.Cancer Immunol Res.2015 May;3(5):557-66).Interestingly, aged and obese mice were shown to be more susceptible to systemic toxicity after immunotherapy such as anti-CD40, further demonstrating that macrophages are the main causative cells in these effects (Bouchlaka MN,Sckisel GD,Chen M, et al. Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy. J Exp Med. 2013 Oct 21;210(11):2223-37; Mirsoian A,Bouchlaka MN,Sckisel GD, et al. Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice. J Exp Med. 2014 Nov 17;211(12):2373-83). Macrophage-mediated hepatotoxicity following anti-CD40 therapy has been shown to be mitigated by subsequent combination therapy with an anti-CSF-1R antibody, blocking CSF-1R signaling that supports monocyte and macrophage differentiation, proliferation, and function (Byrne KT, Vonderheide RH. CD40 Stimulation Obviates Innate Sensors and Drives T Cell Immunity in Cancer. Cell Rep. 2016 Jun 21;15(12):2719-32). Combination therapy with anti-CD40 and anti-CSF-1R is currently being explored in clinical trials (Machiels JP, Gomez-Roca C, Michot JM, et al. Phase Ib study of anti-CSF-1R antibody emactuzumab in combination with CD40 agonist selicrelumab in advanced solid tumor patients. J Immunother Cancer. 2020 Oct;8(2)).

[0132] Immune system cells (e.g., target immune cells) are typically dendritic cells. For example, bispecific polypeptides may be able to induce activation of dendritic cells, which can then internalize tumor-associated debris or extracellular vesicles containing CEA and tumor neoantigens.

[0133] For example, polypeptides are (a) Tumor-specific immune activation, and / or (b) Activation of dendritic cells, and / or (c) Internalization of related tumor debris and / or extracellular vesicles containing CEA as well as tumor neoantigens, and / or (d) Cross-presentation of peptides derived from endogenous tumor antigens in MHC, and / or (e) Priming and activation of effector T cells, and / or (f) It may be possible to induce a direct tumor-killing effect selected from the list consisting of apoptosis, necroptosis, antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

[0134] Those skilled in the art will understand that such activation of dendritic cells may be an increase in the expression of the costimulatory molecules CD40, CD80, or CD86, or increased IL-12 production. Alternatively, dendritic cell activation can be determined by an increased ability to cross-present an antigen, such as a tumor neoantigen, to T cells in MHC class I or II, resulting in enhanced activation of T cells that recognize the antigen by the antigen-presenting cell.

[0135] In one embodiment, a bispecific antibody induces an increase in the uptake of tumor debris or extracellular tumor vesicles by antigen-presenting cells, such as dendritic cells. It will be understood by those skilled in the art that this increase in uptake can be measured by the co-localization or internalization of tumor debris or extracellular tumor vesicles by antigen-presenting cells.

[0136] Increased uptake of tumor debris or tumor extracellular vesicles by antigen-presenting cells subsequently leads to the effective presentation of neoantigens contained within the tumor debris or tumor extracellular vesicles in the context of MHC molecules, in turn leading to a broader tumor-specific T cell repertoire and, therefore, more effective T cell-mediated tumor eradication. Methods for determining the expansion of tumor antigen-specific T cells are well known and include, for example, the use of MHC-peptide multimers, e.g., tetramers or pentamers. Such expansion can be measured by inoculating mice with tumors expressing a specific tumor antigen or tumor model antigens (e.g., ovalbumin), or by inoculating mice with the same cells that have undergone heat shock to induce necrosis, followed by measuring the expansion of tumor antigen-specific T cells using various MHC-tumor (model) antigen peptide tetramers or pentamers by flow cytometry-based methods. Alternatively, such expansion can be measured by culturing dendritic cells with antigen-specific TCR transgenic T cells labeled with a proliferative dye, or with tumor-derived tumor debris or tumor-derived extracellular vesicles transfected with a model antigen (e.g., ovalbumin). The expansion of antigen-specific T cells can be assessed by analyzing the dilution of the proliferative dye using flow cytometry.

[0137] Bispecific polypeptides or binding domains can also be characterized and defined by their binding ability. Standard assays for evaluating the binding ability of ligands to targets are well known in the art and include, for example, ELISA, Western blotting, RIA, and flow cytometry. The binding rate (e.g., binding affinity) of polypeptides can also be evaluated by standard assays known in the art, for example, by surface plasmon resonance analysis or biolayer interferometry.

[0138] The terms "binding activity" and "binding affinity" are intended to refer to the tendency of a polypeptide molecule to bind or not bind to a target. Binding affinity can be quantified by determining the dissociation constant (K D ) of the polypeptide and its target. A lower K D indicates a higher affinity for the target. Similarly, the specificity of binding of a polypeptide to its target can be defined in terms of the comparative dissociation constant (K D ) of the polypeptide for its target compared to the dissociation constants for the polypeptide and another non-target molecule.

[0139] The value of this dissociation constant can be determined directly by well-known methods and can also be calculated for complex mixtures by methods such as those described in Caceci et al., 1984 (this disclosure is incorporated herein by reference). For example, K D can be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman, 1993. Other standard assays for evaluating the binding ability of a ligand such as an antibody to a target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding rate (e.g., binding affinity) of a polypeptide can also be evaluated by standard assays known in the art, such as by surface plasmon resonance analysis (e.g., using a Biacore™ system analysis) or by biolayer interferometry (e.g., using an Octet® system analysis).

[0140] A competitive binding assay can be performed to compare the binding of a polypeptide to its target with the binding of the target by another known ligand of the target, such as another polypeptide. The concentration at which 50% inhibition occurs is known as Ki. Under ideal conditions, Ki is equivalent to K D . Since the measured value of Ki will not be less than K D , the measured value of Ki is K DIt can be conveniently replaced to provide an upper limit.

[0141] Alternative measures of binding affinity include EC50 or IC50. In this context, EC50 indicates the concentration at which the polypeptide achieves 50% of the maximum binding to a fixed amount of the target. IC50 indicates the concentration at which the polypeptide inhibits 50% of the maximum binding to a fixed amount of a competitor to the target. In both cases, lower levels of EC50 or IC50 indicate higher affinity to the target. The EC50 and IC50 values ​​of a ligand for that target can both be determined by well-known methods, such as ELISA. Preferred assays for evaluating the EC50 and IC50 of polypeptides are described in the examples.

[0142] In one embodiment, the bispecific polypeptide can preferably bind to its target with an affinity at least 2, 10, 50, or 100 times greater than its affinity to another non-target molecule.

[0143] In one embodiment, the bispecific polypeptide is (a) Activation of B cells in the presence of CEA (preferably CEACAM5), and / or (b) Activation of dendritic cells in the presence of CEA (preferably CEACAM5), and / or (c) Ability to cross-present dendritic cells with increased neoantigen, and / or (d) It is possible to induce the proliferation of neoantigen-specific T cells.

[0144] In one embodiment, a bispecific polypeptide promotes the uptake of tumor-derived material, which is derived from tumor cells overexpressing CEA (preferably CEACAM5). In a specific embodiment, the uptake of tumor-derived material is carried out by antigen-presenting cells.

[0145] Those skilled in the art will understand that such B cell activation may be characterized by CD86 upregulation and, optionally, by other markers of B cell activation.

[0146] CD40 binding domain The bispecific polypeptide contains a binding domain (B1) that can specifically bind to CD40. Preferably, B1 is an agonist CD40 binding domain.

[0147] The binding domain B1 specifically binds to CD40; that is, it binds to CD40 but not to other molecules, or binds to them with lower affinity. When used herein, the term CD40 typically refers to human CD40. The sequence of human CD40 is shown in GenBank:X60592.1. The binding domain B1 may have some binding affinity to CD40 from other mammals, such as CD40 from non-human primates (e.g., Macaca fascicularis (cynomolgus monkey), Macaca mulatta). Preferably, the binding domain B1 does not bind to mouse CD40 and / or other human TNFR superfamily members, such as human CD137 or OX40.

[0148] Advantageously, the binding domain B1 is 2 × 10 -7 Less than M, or 1.5 × 10 -7 Less than M, or 8.5 × 10 -8 Less than M, or 8×10 -8 Less than M, or 7.5 × 10 -8 Less than M, or 7×10 -8 Less than M, or 9×10 -8 Less than M, or 9×10 -9 Less than M, or 5 × 10 -10 Less than M, or 3 × 10 -10 Less than M, preferably 8.5 × 10 -8 Less than M, comfortable 5x10 -10 Less than M or 3 x 10 -10 Less than M K D It binds to human CD40. Preferably, K D This is measured using Octet and is described, for example, in the examples.

[0149] For example, the binding domain B1 preferably does not bind to mouse CD40, or to any other TNFR superfamily member such as CD137 or OX40. Therefore, typically, the K in the binding domain to human CD40 D This refers to K against other non-target molecules such as mouse CD40, other TNFR superfamily members, or any other unrelated materials or accompanying materials in the environment. D It will be twice, preferably five times, and more preferably less than ten times. More preferably, K D This would be less than 50 times, more preferably less than 100 times, and even more preferably less than 200 times.

[0150] The binding domain B1 can preferably bind to its target with an affinity at least 2, 10, 50, or 100 times greater than its affinity for binding to another non-target molecule.

[0151] Therefore, in summary, the binding domain B1 preferably has the following functional characteristics: a) 2 × 10 -7 Less than M, comfortable 5x10 -10 K is less than M D It binds to human CD40 by value. b) Does not bind to mouse CD40. c) Shows at least one of the other human TNFR superfamily members, e.g., human CD137 or OX40 that do not bind.

[0152] In one embodiment, the binding domain B1 comprises one or more light chain CDR sequences selected from Table C(2) and / or one or more heavy chain CDR sequences selected from Table C(1). Thus, the binding domain B1 is (a) CD40 heavy chain CDR, SEQ ID NOs. 73-89, and / or (b) May contain one or more CDR sequences selected from the group consisting of CD40 light chain CDRs and sequence numbers 90-104.

[0153] In one embodiment, the binding domain B1 includes one, two, or three light chain CDR sequences from a specific row in the individual antibody reference in Table C(2), and / or one, two, or three heavy chain CDR sequences from the corresponding row for the antibody having the same reference in Table C(1). For example, the binding domain B1 may include one or more light chain CDR sequences (SEQ ID NOs. 90, 91, and 92) in 1132 and one or more heavy chain CDR sequences (SEQ ID NOs. 73, 74, and 75) in 1132, or the binding domain B1 may include one or more light chain CDR sequences (SEQ ID NOs. 96, 97, and 98) in 1132 and one or more heavy chain CDR sequences (SEQ ID NOs. 81, 82, and 83) in 1132. Most preferably, B1 includes the CDRs of 1132 and / or the VL and VH. Furthermore, most preferably, B1 includes CDR and / or VL and VH of G12 or G12-mut.

[0154] The CDR of G12-mut is shared by ffAC_05337. Therefore, in a preferred embodiment, B1 includes the CDR of ffAC_05337, which are sequence numbers 81-83 and 96-98.

[0155] A preferred CD40 binding domain may include at least a heavy chain CDR3 defined in any individual row of Table C(1), and / or a light chain CDR3 defined in any individual row of Table C(2).

[0156] Therefore, in one embodiment, the binding domain B1 includes all six CDR sequences in a given antibody (VH / VL) reference, for example, the binding domain B1 may include all six CDR sequences of antibody 1132 or all six CDR sequences of antibody G12 (also present in G12_mut and ffAC_05337).

[0157] In one embodiment, the binding domain B1 includes the VH and / or VL amino acid sequences shown in Table A. In one embodiment, the binding domain B1 includes the VH and VL amino acid sequences shown in Table A in a specific antibody reference. For example, the binding domain B1 may include the VH sequence of 1132 (SEQ ID NO: 3) and / or the VL sequence of 1132 (SEQ ID NO: 1), or the VH sequence of G12 (SEQ ID NO: 19) and / or the VL sequence of G12 (SEQ ID NO: 17), the VH sequence of G12-mut (SEQ ID NO: 29) and / or the VL sequence of G12_mut (SEQ ID NO: 17), or the VH sequence of ffAC_05337 (SEQ ID NO: 431) and / or the VL sequence of ffAC_05337 (SEQ ID NO: 430).

[0158] In a preferred embodiment, B1 includes VL and VH of ffAC_05337, which are sequence numbers 430 and 431.

[0159] In one embodiment, the CD40 binding domain of B1 is selected from 1132, 1150, 1140, 1107, G12, APX005, and 21.4.1. Preferably, the CD40 binding domain of B1 is G12 and / or 1132. Most preferably, the CD40 binding domain of B1 is G12. In an alternative most preferred embodiment, the CD40 binding domain of B1 is G12_mut.

[0160] Therefore, the CDR or VH and VL sequences of binding domain B1 are, (a) 1132 (heavy chain CDR: SEQ ID NOs. 73, 74, and 75; light chain CDR: SEQ ID NOs. 90, 91, and 92; VL: SEQ ID NO. 1; VH: SEQ ID NO. 3) (b) 1150 (heavy chain CDR: SEQ ID NOs. 73, 76, and 77; light chain CDR: SEQ ID NOs. 90, 91, and 93; VL: SEQ ID NO. 5; VH: SEQ ID NO. 7) (c)1140 (Heavy chain CDR: SEQ ID NOs. 73, 78, and 79; Light chain CDR: SEQ ID NOs. 90, 91, and 94; VL: SEQ ID NO. 9; VH: SEQ ID NO. 11) (d)1107 (Heavy chain CDR: SEQ ID NOs. 73, 78, and 80; Light chain CDR: SEQ ID NOs. 90, 91, and 95; VL: SEQ ID NO. 13; VH: SEQ ID NO. 15) (e) G12 (heavy chain CDR: SEQ ID NOs. 81, 82, and 83; light chain CDR: SEQ ID NOs. 96, 97, and 98; VL: SEQ ID NO. 17; VH: SEQ ID NO. 19) (f) APX005 (Heavy chain CDR: SEQ ID NOs: 84, 85, and 86; Light chain CDR: SEQ ID NOs: 99, 100, and 101; VL: SEQ ID NO: 21; VH: SEQ ID NO: 23) (g)21.4.1 (Heavy chain CDR: SEQ ID NOs. 87, 88, and 89; Light chain CDR: SEQ ID NOs. 102, 103, and 104; VL: SEQ ID NOs. 25; VH: SEQ ID NOs. 27) (h)G12_mut(heavy chain CDR: SEQ ID NOs. 81, 82, and 83; light chain CDR: SEQ ID NOs. 96, 97, and 98; VL: SEQ ID NO. 17; VH: SEQ ID NO. 29) (i)ffAC_05337 (Heavy chain CDR: SEQ ID NOs. 81, 82, and 83; Light chain CDR: SEQ ID NOs. 96, 97, and 98; VL: SEQ ID NOs. 431; VH: SEQ ID NOs. 430) Antibodies can be selected from the group consisting of the following:

[0161] The numbering of antibodies (e.g., antibody X / Y) defines the heavy chain variable region (X) and the light chain variable region (Y), respectively (or, if a single number is given, only the heavy chain variable region [X] is defined). As mentioned above, the sequence may be one or more CDR sequences, or VH sequences and / or VL sequences. As mentioned above, the sequence of a bispecific polypeptide may contain certain mutations.

[0162] In one embodiment, the binding domain B1 is specific to CD40, typically human CD40, and independently: (a) A heavy chain CDR1 sequence consisting of the sequence "G, F, T, F, S, S, Y, A", (b) A heavy chain CDR2 sequence having a length of 8 amino acids and containing the consensus sequence: "I, G / S, S / G, Y / S, G / S, G / S, G / Y / S, T", (c) A heavy chain CDR3 sequence having a length of 9 to 12 amino acids and containing the consensus sequence "A, R, Y / R / G, Y / P / V / -, N / S / V, F / Y / W, G / H / S, - / S, - / V, M / F, D, Y", (d) Sequence: Light chain CDR1 sequence consisting of "Q, S, I, S, S, Y", (e) Sequence: Light chain CDR2 sequence consisting of "A, A, S", (f) A light chain CDR3 sequence having a length of 9 amino acids and containing the consensus sequence: "Q, Q, Y / S, G / Y, R / S / V, N / A / Y / T, P, P / F / Y, T" It may contain one, two, three, four, five, or all six of the selected features.

[0163] The use of commas in the context of considering amino acid sequences illustrates lists of amino acids that may include further nomenclature such as slashes. For example, "G, F, T, F, S, S, Y, A" indicates that the amino acid sequence is GFTFSSYA, and "A, R, Y / R / G" indicates that the amino acid sequence could be ARY, ARR, or ARG. The use of hyphens in the context of considering amino acid sequences illustrates that there may be no amino acid present at each of its positions. For example, "- / V, M / F, D" indicates that the amino acid sequence could be VMD, VFD, MD, or FD.

[0164] The binding domain B1 may contain at least the heavy chain CDR3 defined in (c) and / or the light chain CDR3 defined in (f). The binding domain B1 may contain all three heavy chain CDR sequences of (a), (b), and (c), and / or all three light chain CDR sequences of (d), (e), and (f).

[0165] Table A shows examples of complete heavy and light chain variable region amino acid sequences in binding domain B1. Exemplary nucleic acid sequences encoding each amino acid sequence are also shown. The numbering of the VH and VL regions in Table A corresponds to the numbering system used in Tables C(1) and C(2). For example, the amino acid sequence "1132, light chain VL (also known as 1133)" is an example of a complete VL region sequence containing all three CDRs of VL number 1132 (1133) shown in Table C(2), and the amino acid sequence "1132, heavy chain VH" is an example of a complete VH region sequence containing all three CDRs of VH number 1132 shown in Table C(1).

[0166] In an exemplary embodiment, the binding domain B1 is (a) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1132 / 1133 (SEQ ID NOs. 73, 74, and 75, and / or SEQ ID NOs. 90, 91, and 92), (b) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1150 / 1151 (SEQ ID NOs. 73, 76, and 77, and / or SEQ ID NOs. 90, 91, and 93), (c) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1140 / 1135 (SEQ ID NOs. 73, 78, and 79, and / or SEQ ID NOs. 90, 91, and 94), (d) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1107 / 1108 (SEQ ID NOs. 73, 78, and 80, and / or SEQ ID NOs. 90, 91, and 95), (e) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody G12 or G12_mut or ffAC_05337 (SEQ ID NOs. 81, 82, and 83, and / or SEQ ID NOs. 96, 97, and 98), (f) Three CDRs of the heavy chain of antibody APX005, and / or three CDRs of the light chain (SEQ ID NOs. 84, 85, and 86, and / or SEQ ID NOs. 99, 100, and 101), or (g) The antibody 21.4.1 contains three CDRs of the heavy chain and / or three CDRs of the light chain (SEQ ID NOs. 87, 88, and 89, and / or SEQ ID NOs. 102, 103, and 104).

[0167] Therefore, the binding domain B1 is, (a) Heavy chain variable region and / or light chain variable region of antibody 1132 / 1133 (SEQ ID NO: 3 and / or SEQ ID NO: 1), (b) Heavy chain variable region and / or light chain variable region of antibody 1150 / 1151 (SEQ ID NO: 7 and / or SEQ ID NO: 5), (c) Heavy chain variable region and / or light chain variable region of antibody 1140 / 1135 (SEQ ID NO: 11 and / or SEQ ID NO: 9), (d) Heavy chain variable region and / or light chain variable region of antibody 1107 / 1108 (SEQ ID NO: 15 and / or SEQ ID NO: 13), (e) Heavy chain variable region and / or light chain variable region of antibody G12 (SEQ ID NO: 19 and / or SEQ ID NO: 17), (f) Heavy chain variable region and / or light chain variable region of antibody APX005 (SEQ ID NO: 23 and / or SEQ ID NO: 21), (g) Heavy chain variable region and / or light chain variable region of antibody 21.4.1 (SEQ ID NO: 27 and / or SEQ ID NO: 25) (h) The heavy chain variable region and / or light chain variable region of antibody G12_mut (SEQ ID NO: 29 and / or SEQ ID NO: 17), or (h) May include the heavy chain variable region and / or light chain variable region of antibody ffAC_05337 (SEQ ID NO: 431 and / or SEQ ID NO: 430).

[0168] In an exemplary embodiment, the binding domain B1 is The heavy and light antibody chains comprising three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1132 / 1133 (SEQ ID NOs. 73, 74, and 75, and / or SEQ ID NOs. 90, 91, and 92), or exemplary heavy and light chain variable regions (SEQ ID NOs. 3 and SEQ ID NOs. 1), or the CDRs described above.

[0169] In further exemplary embodiments, the binding domain B1 is The heavy and / or light antibody chains comprising three CDRs of the heavy chain and / or three CDRs of the light chain of antibody G12 (SEQ ID NOs: 81, 82, and 83, and / or SEQ ID NOs: 96, 97, and 98), or exemplary heavy and light chain variable regions (SEQ ID NOs: 19 and 17), or the CDRs described above.

[0170] In further exemplary embodiments, the binding domain B1 is The antibody G12_mut comprises three CDRs of the heavy chain and / or three CDRs of the light chain (SEQ ID NOs. 81, 82, and 83, and / or SEQ ID NOs. 96, 97, and 98), or exemplary heavy and light chain variable regions (SEQ ID NOs. 29 and SEQ ID NOs. 17), or heavy and light antibody chains containing the said CDRs as detailed above.

[0171] In a further preferred exemplary embodiment, the binding domain B1 is The antibody ffAC_05337 comprises three CDRs of the heavy chain and / or three CDRs of the light chain (SEQ ID NOs. 81, 82, and 83, and / or SEQ ID NOs. 96, 97, and 98), or exemplary heavy and light chain variable regions (SEQ ID NOs. 431 and 430), or heavy and light antibody chains containing the said CDRs as detailed above.

[0172] The numbering of antibodies (e.g., antibody X / Y) defines the heavy chain variable region (X) and the light chain variable region (Y), respectively (or, if a single number is given, only the heavy chain variable region [X] is defined).

[0173] Those skilled in the art will understand that the bispecific polypeptide may alternatively include variants of the variable region defined above (or variants of the CDR sequence of the B1 and / or B2 binding domain).

[0174] Any variant of any heavy-chain or light-chain amino acid sequence or CDR sequence listed herein may be a substitution, deletion, or addition variant of the sequence. A variant may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the sequence. A “deletion” variant may include the deletion of an individual amino acid, a small group of amino acids, e.g., the deletion of 2, 3, 4, or 5 amino acids, or the deletion of a larger amino acid region, e.g., the deletion of a specific amino acid domain or other feature. A “substitution” variant preferably involves conservative amino acid substitutions, replacing one or more amino acids with the same number of amino acids. For example, an amino acid may be substituted with an alternative amino acid having similar properties, e.g., another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the 20 main amino acids that can be used to select suitable substituents are as follows: [Table 1]

[0175] In this specification, amino acids may be referred to by their full name, a three-letter code, or a one-letter code.

[0176] Preferred "derivatives" or "variants" include those in which the amino acids appearing in the sequence are structural analogs of naturally occurring amino acids. The amino acids used in the sequence may also be derivatized or modified, for example, labeled, as long as the function of the polypeptide is not significantly adversely affected.

[0177] The derivatives and variants described above can be prepared during antibody synthesis, by modification after production, or, if the polypeptide is in recombinant form, by known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0178] Preferably, the variant has an amino acid sequence having more than 60%, or more than 70%, for example 75% or 80%, preferably more than 85%, for example 90% or 95%, amino acid identity with respect to the sequence shown in the sequence disclosed herein. This level of amino acid identity may be observed over the entire length of the sequence of the relevant SEQ ID NO: or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0179] In relation to amino acid sequences, "sequence identity" refers to sequences having the values ​​described when evaluated using ClustalW (Thompson et al., 1994, the disclosure of which is incorporated herein by reference) and the following parameters. Pairwise alignment parameters - Method: Accurate, Matrix: PAM, Gap Open Penalty: 10.00, Gap Extension Penalty: 0.10.

[0180] Multiple alignment parameters - Matrix: PAM, Gap Open Penalty: 10.00, Identity % for Delay: 30, End Gap Penalty: On, Gap Separation Distance: 0, Negative Matrix: None, Gap Extension Penalty: 0.20, Residue-Specific Gap Penalty: On, Hydrophilic Gap Penalty: On, Hydrophilic Residue: GPSNDQEKR. Sequence identity at specific residues is intended to include only identical residues that have been derivatized.

[0181] In one embodiment, the binding domain B1 includes the light chain of antibody 1132 / 1133 (SEQ ID NO: 372 or 379) and / or the heavy chain of antibody 1132 / 1133 (SEQ ID NO: 371 or 378).

[0182] In one embodiment, the binding domain B1 includes the light chain of antibody G12 (SEQ ID NO: 381) and / or the heavy chain of antibody G12 (SEQ ID NO: 380).

[0183] In one embodiment, the binding domain B1 includes the light chain of antibody G12_mut (SEQ ID NO: 383) and / or the heavy chain of antibody G12_mut (SEQ ID NO: 382).

[0184] Those skilled in the art will understand, and are included herein, that the mutations described herein for the RUBY® format and / or optimized RUBY® format may be applied to the above light and / or heavy chain sequences of G12 and / or G12_mut.

[0185] Accordingly, in one embodiment, the binding domain B1 may include one or more variants of the light chain variable region and / or the heavy chain variable region (and / or the light chain and / or the heavy chain) as defined above, having at least 90% sequence identity thereto, or 95% sequence identity thereto, or 99% sequence identity thereto. The binding domain B1 may also include variants of the CDR sequence specified herein, for example, variants in which up to 1, 2, 3, 4, or 5 amino acid residues are substituted, deleted, or added compared to the specified reference sequence.

[0186] For reference, the antibody reference used in this application, possible alternative names for the same antibody / binding domain, and the target of the antibody / binding domain are expanded in Table i below. [Table 2]

[0187] The "G12_mut" antibody largely corresponds to the "G12" sequence, but G12_mut contains three mutations in the VH framework. The CDR and VL sequences of G12_mut are the same as those of G12.

[0188] Carcinoembryonic antigen (CEA) binders The bispecific polypeptide further contains a binding domain (B2) that can specifically bind to carcinoembryonic antigen (CEA).

[0189] The binding domain B2 specifically binds to CEA, meaning it binds to CEA but not to other molecules, or binds to them with lower affinity. The term CEA, as used herein, typically refers to human CEA. The binding domain B2 may have some binding affinity to CEA from other mammals, such as CEA from non-human primates (e.g., Macaca fascicularis (cynomolgus monkey), Macaca mulatta). Preferably, the binding domain B2 does not bind to non-target molecules such as CTLA-4-Fc and / or human ubiquitin.

[0190] In one embodiment, the CEA is a tumor-associated CEA. "Tumor-associated CEA" includes members of the CEA family whose presence and / or overexpression correlate with the presence of cancer and / or tumors, e.g., CEAs known or suspected to be overexpressed by cancer and / or tumor cells. Members of the tumor and / or cancer-associated CEA family are known to those skilled in the art, e.g., CEACAM1, CEACAM6, CEACAM7, and / or CEACAM5.

[0191] In one embodiment, CEA is a carcinoembryonic antigen-associated cell adhesion molecule (CEACAM).

[0192] In one embodiment, CEACAM is CEACAM1 (GenBank:NG_029051.2, etc.), CEACAM3 (GenBank:D90278.1, etc.), CEACAM4 (GenBank:D90276.1, etc.), CEACAM5 (GenBank:M17303.1, etc.), CEACAM6 (GenBank:M29541.1, etc.), CEACAM7 (GenBank:L31792.1, etc.), CEACAM8 (G It is one or more selected from the enumeration consisting of enBank:X52378.1, CEACAM16 (GenBank:EU021223.1, etc.), CEACAM18 (GenBank:AC020914.9, etc.), CEACAM19 (GenBank:BC083499.1, etc.), CEACAM20 (GenBank:AY358129.1, etc.), and CEACAM21 (GenBank:BC106727.1, etc.). The aforementioned references to CEACAM molecules should be understood to include splice variants.

[0193] Preferably, CEACAM is one or more selected from the enumeration consisting of CEACAM1, CEACAM5, and CEACAM6. Preferably, CEACAM is CEACAM1. Most preferably, CEACAM is CEACAM5.

[0194] In a preferred embodiment, B2 can specifically bind to CEACAM5 but cannot bind to other CEACAMs, particularly CEACAM1.

[0195] In one embodiment, B2 can specifically bind to CEA in target cells. Preferably, the target cells are cancer cells and / or tumor cells.

[0196] Preferably, the CEA levels in the target cells are intermediate or high.

[0197] In one embodiment, intermediate levels of CEA expression are approximately 10,000 or more CEA receptors per target cell, for example, approximately 11,000 or more, approximately 12,000 or more, approximately 13,000 or more, approximately 14,000 or more, approximately 15,000 or more, approximately 16,000 or more, approximately 17,000 or more, approximately 18,000 or more, approximately 19,000 or more, approximately 20,000 or more, approximately 2 The target cells are characterized by expressing 5,000 or more, approximately 30,000 or more, approximately 35,000 or more, approximately 40,000 or more, approximately 50,000 or more, approximately 60,000 or more, approximately 70,000 or more, approximately 80,000 or more, approximately 90,000 or more, approximately 100,000 or more, approximately 125,000 or more, approximately 150,000 or more, or approximately 175,000 or more CEA receptors. In another embodiment, intermediate levels of CEA expression are characterized by target cells expressing approximately 10,000 to 200,000 CEA receptors per target cell, for example, approximately 20,000 to 175,000 CEA receptors per target cell, or approximately 20,000 to 200,000 CEA receptors per target cell, or approximately 50,000 to 175,000 CEA receptors per target cell, or approximately 50,000 to 200,000 CEA receptors per target cell. Preferably, the CEA receptor is the CEACAM5 receptor.

[0198] In one embodiment, high levels of CEA expression are defined as approximately 200,000 or more CEA receptors per target cell, for example, approximately 225,000 or more, approximately 250,000 or more, approximately 275,000 or more, approximately 300,000 or more, approximately 325,000 or more, approximately 350,000 or more, approximately 375,000 or more, approximately 400,000 or more, approximately 425,0 The target cells are characterized by expressing 00 or more, approximately 450,000 or more, approximately 475,000 or more, approximately 500,000 or more, approximately 600,000 or more, approximately 700,000 or more, approximately 800,000 or more, approximately 900,000 or more, or approximately 1,000,000 CEA receptors, preferably more than approximately 300,000 CEA receptors per target cell. In another embodiment, high levels of CEA expression are characterized by target cells expressing approximately 200,000 to approximately 1,000,000 CEA receptors per target cell, for example, approximately 200,000 to approximately 500,000 CEA receptors per target cell, or approximately 300,000 to approximately 500,000 CEA receptors per target cell. Preferably, the CEA receptor is the CEACAM5 receptor.

[0199] In one embodiment, B2 cannot specifically bind to cells that do not express CEA or have low levels of CEA expression. In one embodiment, low levels of CEA expression are characterized by cells expressing approximately 10,000 or fewer CEA receptors per cell, for example, approximately 9,000 or fewer, approximately 8,000 or fewer, approximately 7,000 or fewer, approximately 6,000 or fewer, approximately 5,000 or fewer, approximately 4,000 or fewer, approximately 3,000 or fewer, approximately 2,000 or fewer, or approximately 1,000 or fewer CEA receptors per cell.

[0200] Advantageously, the binding domain B2 is 2 × 10 -6 Less than M, or 1.5 × 10 -8 Less than M, or 2.5 × 10 -9 Less than M, or 2 × 10 -9 Less than M, or 1.5 × 10 -12 Less than M, or 1 × 10 -12 Less than M, preferably 1.5 × 10 -8 Less than M, or 2.5 × 10-9 Less than M, or 1.5 × 10 -12 Less than M K D It binds to human CEA. Preferably, K D This is measured using Octet and is described, for example, in the examples.

[0201] For example, the binding domain B2 preferably does not bind to non-target molecules such as CTLA-4-Fc and / or human ubiquitin. In certain embodiments relating to a particular CEACAM, the non-target molecule may be a different CEACAM; for example, for CEACAM5, the non-target molecule may be a CEACAM, and vice versa. Therefore, typically, the K in the binding domain for human CEA D This is K against CTLA-4-Fc and / or human ubiquitin or any other unrelated material or accompanying material in the environment, or other non-target molecules. D It will be twice, preferably five times, and more preferably less than ten times. More preferably, K D This would be less than 50 times, more preferably less than 100 times, and even more preferably less than 200 times.

[0202] The binding domain B2 can preferably bind to its target with an affinity at least 2, 10, 50, or 100 times greater than its affinity to bind to another non-target molecule.

[0203] Therefore, in summary, the binding domain B2 preferably has the following functional characteristics: a) 2 × 10 -6 Less than M, more preferably 2.5 × 10 -9 Less than M, or 1.5 × 10 -12 Less than M, more comfortable 1.5 × 10 -12 K is less than M D It binds to human CEA by value. b) Does not bind to CTLA-4-Fc and / or non-target molecules such as human ubiquitin, exhibiting at least one of these characteristics.

[0204] In one embodiment, the binding domain B2 preferentially binds to CEA on cells rather than soluble CEA. "Preferentially binding to CEA on cells rather than soluble CEA" means that, in the presence of CEA on cells (such as on the cell surface) and soluble CEA, B2 is more likely to bind to CEA on cells than to soluble CEA.

[0205] In one embodiment, the binding domain B2 includes one or more light chain CDR sequences selected from Table D(2), and / or one or more heavy chain CDR sequences selected from Table D(1a) and / or Table D(1b). Therefore, the binding domain B2 is (a) CEA heavy chain CDRs, SEQ ID NOs: 216-310, 335, and / or (b) May contain one or more CDR sequences selected from the group consisting of CEA light chain CDRs, SEQ ID NOs: 90, 91, 94, 311-334.

[0206] In one embodiment, the binding domain B2 includes one, two, or three light chain CDR sequences from a specific row for an individual antibody reference in Table D(2), and / or one, two, or three heavy chain CDR sequences from the corresponding row for an antibody having the same reference in Table D(1a) and / or Table D(1b). For example, the binding domain B2 includes one or more light chain CDR sequences of AC_05059 (sequence numbers 90, 91, and 311) and one or more heavy chain CDR sequences of AC_05059 (sequence numbers 216, 217, and 218, or 280, 281, and 218), or one or more light chain CDR sequences of AC_05060 (sequence numbers 312, 91, and 313) and heavy chain CDR sequences of AC_05060 (sequence numbers 219, 220, and 221, or 282, One or more of 283 and 221), or one or more of the light chain CDR sequences of AC_05061 (sequence numbers 90, 91 and 314) and one or more of the heavy chain CDR sequences of AC_05061 (sequence numbers 222, 223 and 224, or 284, 285 and 224), or one or more of the light chain CDR sequences of AC_05062 (sequence numbers 315, 316 and 94) and the heavy chain CDR sequences of AC_05062 (sequence numbers 222, 223 and 225, One or more of AC_05064 (284, 285, and 225), or one or more of the light chain CDR sequences of AC_05064 (sequence numbers 90, 91, and 317) and one or more of the heavy chain CDR sequences of AC_05064 (sequence numbers 222, 223, and 226, or 284, 285, and 226), or one or more of the light chain CDR sequences of AC_05079 (sequence numbers 90, 91, and 311) and the heavy chain CDR sequences of AC_05079 (sequence numbers 216, 217, and One or more of 227, or 280, 281, and 227) or one or more of the light chain CDR sequences of AC_05081 (sequence numbers 90, 91, and 311) and one or more of the heavy chain CDR sequences of AC_05081 (sequence numbers 216, 217, and 229, or 280, 281, and 229) or one or more of the light chain CDR sequences of AC_05088 (sequence numbers 90, 91, and 311) and the heavy chain CDR sequence of AC_05088 (sequence number 216,One or more of the following (217, and 237, or 280, 281, and 237), or one or more of the light chain CDR sequences of AC_05089 (sequence numbers 90, 91, and 311) and one or more of the heavy chain CDR sequences of AC_05089 (sequence numbers 216, 217, and 238, or 280, 281, and 238), or one or more of the light chain CDR sequences of AC_05090 (sequence numbers 90, 91, and 311) and heavy chain CDR sequences of AC_05090 or ffAC_05337 (sequence numbers 216, 217, and 239, and (280, 281, and 239) or one or more of the light chain CDR sequences of AC_05091 (sequence numbers 90, 91, and 311) and one or more of the heavy chain CDR sequences of AC_05091 (sequence numbers 216, 217, and 240, or 280, 281, and 240), or one or more of the light chain CDRs of AC_05093 (sequence numbers 90, 91, and 311) and one or more of the heavy chain CDR sequences of AC_05093 (sequence numbers 216, 217, and 241, or 280, 281, and 241), or A One or more light chain CDR sequences of C_05094 (sequence numbers 90, 91, and 311) and one or more heavy chain CDR sequences of AC_05094 (sequence numbers 216, 217, and 242, or 280, 281, and 242), or one or more light chain CDR sequences of AC_05096 (sequence numbers 90, 91, and 311) and one or more heavy chain CDR sequences of AC_05096 (sequence numbers 216, 217, and 244, or 280, 281, and 244), and light chain CDR sequences of AC_05097 (sequence numbers 90, 91, and 311). 11) One or more of the above and one or more of the heavy chain CDR sequences of AC_05097 (sequence numbers 216, 217, and 245, or 280, 281, and 245), or one or more of the light chain CDR sequences of Fab1 (sequence numbers 90, 91, and 322) and one or more of the heavy chain CDR sequences of Fab1 (sequence numbers 248, 249, and 250, or 289, 290, and 250), or one or more of the light chain CDR sequences of Fab3 (sequence numbers 324, 325, and 326) and the heavy chain CDR sequences of Fab3 (sequence numbers 254, 255,and 256, or 293, 294, and 256) and may include one or more of them. Most preferably, B2 includes the CDRs of AC_05088, AC_05090 / ffAC_05337, AC_05093, AC_05097, Fab1, and / or Fab3, and / or VL and VH.,

[0207] In a preferred embodiment, B2 includes the CDR of ffAC_05337, and / or VL and VH, that is, the three heavy-chain CDRs of SEQ ID NOs: 216, 217, and 239, and the three light-chain CDRs of SEQ ID NOs: 90, 91, and 311, and / or the VL sequence of 430 and the VH sequence of SEQ ID NO: 431.

[0208] As further explained in the examples, the reference to an exemplary B2 polypeptide (such as "Fab1") is a nomenclature based on a library in which a specific binder has been identified, and is not a specific reference to a particular type or fragment of an antibody. In other words, "Fab1" is not necessarily a Fab fragment. Therefore, the CDR, VL, and VH amino acid sequences defined for each of the exemplary B2 polypeptides can be used in any compatible antibody format or fragment thereof.

[0209] Preferred CEA-binding domains may include at least heavy-chain CDR3 defined in any individual row of Table D(1a), and / or light-chain CDR3 defined in any individual row of Table D(2).

[0210] Thus, in one embodiment, the binding domain B2 includes all six CDR sequences in a given antibody (VH / VL) reference. For example, the binding domain B2 may include AC_05059, AC_05060, AC_05061, AC_05062, AC_05064, AC_05079, AC_05080, AC_05081, AC_05082, AC_05083, AC_05084, AC_05085, AC_05086, AC_05087, AC_05088, AC_05089, AC_05090, AC_05091, AC_05092, AC_05093, AC_05094, AC_05095, AC_05096, AC_05097, AC_05098, AC_05099, AC_05100, Fab^{1}, Fab^{2}, Fab^{3}, Fab^{4}, Fab^{5}, Fab^{6}, Fab^{7}, Fab^{8}, Fab^{9}, Fab^{10}, Fab^{11}, ffAC_05337, and mAb^{2}, preferably, all six CDR sequences of an antibody selected from the list consisting of AC_05059, AC_05060, AC_05061, AC_05062, AC_05064, AC_05079, AC_05081, AC_05088, AC_05089, AC_05090, AC_05091, AC_05093, AC_05094, AC_05096, AC_05097, Fab^{1}, ffAC_05337, and Fab^{3}, most preferably, AC_05088, AC_05090, the CEA binding domain of ffAC_05337, AC_05093, AC_05097, Fab^{1}, and Fab^{3}.

[0211] In one embodiment, binding domain B2 includes the VH and / or VL amino acid sequences shown in Table B. In one embodiment, binding domain B2 includes the VH and VL amino acid sequences shown in Table B in a specific antibody reference. For example, binding domain B2 may include the VH sequence of AC_05059 (SEQ ID NO: 33) and / or the VL sequence of AC_05059 (SEQ ID NO: 31), or binding domain B2 may include the VH sequence of AC_05060 (SEQ ID NO: 37) and / or the VL sequence of AC_05060 (SEQ ID NO: 35), or binding domain B2 may include the VH sequence of AC_05062 (SEQ ID NO: 45) and / or the VL sequence of AC_05062 (SEQ ID NO: 43), or binding Domain B2 may contain the VH sequence (sequence number 49) of AC_05064 and / or the VL sequence (sequence number 47) of AC_05064, or the binding domain B2 may contain the VH sequence (sequence number 53) of AC_05079 and / or the VL sequence (sequence number 51) of AC_05079, or the binding domain B2 may contain the VH sequence (sequence number 61) of AC_05081 and / or the VL sequence (sequence number 59) of AC_05081, or the binding domain B2 may contain , may contain the VH sequence (sequence number 122) of AC_05088 and / or the VL sequence (sequence number 120) of AC_05088, or the binding domain B2 may contain the VH sequence (sequence number 126) of AC_05089 and / or the VL sequence (sequence number 124) of AC_05089, or the binding domain B2 may contain the VH sequence (sequence number 130) of AC_05090 and / or the VL sequence (sequence number 128) of AC_05090, or the binding domain B2 is , may contain the VH sequence (sequence number 134) of AC_05091 and / or the VL sequence (sequence number 132) of AC_05091, or the binding domain B2 may contain the VH sequence (sequence number 142) of AC_05093 and / or the VL sequence (sequence number 140) of AC_05093, or the binding domain B2 may contain the VH sequence (sequence number 146) of AC_05094 and / or the VL sequence (sequence number 144) of AC_05094, or the binding domain B2 may contain,The binding domain B2 may contain the VH sequence (sequence number 154) of AC_05096 and / or the VL sequence (sequence number 152) of AC_05096, or the binding domain B2 may contain the VH sequence (sequence number 158) of AC_05097 and / or the VL sequence (sequence number 156) of AC_05097, or the binding domain B2 may contain the VH sequence (sequence number 174) of Fab1 and / or the VL sequence (sequence number 172) of Fab1, or the binding domain B2 may contain the VH sequence (sequence number 182) of Fab3 and / or the VL sequence (sequence number 180) of Fab3, or the binding domain B2 may contain the VH sequence (sequence number 433) of ffAC_05337 and / or the VL sequence (sequence number 432) of ffAC_05337.

[0212] In one embodiment, the CEA binding domains of B2 are AC_05059, AC_05060, AC_05061, AC_05062, AC_05064, AC_05079, AC_05080, AC_05081, AC_05082, AC_05083, AC_05084, AC_05085, AC_05086, AC_05087, AC_0 5088, AC_05089, AC_05090, AC_05091, AC_05092, AC_05093, AC_05094, AC_05095, AC_05096 , AC_05097, AC_05098, AC_05099, AC_05100, Fab1, Fab2, Fab3, Fab4, Fab5, Fab6, Fab7, Fab8, Fab9, Fab10, Fab11, the CEA binding domain of ffAC_05337, and mAb2, preferably selected from AC_05059, AC_05060, AC_05061, AC_05062, AC_05064, AC_05079, AC_05081, AC_05088, AC_05089, AC_05090, AC_05091, AC_05093, AC_05094, AC_05096, AC_05097, Fab1, the CEA binding domain of ffAC_05337, and Fab3, most preferably selected from AC_05088, AC_05090, the CEA binding domain of ffAC_05337, AC_05093, AC_05097, Fab1, and Fab3.

[0213] Therefore, the CDR or VH and VL sequences of binding domain B2 are, (a) AC_05059 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 218, or 280, 281, and 218; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 31; VH: SEQ ID NO. 33) (b) AC_05060 (Heavy chain CDRs: SEQ ID NOs. 219, 220, and 221, or 282, 283, and 221; Light chain CDRs: SEQ ID NOs. 312, 91, and 313; VL: SEQ ID NO. 35; VH: SEQ ID NO. 37) (c)AC_05061 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 224, or 284, 285, and 224; Light chain CDR: SEQ ID NOs. 90, 91, and 314; VL: SEQ ID NO. 39; VH: SEQ ID NO. 41) (d)AC_05062 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 225, or 284, 285, and 225; Light chain CDR: SEQ ID NOs. 315, 316, and 94; VL: SEQ ID NO. 43; VH: SEQ ID NO. 45) (e)AC_05064 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 226, or 284, 285, and 226; Light chain CDR: SEQ ID NOs. 90, 91, and 317; VL: SEQ ID NO. 47; VH: SEQ ID NO. 49) (f)AC_05079 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 227, or 280, 281, and 227; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 51; VH: SEQ ID NO. 53) (g)AC_05080 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 228, or 280, 281, and 228; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NOs. 55; VH: SEQ ID NOs. 57) (h)AC_05081 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 229, or 280, 281, and 229; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 59; VH: SEQ ID NO. 61) (i) AC_05082 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 230, or 284, 285, and 230; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 63; VH: SEQ ID NO. 65) (j)AC_05083 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 231, or 284, 285, and 231; Light chain CDR: SEQ ID NOs. 318, 91, and 319; VL: SEQ ID NO. 67; VH: SEQ ID NO. 69) (k)AC_05084 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 232, or 284, 285, and 232; Light chain CDR: SEQ ID NOs. 90, 91, and 320; VL: SEQ ID NOs. 71; VH: SEQ ID NOs. 106) (l)AC_05085 (Heavy chain CDR: SEQ ID NOs. 219, 233, and 234, or 286, 287, and 234; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 108; VH: SEQ ID NO. 110) (m)AC_05086 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 235, or 280, 281, and 235; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 112; VH: SEQ ID NO. 114) (n)AC_05087 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 236, or 280, 281, and 236; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 116; VH: SEQ ID NO. 118) (o)AC_05088 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 237, or 280, 281, and 237; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 120; VH: SEQ ID NO. 122) (p)AC_05089 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 238, or 280, 281, and 238; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 124; VH: SEQ ID NO. 126) (q)AC_05090 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 239, or 280, 281, and 239; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 128; VH: SEQ ID NO. 130) (r)AC_05091 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 240, or 280, 281, and 240; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 132; VH: SEQ ID NO. 134) (s)AC_05092 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 218, or 280, 281, and 218; Light chain CDR: SEQ ID NOs. 321, 91, and 311; VL: SEQ ID NOs. 136; VH: SEQ ID NOs. 138) (t)AC_05093 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 241, or 280, 281, and 241; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 140; VH: SEQ ID NO. 142) (u)AC_05094 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 242, or 280, 281, and 242; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 144; VH: SEQ ID NO. 146) (v)AC_05095 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 243, or 280, 281, and 243; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 148; VH: SEQ ID NO. 150) (w)AC_05096 (Heavy chain CDR: SEQ ID NOs. 216, 217, and 244, or 280, 281, and 244; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 152; VH: SEQ ID NO. 154) (x)AC_05097 (Heavy chain CDR: SEQ ID NOs. 217, 216, and 245, or 280, 281, and 245; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 156; VH: SEQ ID NO. 158) (y)AC_05098 (Heavy chain CDR: SEQ ID NOs. 219, 220, and 246, or 282, 283, and 246; Light chain CDR: SEQ ID NOs. 312, 91, and 313; VL: SEQ ID NO. 160; VH: SEQ ID NO. 162) (z)AC_05099 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 224, or 288, 285, and 224; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 164; VH: SEQ ID NO. 166) (aa)AC_05100 (Heavy chain CDR: SEQ ID NOs. 222, 223, and 247, or 288, 285, and 247; Light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NO. 168; VH: SEQ ID NO. 170) (ab)Fab1 (Heavy chain CDR: SEQ ID NOs: 248, 249, and 250, or 289, 290, and 250; Light chain CDR: SEQ ID NOs: 90, 91, and 322; VL: SEQ ID NO: 172; VH: SEQ ID NO: 174) (ac)Fab2 (heavy chain CDR: SEQ ID NOs. 251, 252, and 253, or 291, 292, and 253; light chain CDR: SEQ ID NOs. 90, 91, and 323; VL: SEQ ID NO. 176; VH: SEQ ID NO. 178) (ad)Fab3 (heavy chain CDR: SEQ ID NOs. 254, 255, and 256, or 293, 294, and 256; light chain CDR: SEQ ID NOs. 324, 325, and 326; VL: SEQ ID NOs. 180; VH: SEQ ID NOs. 182) (ae)Fab4 (heavy chain CDR: SEQ ID NOs. 257, 258, and 259, or 295, 296, and 259; light chain CDR: SEQ ID NOs. 90, 91, and 327; VL: SEQ ID NO. 184; VH: SEQ ID NO. 186) (af)Fab5 (Heavy chain CDR: SEQ ID NOs: 260, 261, and 262, or 297, 298, and 262; Light chain CDR: SEQ ID NOs: 324, 325, and 328; VL: SEQ ID NO: 188; VH: SEQ ID NO: 190) (ag)Fab6 (heavy chain CDR: SEQ ID NOs. 263, 264, and 265, or 299, 300, and 265; light chain CDR: SEQ ID NOs. 324, 325, and 329; VL: SEQ ID NO. 192; VH: SEQ ID NO. 194) (ah)Fab7 (heavy chain CDR: SEQ ID NOs. 266, 267, and 268, or 301, 302, and 268; light chain CDR: SEQ ID NOs. 90, 91, and 330; VL: SEQ ID NOs. 196; VH: SEQ ID NOs. 198) (ai)Fab8 (Heavy chain CDR: SEQ ID NOs. 269, 270, and 271, or 303, 304, and 271; Light chain CDR: SEQ ID NOs. 90, 91, and 331; VL: SEQ ID NOs. 200; VH: SEQ ID NOs. 202) (aj)Fab9 (heavy chain CDR: SEQ ID NOs: 272, 335, and 273, or 305, 306, and 273; light chain CDR: SEQ ID NOs: 90, 91, and 332; VL: SEQ ID NO: 204; VH: SEQ ID NO: 206) (ak)Fab10 (Heavy chain CDR: SEQ ID NOs. 274, 275, and 276, or 307, 308, and 276; Light chain CDR: SEQ ID NOs. 90, 91, and 333; VL: SEQ ID NOs. 208; VH: SEQ ID NOs. 210) (al)Fab11 (heavy chain CDR: SEQ ID NOs. 277, 278, and 279, or 309, 310, and 279; light chain CDR: SEQ ID NOs. 324, 325, and 334; VL: SEQ ID NOs. 212; VH: SEQ ID NOs. 214), and / or Antibodies can be selected from the group consisting of (am)ffAC_05337 (heavy chain CDR: SEQ ID NOs. 216, 217, and 239; light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NOs. 432; VH: SEQ ID NOs. 433), preferably (am)ffAC_05337 (heavy chain CDR: SEQ ID NOs. 216, 217, and 239; light chain CDR: SEQ ID NOs. 90, 91, and 311; VL: SEQ ID NOs. 432; VH: SEQ ID NOs. 433).

[0214] The numbering of an antibody (e.g., antibody X / Y) defines the heavy chain variable region (X) and the light chain variable region (Y), respectively (or, if a single number is indicated, only the heavy chain variable region [X] is defined). As described above, the sequence can be one or more CDR sequences, or a VH sequence and / or a VL sequence. As described above, the sequence of a bispecific polypeptide can contain certain mutations.

[0215] Any variant of the amino acid sequence or CDR sequence of a heavy chain or light chain listed herein can be a substitution, deletion, or addition variant of the sequence. The variant can include 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the sequence. A "deletion" variant can include the deletion of an individual amino acid, a small group of amino acids, e.g., 2, 3, 4, or 5 amino acids, or the deletion of a larger amino acid region, e.g., the deletion of a particular amino acid domain or other feature. A "substitution" variant preferably involves replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid can be replaced with an alternative amino acid having similar properties, e.g., another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of several of the 20 major amino acids that can be used to select suitable substituents are as follows.

Table 3

[0216] Amino acids herein may be referred to by their full name, three-letter code, or one-letter code.

[0217] Preferred "derivatives" or "variants" include those in which the amino acids appearing in the sequence are structural analogs of naturally occurring amino acids. The amino acids used in the sequence may also be derivatized or modified, for example, labeled, as long as the function of the polypeptide is not significantly adversely affected.

[0218] The derivatives and variants described above can be prepared during antibody synthesis, by modification after production, or, if the polypeptide is in recombinant form, by known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0219] Preferably, the variant has an amino acid sequence having more than 60%, or more than 70%, for example 75% or 80%, preferably more than 85%, for example 90% or 95%, amino acid identity with respect to the sequence shown in the sequence disclosed herein. This level of amino acid identity may be observed over the entire length of the sequence of the relevant SEQ ID NO: or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0220] In one embodiment, binding domain B2 is specific to CEA, typically human CEA, and independently: (a) Sequence: A heavy chain CDR1 sequence consisting of "G, F, T, F, S, S, S, Y" or containing the consensus sequence "G, F, T, F, G / S, S, Y, Y / A", (b) Sequence: A heavy chain CDR2 sequence consisting of "I, G, S, G, S, Y, S, T" or containing the consensus sequence "I, S, G, Y / S, G, Y / G, S, T", (c) A heavy chain CDR3 sequence containing the consensus sequence "A, R, Y, P, S, V, P / L, F, P, Q, S, P / H / L, H / P / L, L / F / V / W, D, Y" or the consensus sequence "A, R, H / Y, G, Y, G / S / T, V / H, L / F, D, Y", (d) Sequence: A light chain CDR1 sequence consisting of "Q, S, I, S, S, Y" or containing the consensus sequence "Q, S, I, R / S, S, Y", (e) Sequence: Light chain CDR2 sequence consisting of "A, A, S", (f) Sequence: A light chain CDR3 sequence consisting of "Q, Q, A, G, N, P, H, T" or containing any one, two, three, four, five, or all six features selected from the consensus sequence "Q, Q, G / Y, T / P / A, W / -, Y / -, F / V, P, F / Y, T".

[0221] In an alternative embodiment, the binding domain B2 is specific to CEA, typically human CEA, and is as follows: (a) Sequence: A heavy chain CDR1 sequence consisting of "G, F, T, F, S, S, S, Y", (b) Sequence: A heavy chain CDR2 sequence consisting of "I, G, S, G, S, Y, S, T", (c) Heavy chain CDR3 sequence containing the consensus sequence "A, R, Y, P, S, V, P / L, F, P, Q, S, P / H / L, H / P / L, L / F / V / W, D, Y", (d) Sequence: Light chain CDR1 sequence consisting of "Q, S, I, S, S, Y", (e) Sequence: Light chain CDR2 sequence consisting of "A, A, S", (f) Sequence: May contain any one, two, three, four, five, or all six features independently selected from the light chain CDR3 sequence consisting of "Q, Q, A, G, N, P, H, T". The binding domain B2 may contain at least the heavy chain CDR3 defined in (c) and / or the light chain CDR3 defined in (f) of this embodiment. The binding domain B2 may contain all three heavy chain CDR sequences of (a), (b), and (c), and / or all three light chain CDR sequences of (d), (e), and (f).

[0222] In further alternative embodiments, the binding domain B2 is specific to CEA, typically human CEA, and independently: (a) A heavy chain CDR1 sequence containing the consensus sequence "G, F, T, F, G / S, S, Y, Y / A", (b) Heavy chain CDR2 sequence containing the consensus sequence "I, S, G, Y / S, G, Y / G, S, T", (c) Heavy chain CDR3 sequence containing the consensus sequence "A, R, H / Y, G, Y, G / S / T, V / H, L / F, D, Y", (d) Light chain CDR1 sequence containing the consensus sequence "Q, S, I, R / S, S, Y", (e) Sequence: Light chain CDR2 sequence consisting of "A, A, S", (f) The light chain CDR3 sequence may contain any one, two, three, four, five, or all six features selected from the consensus sequence "Q, Q, G / Y, T / P / A, W / -, Y / -, F / V, P, F / Y, T".

[0223] The binding domain B2 may include at least the heavy chain CDR3 defined in (c) and / or the light chain CDR3 defined in (f) of this embodiment. The binding domain B2 may include all three heavy chain CDR sequences of (a), (b), and (c), and / or all three light chain CDR sequences of (d), (e), and (f).

[0224] Table B shows examples of complete heavy and light chain variable region amino acid sequences in binding domain B2. Exemplary nucleic acid sequences encoding each amino acid sequence are also shown. The numbering of the VH and VL regions in Table B corresponds to the numbering system used in Tables D(1a), D(1b), and / or D(2). For example, the amino acid sequence "AC_05088, light chain VL" is an example of a complete VL region sequence containing all three CDRs of VL number AC_05088 shown in Table D(2), and the amino acid sequence "AC_05088, heavy chain VH" is an example of a complete VH region sequence containing all three CDRs of VH number AC_05088 shown in Tables D(1a) and / or D(1b).

[0225] In an exemplary embodiment, the binding domain B2 is (a) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05059 (SEQ ID NOs. 216, 217, and 218, or 280, 281, and 218, and / or SEQ ID NOs. 90, 91, and 311) (b) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05060 (SEQ ID NOs. 219, 220, and 221, or 282, 283, and 221, and / or SEQ ID NOs. 312, 91, and 313) (c) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05061 (SEQ ID NOs. 222, 223, and 224, or 284, 285, and 224, and / or SEQ ID NOs. 90, 91, and 314) (d) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05062 (SEQ ID NOs. 222, 223, and 225, or 284, 285, and 225, and / or SEQ ID NOs. 315, 316, and 94) (e) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05064 (SEQ ID NOs. 222, 223, and 226, or 284, 285, and 226, and / or SEQ ID NOs. 90, 91, and 317) (f) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05079 (SEQ ID NOs. 216, 217, and 227, or 280, 281, and 227, and / or SEQ ID NOs. 90, 91, and 311) (g) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05080 (SEQ ID NOs. 216, 217, and 228, or 280, 281, and 228, and / or SEQ ID NOs. 90, 91, and 311) (h) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05081 (SEQ ID NOs. 216, 217, and 229, or 280, 281, and 229, and / or SEQ ID NOs. 90, 91, and 311) (i) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05082 (SEQ ID NOs. 222, 223, and 230, or 284, 285, and 230, and / or SEQ ID NOs. 90, 91, and 311) (j) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05083 (SEQ ID NOs. 222, 223, and 231, or 284, 285, and 231, and / or SEQ ID NOs. 318, 91, and 319) (k) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05084 (SEQ ID NOs. 222, 223, and 232, or 284, 285, and 232, and / or SEQ ID NOs. 90, 91, and 320) (l) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05085 (SEQ ID NOs. 219, 233, and 234, or 286, 287, and 234, and / or SEQ ID NOs. 90, 91, and 311) (m) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05086 (SEQ ID NOs. 216, 217, and 235, or 280, 281, and 235, and / or SEQ ID NOs. 90, 91, and 311) (n) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05087 (SEQ ID NOs. 216, 217, and 236, or 280, 281, and 236, and / or SEQ ID NOs. 90, 91, and 311) (o) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05088 (SEQ ID NOs. 216, 217, and 237, or 280, 281, and 237, and / or SEQ ID NOs. 90, 91, and 311) (p) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05089 (SEQ ID NOs. 216, 217, and 238, or 280, 281, and 238, and / or SEQ ID NOs. 90, 91, and 311) (q) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05090 / ffAC_05337 (SEQ ID NOs. 216, 217, and 239, or 280, 281, and 239, and / or SEQ ID NOs. 90, 91, and 311) (r) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05091 (SEQ ID NOs. 216, 217, and 240, or 280, 281, and 240, and / or SEQ ID NOs. 90, 91, and 311) (s) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05092 (SEQ ID NOs. 216, 217, and 218, or 280, 281, and 218, and / or SEQ ID NOs. 321, 91, and 311) (t) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05093 (SEQ ID NOs. 216, 217, and 241 or 280, 281, and 241, and / or SEQ ID NOs. 90, 91, and 311) (u) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05094 (SEQ ID NOs. 216, 217, and 242, or 280, 281, and 242, and / or SEQ ID NOs. 90, 91, and 311) (v) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05095 (SEQ ID NOs. 216, 217, and 243, or 280, 281, and 243, and / or SEQ ID NOs. 90, 91, and 311) (w) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05096 (SEQ ID NOs. 216, 217, and 244, or 280, 281, and 244, and / or SEQ ID NOs. 90, 91, and 311) (x) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05097 (SEQ ID NOs. 217, 216, and 245, or 280, 281, and 245, and / or SEQ ID NOs. 90, 91, and 311) (y) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05098 (SEQ ID NOs. 219, 220, and 246, or 282, 283, and 246, and / or SEQ ID NOs. 312, 91, and 313) (z) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05099 (SEQ ID NOs. 222, 223, and 224, or 288, 285, and 224, and / or SEQ ID NOs. 90, 91, and 311) (aa) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05100 (SEQ ID NOs. 222, 223, and 247, or 288, 285, and 247, and / or SEQ ID NOs. 90, 91, and 311) (ab) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab1 (SEQ ID NOs: 248, 249, and 250, or 289, 290, and 250, and / or SEQ ID NOs: 90, 91, and 322) (ac) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab2 (SEQ ID NOs. 251, 252, and 253, or 291, 292, and 253, and / or SEQ ID NOs. 90, 91, and 323) (ad) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab3 (SEQ ID NOs. 254, 255, and 256, or 293, 294, and 256, and / or SEQ ID NOs. 324, 325, and 326) (ae) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab4 (SEQ ID NOs. 257, 258, and 259, or 295, 296, and 259, and / or SEQ ID NOs. 90, 91, and 327) (af) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab5 (SEQ ID NOs: 260, 261, and 262, or 297, 298, and 262, and / or SEQ ID NOs: 324, 325, and 328) (ag) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab6 (SEQ ID NOs: 263, 264, and 265, or 299, 300, and 265, and / or SEQ ID NOs: 324, 325, and 329) (ah) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab7 (SEQ ID NOs. 266, 267, and 268, or 301, 302, and 268, and / or SEQ ID NOs. 90, 91, and 330) (ai) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab8 (SEQ ID NOs: 269, 270, and 271, or 303, 304, and 271, and / or SEQ ID NOs: 90, 91, and 331) (aj) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab9 (SEQ ID NOs: 272, 335, and 273, or 305, 306, and 273, and / or SEQ ID NOs: 90, 91, and 332) (ak) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab10 (SEQ ID NOs. 274, 275, and 276, or 307, 308, and 276, and / or SEQ ID NOs. 90, 91, and 333) and / or (al) Contains three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab11 (SEQ ID NOs: 277, 278, and 279, or 309, 310, and 279, and / or SEQ ID NOs: 324, 325, and 334).

[0226] Therefore, binding domain B2 is, (a) Heavy chain variable region and / or light chain variable region of antibody AC_05059 (SEQ ID NO: 33 and / or SEQ ID NO: 31) (b) Heavy chain variable region and / or light chain variable region of antibody AC_05060 (SEQ ID NO: 37 and / or SEQ ID NO: 35) (c) Heavy chain variable region and / or light chain variable region of antibody AC_05061 (SEQ ID NO: 41 and / or SEQ ID NO: 39) (d) Heavy chain variable region and / or light chain variable region of antibody AC_05062 (SEQ ID NO: 45 and / or SEQ ID NO: 43) (e) Heavy chain variable region and / or light chain variable region of antibody AC_05064 (SEQ ID NO: 49 and / or SEQ ID NO: 47) (f) Heavy chain variable region and / or light chain variable region of antibody AC_05079 (SEQ ID NO: 53 and / or SEQ ID NO: 51) (g) Heavy chain variable region and / or light chain variable region of antibody AC_05080 (SEQ ID NO: 57 and / or SEQ ID NO: 55) (h) Heavy chain variable region and / or light chain variable region of antibody AC_05081 (SEQ ID NO: 61 and / or SEQ ID NO: 59) (i) Heavy chain variable region and / or light chain variable region of antibody AC_05082 (SEQ ID NO. 65 and / or SEQ ID NO. 63) (j) Heavy chain variable region and / or light chain variable region of antibody AC_05083 (SEQ ID NO: 69 and / or SEQ ID NO: 67) (k) Heavy chain variable region and / or light chain variable region of antibody AC_05084 (SEQ ID NO: 106 and / or SEQ ID NO: 71) (l) Heavy chain variable region and / or light chain variable region of antibody AC_05085 (SEQ ID NO: 110 and / or SEQ ID NO: 108) (m) Heavy chain variable region and / or light chain variable region of antibody AC_05086 (SEQ ID NO: 114 and / or SEQ ID NO: 112) (n) Heavy chain variable region and / or light chain variable region of antibody AC_05087 (SEQ ID NO: 118 and / or SEQ ID NO: 116) (o) Heavy chain variable region and / or light chain variable region of antibody AC_05088 (SEQ ID NO: 122 and / or SEQ ID NO: 120) (p) Heavy chain variable region and / or light chain variable region of antibody AC_05089 (SEQ ID NO: 126 and / or SEQ ID NO: 124) (q) Heavy chain variable region and / or light chain variable region of antibody AC_05090 (SEQ ID NO: 130 and / or SEQ ID NO: 128) (r) Heavy chain variable region and / or light chain variable region of antibody AC_05091 (SEQ ID NO: 134 and / or SEQ ID NO: 132) (s) Heavy chain variable region and / or light chain variable region of antibody AC_05092 (SEQ ID NO: 138 and / or SEQ ID NO: 136) (t) Antibody AC_05093 heavy chain variable region and / or light chain variable region (SEQ ID NO: 142 and / or SEQ ID NO: 140) (u) Heavy chain variable region and / or light chain variable region of antibody AC_05094 (SEQ ID NO: 146 and / or SEQ ID NO: 144) (v) Heavy chain variable region and / or light chain variable region of antibody AC_05095 (SEQ ID NO: 150 and / or SEQ ID NO: 148) (w) Heavy chain variable region and / or light chain variable region of antibody AC_05096 (SEQ ID NO: 154 and / or SEQ ID NO: 152) (x) Heavy chain variable region and / or light chain variable region of antibody AC_05097 (SEQ ID NO: 158 and / or SEQ ID NO: 156) (y) Heavy chain variable region and / or light chain variable region of antibody AC_05098 (SEQ ID NO: 162 and / or SEQ ID NO: 160) (z) Heavy chain variable region and / or light chain variable region of antibody AC_05099 (SEQ ID NO: 166 and / or SEQ ID NO: 164) (aa) Heavy chain variable region and / or light chain variable region of antibody AC_05100 (SEQ ID NO: 170 and / or SEQ ID NO: 168) (ab) Heavy chain variable region and / or light chain variable region of antibody Fab1 (SEQ ID NO: 174 and / or SEQ ID NO: 172) (ac) Heavy chain variable region and / or light chain variable region of antibody Fab2 (SEQ ID NO: 178 and / or SEQ ID NO: 176) (ad) Heavy chain variable region and / or light chain variable region of antibody Fab3 (SEQ ID NO: 182 and / or SEQ ID NO: 180) (ae) Heavy chain variable region and / or light chain variable region of antibody Fab4 (SEQ ID NO: 186 and / or SEQ ID NO: 184) (af) Heavy chain variable region and / or light chain variable region of antibody Fab5 (SEQ ID NO: 190 and / or SEQ ID NO: 188) (ag) Heavy chain variable region and / or light chain variable region of antibody Fab6 (SEQ ID NO: 194 and / or SEQ ID NO: 192) (ah) Heavy chain variable region and / or light chain variable region of antibody Fab7 (SEQ ID NO: 198 and / or SEQ ID NO: 196) (ai) Heavy chain variable region and / or light chain variable region of antibody Fab8 (SEQ ID NO: 202 and / or SEQ ID NO: 200) (aj) Heavy chain variable region and / or light chain variable region of antibody Fab9 (SEQ ID NO: 206 and / or SEQ ID NO: 204) (ak) Heavy chain variable region and / or light chain variable region of antibody Fab10 (SEQ ID NO: 210 and / or SEQ ID NO: 208) (al) Heavy chain variable region and / or light chain variable region of antibody Fab11 (SEQ ID NO: 214 and / or SEQ ID NO: 212) (am) Heavy chain variable region and / or light chain variable region of antibody mAb2 (SEQ ID NO: 387 and / or SEQ ID NO: 385) and / or (an) May include the heavy chain variable region and / or light chain variable region of antibody ffAC_05337 (SEQ ID NO: 433 and / or SEQ ID NO: 432).

[0227] In one embodiment, the binding domain B2 is (a) Light and / or heavy chains of antibody AC_05059 (SEQ ID NO: 388 and / or SEQ ID NO: 389) (b) Light and / or heavy chain of antibody AC_05060 (SEQ ID NO: 390 and / or SEQ ID NO: 391) (c) Light and / or heavy chains of antibody AC_05061 (SEQ ID NO: 392 and / or (SEQ ID NO: 393) (d) Light and / or heavy chains of antibody AC_05062 (SEQ ID NO: 394 and / or SEQ ID NO: 395) (e) Light and / or heavy chains of antibody AC_05064 (SEQ ID NO: 396 and / or SEQ ID NO: 397) (f) Light and / or heavy chains of antibody AC_05079 (SEQ ID NO: 398 and / or SEQ ID NO: 399) (g) Light and / or heavy chains of antibody AC_05081 (SEQ ID NO: 400 and / or SEQ ID NO: 401) (h) Light and / or heavy chains of antibody AC_05088 (SEQ ID NO: 402 and / or SEQ ID NO: 403) (i) Light and / or heavy chain of antibody AC_05089 (SEQ ID NO: 404 and / or SEQ ID NO: 405) (j) Light and / or heavy chain of antibody AC_05090 (SEQ ID NO: 406 and / or SEQ ID NO: 407) (k) Light and / or heavy chain of antibody AC_05091 (SEQ ID NO: 408 and / or SEQ ID NO: 409) (l) Light and / or heavy chain of antibody AC_05093 (SEQ ID NO: 410 and / or SEQ ID NO: 411) (m) Light and / or heavy chain of antibody AC_05094 (SEQ ID NO: 412 and / or SEQ ID NO: 413) (n) Light and / or heavy chains of antibody AC_05096 (SEQ ID NO: 414 and / or SEQ ID NO: 415) (o) Light and / or heavy chain of antibody AC_05097 (SEQ ID NO: 416 and / or SEQ ID NO: 417) (p) Light chain and / or heavy chain of antibody Fab1 (SEQ ID NO: 418 and / or SEQ ID NO: 419) and / or (q) The light chain and / or heavy chain of antibody Fab3 may include (SEQ ID NO: 420 and / or (SEQ ID NO: 421)).

[0228] Those skilled in the art will understand, and are included herein, that the mutations described herein for the RUBY® format and / or optimized RUBY® format may be applied to the above light and / or heavy chain sequences of binding domain B2.

[0229] In one embodiment, the binding domain B2 may include one or more variants of the light chain variable region and / or the heavy chain variable region (and / or light chain and / or heavy chain) as defined above, having at least 90% sequence identity thereto, or 95% sequence identity thereto, or 99% sequence identity thereto. The binding domain B2 may also include variants of the CDR sequence specified herein, for example, variants in which up to 1, 2, 3, 4, or 5 amino acid residues are substituted, deleted, or added compared to the specified reference sequence.

[0230] In one embodiment, the CEA binding domains of B2 are AC_05059, AC_05060, AC_05061, AC_05062, AC_05064, AC_05079, AC_05080, AC_05081, AC_05082, AC_05083, AC_05084, AC_05085, AC_05086, AC_05087, A The CEA binding domains of C_05088, AC_05089, AC_05090, AC_05091, AC_05092, AC_05093, AC_05094, AC_05095, AC_05096, AC_05097, AC_05098, AC_05099, ffAC_05337, and AC_05100, Fab1, Fab2, Selected from Fab3, Fab4, Fab5, Fab6, Fab7, Fab8, Fab9, Fab10, Fab11, and mAb2, preferably AC_05059, AC_05060, AC_05061, AC_05062, AC_05064, AC_05079, AC_05081, AC_05088, AC_05089, AC_05090, AC_05091, AC_05093, AC_05094, AC_05096, and AC_05097, Fab1, and Fab3, most preferably AC_05088, AC_05090, AC_05093, and AC_05097, the CEA binding domain of Fab1, ffAC_05337, and Fab3. The CDR, VL, VH, light chain, and / or heavy chain of the antibody described in the first aspect of the present invention are related to and included in the second aspect of the present invention.

[0231] Advantageously, the binding domain B2 is 2 × 10 -6 Less than M, or 1.5 × 10 -7 Less than M, or 1.5 × 10 -8 Less than M, or 2.5 × 10 -8 Less than M, or 4.5 × 10 -8 Less than M, or 5.5 × 10 -8 Less than M, or 6.5 × 10 -9 Less than M, or 2.5 × 10 -9 Less than M, or 2 × 10 -9 Less than M, or 9.5 × 10 -10 Less than M, or 4.5 × 10 -10 Less than M, or 7.5 × 10 -11 Less than M, or 8.5 × 10 -12 Less than M, or 1.5 × 10 -12 Less than M, or 1 × 10 -12 Less than M, preferably 1.5 × 10 -8 Less than M, or 2.5 × 10 -9 Less than M, or 1.5 × 10 -12 Less than M K D It binds to human CEA. Preferably, K D This is measured with Octet and is described, for example, in the examples. Preferably, K D This is measured using Octet and is described, for example, in the examples.

[0232] In preferred embodiments, the bispecific polypeptide is a bispecific antibody, for example, a bispecific antibody in RUBY® format or optimized RUBY® format (both described herein).

[0233] Exemplary CD40-CEA bispecific antibody In one embodiment of a bispecific polypeptide, binding domain B1 is IgG and binding domain B2 is scFv. Conversely, binding domain B1 may be scFv and binding domain B2 may be IgG.

[0234] In one embodiment, binding domain B1 is an immunoglobulin and binding domain B2 is Fab. Conversely, binding domain B1 may be Fab and binding domain B2 may be an immunoglobulin. The bispecific polypeptide may optionally be in RUBY® format or optimized RUBY® format, both of which are described herein. The format of the bispecific polypeptide is as described above and is shown in Figure 23, and the bispecific polypeptide may include certain mutations as described above.

[0235] In one embodiment, the bispecific polypeptide may contain a CDR of any light chain of any of the B1 domains (as shown in Table C(2) below) and / or a CDR of any heavy chain of any of the B1 domains (as shown in Table C(1) below) in combination with any of the CDRs of any light chain of any of the B2 domains (as shown in Table D(2)) and / or a CDR of any heavy chain of any of the B2 domains (as shown in Table D(1a) and / or Table D(1b) above).

[0236] In a preferred embodiment, the bispecific polypeptide comprises the CDR sequence of ATOR-4066 (also referred to herein as ffAC_05337) as follows: (a) The binding domain B1 contains three heavy chain CDRs of sequence numbers 81, 82, and 83, and three light chain CDRs of sequence numbers 96, 97, and 98, (b) The binding domain B2 contains three heavy chain CDRs of sequence numbers 216, 217, and 239, and three light chain CDRs of sequence numbers 90, 91, and 311.

[0237] In one embodiment, the bispecific polypeptide may contain any light chain variable region of the B1 domain (as shown in Table A below) and / or any heavy chain variable region of the B1 domain (as shown in Table A below) in combination with any of the light chain variable regions of the B2 domain (as shown in Table B) and / or any heavy chain variable region of the B2 domain (as shown in Table B).

[0238] Therefore, in certain embodiments, B1 and B2 each include the variable regions containing the CDRs identified above. For example, B1 may include the heavy chain variable region and / or light chain variable region of antibody G12 (SEQ ID NO: 19 and / or SEQ ID NO: 17) or the heavy chain variable region and / or light chain variable region of G12_mut (SEQ ID NO: 29 and / or SEQ ID NO: 17), and B2 may include the heavy chain variable region and / or light chain variable region of any of the reference CEA antibodies: (a) Heavy chain variable region and / or light chain variable region of antibody AC_05059 (SEQ ID NO: 33 and / or SEQ ID NO: 31) (b) Heavy chain variable region and / or light chain variable region of antibody AC_05060 (SEQ ID NO: 37 and / or SEQ ID NO: 35) (c) Heavy chain variable region and / or light chain variable region of antibody AC_05061 (SEQ ID NO: 41 and / or SEQ ID NO: 39) (d) Heavy chain variable region and / or light chain variable region of antibody AC_05062 (SEQ ID NO: 45 and / or SEQ ID NO: 43) (e) Heavy chain variable region and / or light chain variable region of antibody AC_05064 (SEQ ID NO: 49 and / or SEQ ID NO: 47) (f) Heavy chain variable region and / or light chain variable region of antibody AC_05079 (SEQ ID NO: 53 and / or SEQ ID NO: 51) (g) Heavy chain variable region and / or light chain variable region of antibody AC_05080 (SEQ ID NO: 57 and / or SEQ ID NO: 55) (h) Heavy chain variable region and / or light chain variable region of antibody AC_05081 (SEQ ID NO: 61 and / or SEQ ID NO: 59) (i) Heavy chain variable region and / or light chain variable region of antibody AC_05082 (SEQ ID NO. 65 and / or SEQ ID NO. 63) (j) Heavy chain variable region and / or light chain variable region of antibody AC_05083 (SEQ ID NO: 69 and / or SEQ ID NO: 67) (k) Heavy chain variable region and / or light chain variable region of antibody AC_05084 (SEQ ID NO: 106 and / or SEQ ID NO: 71) (l) Heavy chain variable region and / or light chain variable region of antibody AC_05085 (SEQ ID NO: 110 and / or SEQ ID NO: 108) (m) Heavy chain variable region and / or light chain variable region of antibody AC_05086 (SEQ ID NO: 114 and / or SEQ ID NO: 112) (n) Heavy chain variable region and / or light chain variable region of antibody AC_05087 (SEQ ID NO: 118 and / or SEQ ID NO: 116) (o) Heavy chain variable region and / or light chain variable region of antibody AC_05088 (SEQ ID NO: 122 and / or SEQ ID NO: 120) (p) Heavy chain variable region and / or light chain variable region of antibody AC_05089 (SEQ ID NO: 126 and / or SEQ ID NO: 124) (q) Heavy chain variable region and / or light chain variable region of antibody AC_05090 (SEQ ID NO: 130 and / or SEQ ID NO: 128) (r) Heavy chain variable region and / or light chain variable region of antibody AC_05091 (SEQ ID NO: 134 and / or SEQ ID NO: 132) (s) Heavy chain variable region and / or light chain variable region of antibody AC_05092 (SEQ ID NO: 138 and / or SEQ ID NO: 136) (t) Antibody AC_05093 heavy chain variable region and / or light chain variable region (SEQ ID NO: 142 and / or SEQ ID NO: 140) (u) Heavy chain variable region and / or light chain variable region of antibody AC_05094 (SEQ ID NO: 146 and / or SEQ ID NO: 144) (v) Heavy chain variable region and / or light chain variable region of antibody AC_05095 (SEQ ID NO: 150 and / or SEQ ID NO: 148) (w) Heavy chain variable region and / or light chain variable region of antibody AC_05096 (SEQ ID NO: 154 and / or SEQ ID NO: 152) (x) Heavy chain variable region and / or light chain variable region of antibody AC_05097 (SEQ ID NO: 158 and / or SEQ ID NO: 156) (y) Heavy chain variable region and / or light chain variable region of antibody AC_05098 (SEQ ID NO: 162 and / or SEQ ID NO: 160) (z) Heavy chain variable region and / or light chain variable region of antibody AC_05099 (SEQ ID NO: 166 and / or SEQ ID NO: 164) (aa) Heavy chain variable region and / or light chain variable region of antibody AC_05100 (SEQ ID NO: 170 and / or SEQ ID NO: 168) (ab) Heavy chain variable region and / or light chain variable region of antibody Fab1 (SEQ ID NO: 174 and / or SEQ ID NO: 172) (ac) Heavy chain variable region and / or light chain variable region of antibody Fab2 (SEQ ID NO: 178 and / or SEQ ID NO: 176) (ad) Heavy chain variable region and / or light chain variable region of antibody Fab3 (SEQ ID NO: 182 and / or SEQ ID NO: 180) (ae) Heavy chain variable region and / or light chain variable region of antibody Fab4 (SEQ ID NO: 186 and / or SEQ ID NO: 184) (af) Heavy chain variable region and / or light chain variable region of antibody Fab5 (SEQ ID NO: 190 and / or SEQ ID NO: 188) (ag) Heavy chain variable region and / or light chain variable region of antibody Fab6 (SEQ ID NO: 194 and / or SEQ ID NO: 192) (ah) Heavy chain variable region and / or light chain variable region of antibody Fab7 (SEQ ID NO: 198 and / or SEQ ID NO: 196) (ai) Heavy chain variable region and / or light chain variable region of antibody Fab8 (SEQ ID NO: 202 and / or SEQ ID NO: 200) (aj) Heavy chain variable region and / or light chain variable region of antibody Fab9 (SEQ ID NO: 206 and / or SEQ ID NO: 204) (ak) Heavy chain variable region and / or light chain variable region of antibody Fab10 (SEQ ID NO: 210 and / or SEQ ID NO: 208) (al) Heavy chain variable region and / or light chain variable region of antibody Fab11 (SEQ ID NO: 214 and / or SEQ ID NO: 212) and / or (am) May contain the heavy chain variable region and / or light chain variable region of antibody mAb2 (SEQ ID NO: 387 and / or SEQ ID NO: 385).

[0239] In a preferred embodiment, B1 may include the heavy chain variable region and / or the light chain variable region of the antibody ffAC_05337 (SEQ ID NO: 430 and / or SEQ ID NO: 431), and B2 may include the heavy chain variable region and / or the light chain variable region of the antibody ffAC_05337 (SEQ ID NO: 433 and / or SEQ ID NO: 432).

[0240] The B1 domain may include the light chain variable region and / or heavy chain variable region of any of the B1 domains described above, and the B2 domain may include the light chain variable region and / or heavy chain variable region of any of the B2 domains described above, or variants of said light chain variable region and / or heavy chain variable region having at least 90% sequence identity thereto.

[0241] Typically, a bispecific polypeptide includes a constant region sequence in addition to the variable region sequence defined above. The bispecific polypeptide can be in any preferred format. For example, it may be in the RUBY® format or the optimized RUBY® format (described above and shown in Figure 23), or the Morrison format.

[0242] An exemplary heavy chain constant region amino acid sequence that can be combined with any VH region sequence disclosed herein (to form a complete heavy chain) is the following IgG1 heavy chain constant region sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 349)

[0243] Similarly, an exemplary light chain constant region amino acid sequence that can be combined with any VL region sequence disclosed herein (to form a complete light chain) is the kappa chain constant region sequence reproduced below. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 350)

[0244] Other light chain constant region sequences are known in the art and can be combined with any VL regions disclosed herein.

[0245] In one embodiment, the polypeptide may include the following constant region amino acid sequence. (a) Reference array CH1 (sequence number 354): [ka] (The bolded and underlined parts are part of the hinge region, but are present in the Fab fragment.) and / or (b) Reference array C kappa (sequence number 355): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC and / or Reference array C lambda (sequence number 356) GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS and / or Reference array C lambda (sequence number 357) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS and / or Reference array C lambda (sequence number 358) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0246] As described above, these reference sequences may contain one or more mutations to prevent the formation of aggregates and / or Fab byproducts. Such mutation sites (as previously identified herein) may be given for any of the above-mentioned constant region sequences.

[0247] In one embodiment, the bispecific polypeptide is in RUBY® format or optimized RUBY® format and comprises an immunoglobulin and a Fab fragment, the Fab fragment being fused to the C-terminus of the heavy chain of the immunoglobulin via the light chain of the Fab fragment.

[0248] Therefore, in one embodiment, binding domain B1 is an immunoglobulin, and binding domain B2 is a Fab fragment, which is fused to the C-terminus of the heavy chain of the immunoglobulin via the light chain of the Fab fragment. In an alternative embodiment, binding domain B2 is an immunoglobulin, and binding domain B1 is a Fab fragment, which is fused to the C-terminus of the heavy chain of the immunoglobulin via the light chain of the Fab fragment. In addition, the bispecific polypeptide includes one or more mutations selected from the above for the RUBY® format and / or optimized RUBY® format.

[0249] One-instance, bispecific polypeptides are Multi1, Multi2, Multi3, Multi4, Multi5, Multi6, Multi7, Multi8, Multi9, Multi10, Multi11, Multi12, Multi13, Multi14, Multi17, Multi18, Multi19, Multi20, Multi23, Multi24, Multi25, Multi26, Multi27, Multi28, Multi29, Multi30, Multi31, Multi32, Multi33, Multi34, Multi35, Multi37, Multi38, Multi39, Multi The antibody comprises a light chain CDR and / or heavy chain CDR, and / or a binding domain B1 and / or binding domain 2, which include a format, selected from the list consisting of ti40, Multi41, Multi42, Multi44, Multi46, Multi47, Multi48, Multi49, AC_05333, AC_05334, AC_05336, AC_05337, AC_05338, AC_05339, AC_05341, ffAC_05337, ffAC_05339, and / or AC_05355, preferably Multi34, Multi42, Multi46, and / or ffAC_05337.

[0250] One-instance, bispecific polypeptides include Multi1, Multi2, Multi3, Multi4, Multi5, Multi6, Multi7, Multi8, Multi9, Multi10, Multi11, Multi12, Multi13, Multi14, Multi17, Multi18, Multi19, Multi20, Multi23, Multi24, Multi25, Multi26, Multi27, Multi28, Multi29, Multi30, Multi31, Multi32, Multi33, Multi34, Multi35, Multi37, Multi38, Multi39, Multi The antibody comprises a light chain variable region and / or heavy chain variable region of an antibody selected from the list consisting of i40, Multi41, Multi42, Multi44, Multi46, Multi47, Multi48, Multi49, AC_05333, AC_05334, AC_05336, AC_05337, AC_05338, AC_05339, AC_05341, ffAC_05337, ffAC_05339, and / or AC_05355, preferably Multi34, Multi42, Multi46, and / or ffAC_05337, and / or a binding domain B1 and / or binding domain 2 including a format.

[0251] One-instance, bispecific polypeptides are Multi1, Multi2, Multi3, Multi4, Multi5, Multi6, Multi7, Multi8, Multi9, Multi10, Multi11, Multi12, Multi13, Multi14, Multi17, Multi18, Multi19, Multi20, Multi23, Multi24, Multi25, Multi26, Multi27, Multi28, Multi29, Multi30, Multi31, Multi32, Multi33, Multi34, Multi35, Multi37, Multi38, Multi39, The antibody comprises a light chain and / or heavy chain selected from the list consisting of Multi40, Multi41, Multi42, Multi44, Multi46, Multi47, Multi48, Multi49, AC_05333, AC_05334, AC_05336, AC_05337, AC_05338, AC_05339, AC_05341, ffAC_05337, ffAC_05339, and / or AC_05355, preferably Multi34, Multi42, Multi46, and / or ffAC_05337, and / or a binding domain B1 and / or a binding domain 2 including a format.

[0252] In one embodiment, the bispecific polypeptide comprises a chain H1 containing a sequence selected from the enumeration consisting of SEQ ID NO: 359, SEQ ID NO: 362, SEQ ID NO: 365, and / or SEQ ID NO: 367.

[0253] In one embodiment, the bispecific polypeptide comprises a chain L1 containing a sequence selected from the enumeration consisting of SEQ ID NO: 360, SEQ ID NO: 363, SEQ ID NO: 372, and / or SEQ ID NO: 368.

[0254] In one embodiment, the bispecific polypeptide comprises a chain H2 containing a sequence selected from the enumeration consisting of SEQ ID NO: 361, SEQ ID NO: 364, SEQ ID NO: 366, and / or SEQ ID NO: 369.

[0255] In one embodiment, the bispecific polypeptide is ●Includes a chain H1 containing an array selected from the enumeration consisting of SEQ ID NOs. 359, SEQ ID NOs. 362, SEQ ID NOs. 365, and / or SEQ ID NOs. 367, and / or ●Includes a chain L1 containing an array selected from the enumeration consisting of SEQ ID NOs. 360, SEQ ID NOs. 363, SEQ ID NOs. 372, and / or SEQ ID NOs. 368, and / or ●Contains a strand H2 containing an array selected from the enumeration consisting of SEQ ID NOs. 361, 364, 366, and / or 369.

[0256] In one embodiment, the bispecific polypeptide is ● Each contains two strands H1 containing an array selected from the enumeration consisting of SEQ ID NOs. 359, 362, 365, and / or 367, and / or ● Each of the two strands L1 contains an array selected from the enumeration consisting of SEQ ID NOs. 360, SEQ ID NOs. 363, SEQ ID NOs. 372, and / or SEQ ID NOs. 368, and / or ●Each contains two strands H2 containing an array selected from an enumeration consisting of SEQ ID NOs. 361, 364, 366, and / or 369.

[0257] In one embodiment, the bispecific polypeptide may comprise one or more variants of the chain H1, chain L1, and / or chain H2 as defined above, having at least 90% sequence identity to them, or 95% sequence identity to them, or 99% sequence identity to them.

[0258] In one embodiment, the bispecific polypeptide is ●Contains chain H1 containing sequence number 359, and / or ●Contains chain L1 containing sequence number 360, and / or ●Contains chain H2 including sequence number 361.

[0259] In one embodiment, the bispecific polypeptide is ●Contains chain H1 containing sequence number 362, and / or ●Includes chain L1 containing sequence number 363, and / or ●Contains chain H2 including sequence number 364.

[0260] In one embodiment, the bispecific polypeptide is ●Contains chain H1 containing sequence number 365, and / or ●Contains chain L1 containing sequence number 372, and / or ●Contains chain H2 including sequence number 366.

[0261] In one embodiment, the bispecific polypeptide is ●Contains chain H1 including sequence number 367, and / or ●Contains chain L1 containing sequence number 368, and / or ●Contains chain H2 including sequence number 369.

[0262] In one embodiment, the bispecific polypeptide is ●Contains two strands of H1 including sequence number 359, and / or ●Contains two L1 chains including sequence number 360, and / or ●Contains two strands of H2, including sequence number 361.

[0263] In one embodiment, the bispecific polypeptide is ●Contains two strands of H1 including sequence number 362, and / or ●Contains two L1 chains including sequence number 363, and / or ●Contains two strands of H2, including sequence number 364.

[0264] In one embodiment, the bispecific polypeptide is ●Contains two strands of H1 including sequence number 365, and / or ●Contains two L1 chains including sequence number 372, and / or ●Contains two H2 chains, including sequence number 366.

[0265] In one embodiment, the bispecific polypeptide is ●Contains two strands of H1 including sequence number 367, and / or ●Contains two L1 chains including sequence number 368, and / or ●Contains two strands of H2, including sequence number 369.

[0266] One-instance, bispecific polypeptides are Multi1, Multi2, Multi3, Multi4, Multi5, Multi6, Multi7, Multi8, Multi9, Multi10, Multi11, Multi12, Multi13, Multi14, Multi17, Multi18, Multi19, Multi20, Multi23, Multi24, Multi25, Multi26, Multi27, Multi28, Multi29, Multi30, Multi31, Multi32, Multi33, Multi34, Multi3 5. An antibody selected from the list consisting of Multi37, Multi38, Multi39, Multi40, Multi41, Multi42, Multi44, Multi46, Multi47, Multi48, Multi49, AC_05333, AC_05334, AC_05336, AC_05337, AC_05338, AC_05339, AC_05341, ffAC_05337, ffAC_05339, and / or AC_05355, preferably Multi34, Multi42, Multi46, and / or ffAC_05337.

[0267] One-instance, bispecific polypeptides are defined as Multi1, Multi2, Multi3, Multi4, Multi5, Multi6, Multi7, Multi8, Multi9, Multi10, Multi11, Multi12, Multi13, Multi14, Multi17, Multi18, Multi19, Multi20, Multi23, Multi24, Multi25, Multi26, Multi27, Multi28, Multi29, Multi30, Multi31, Multi32, Multi33, Multi34, Multi35, Multi37 , may include one or more variants of Multi38, Multi39, Multi40, Multi41, Multi42, Multi44, Multi46, Multi47, Multi48, Multi49, AC_05333, AC_05334, AC_05336, AC_05337, AC_05338, AC_05339, AC_05341, ffAC_05337, ffAC_05339, and / or AC_05355, which have at least 90% sequence identity to them, or 95% sequence identity to them, or 99% sequence identity to them.

[0268] Exemplary full-heavy and light-chain sequences Joining domain B1: 1132 / 1133 heavy chain (VH:Q44R, CH1:H168A, F170G, CH2:L234A,L235A) (SEQ ID NO: 371), including the RUBY mutation: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRRAPGKGLEWVSIGSYGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVNFGMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1132 / 1133 light chain containing the RUBY mutation (VL:Q44E, C kappa L135Y, S176W) (SEQ ID NO: 372): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQEKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCYLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLWSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 1132 / 1133 heavy chain (SEQ ID NO: 378): EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSIGSYGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVNFGMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1132 / 1133 light chain (Sequence ID 379): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC G12 heavy chain (SEQ ID NO: 380): EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWLSYISGGSSYIFYADSVRGRFTISRDNSENALYLQMNSLRAEDTAVYYCARILRGGSGMDLWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G12 light chain (Sequence ID 381): QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVYWYQQLPGTAPKLLIYGNINRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDKSISGLVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS G12_mut heavy chain (SEQ ID NO: 382): EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWLSYISGGSSYIFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARILRGGSGMDLWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G12_mut light chain (SEQ ID NO: 383): QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVYWYQQLPGTAPKLLIYGNINRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDKSISGLVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Joining domain B2: AC_05059, light chain (SEQ ID NO: 388): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05059, heavy chain (SEQ ID NO: 389): EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFPPHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05060, light chain (sequence number 390): DIQMTQSPSSLSASVGDRVTITCRASQSIRDYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGTFPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05060, heavy chain (SEQ ID NO: 391): EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYYMSWVRQAPGKGLEWVSGISGYGYYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGYGVIDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05061, light chain (SEQ ID NO: 392): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGAYVPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05061, heavy chain (SEQ ID NO: 393): EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGYTHFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05062, light chain (SEQ ID NO: 394): DIQMTQSPSSLSASVGDRVTITCRASQAISGYLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05062, heavy chain (SEQ ID NO: 395): EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYRWHGSVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05064, light chain (SEQ ID NO: 396): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYPWYFPYTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05064, heavy chain (SEQ ID NO: 397): EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGYSVLDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05079, light chain (SEQ ID NO: 398): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05079, heavy chain (SEQ ID NO: 399): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFPPPLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05081, light chain (SEQ ID NO: 400): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05081, heavy chain (SEQ ID NO: 401): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFQPHLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05088, light chain (Sequence ID 402): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05088, heavy chain (SEQ ID NO: 403): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVLFPPHLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05089, light chain (SEQ ID NO: 404): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05089, heavy chain (SEQ ID NO: 405): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFPPHHLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05090, light chain (sequence number 406): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05090, heavy chain (SEQ ID NO: 407): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFPLHLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05091, light chain (SEQ ID NO: 408): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05091, heavy chain (SEQ ID NO: 409): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFPPHFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05093, light chain (SEQ ID NO: 410): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05093, heavy chain (SEQ ID NO: 411): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFPPHVDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05094, light chain (SEQ ID NO: 412): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05094, heavy chain (SEQ ID NO: 413): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFPPHWDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05096, light chain (SEQ ID NO: 414): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05096, heavy chain (SEQ ID NO: 415): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFRPHLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AC_05097, light chain (SEQ ID NO: 416): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQAGNPHTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC AC_05097, heavy chain (SEQ ID NO: 417): EVQLLESGGGLVQPGGSLRLSCAASGTFSSSYMGWVRQAPGKGLEWVSSIGSGSYSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPSVPFSPHLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fab1, light chain (SEQ ID NO: 418): DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSHGPLLTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Fab1, heavy chain (SEQ ID NO: 419): QVQLVQSGAEVKKPGSSVKVSCKASGGTFGYYAIHWVRQAPGQGLEWMGGIGSIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARAWSSDHMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fab3, light chain (SEQ ID NO: 420): EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYWYPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Fab3, heavy chain (SEQ ID NO: 421): QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSSSIHWVRQAPGQGLEWMGHIYPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARHSGSRFFSPMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0269] As discussed above, methods for producing bispecific polypeptides are well known in the relevant field.

[0270] Conveniently, bispecific polypeptides are recombinant polypeptides or contain recombinant polypeptides. Suitable methods for producing such recombinant polypeptides are well known in the art, for example, expression in prokaryotic or eukaryotic host cells (see, for example, Green & Sambrook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor, New York, relevant disclosures in the literature are incorporated herein by reference).

[0271] The polypeptides described can also be produced using commercially available in vitro translation systems such as rabbit reticulocyte lysate or wheat germ lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system can be coupled to a transcription system such as the TNT transcription-translation system (Promega). This system has the advantage of producing suitable mRNA transcripts from coding DNA polynucleotides in the same reaction as translation.

[0272] Those skilled in the art will understand that bispecific polypeptides can be artificially synthesized, for example, using well-known liquid-phase or solid-phase synthesis techniques (such as t-Boc or Fmoc solid-phase peptide synthesis).

[0273] PD-1 inhibitors The combination therapy, pharmaceutical composition, use, and method of the present invention comprises a PD-1 inhibitor. The PD-1 inhibitor may be effective in treating cancer and / or may specifically bind to PD-1 or PD-L1. It will be understood that the therapeutic benefit of a PD-1 inhibitor may be mediated by attenuating the function of the inhibitory immune checkpoint molecule PD-1.

[0274] Therefore, in one embodiment of the present invention, a PD-1 inhibitor is an effective immunotherapy agent for treating cancer.

[0275] The term “immunotherapy agent” is intended to include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to produce an immune response against a tumor or cancer in a subject. Various immunotherapy agents are useful in the compositions and methods described herein. In one embodiment, the immunotherapy agent is an antibody, such as an anti-PD-1 antibody that can specifically bind to PD-1 or an anti-PD-L1 antibody that can specifically bind to PD-L1, or an antigen-binding fragment thereof.

[0276] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cytotoxicity. In addition, the term immune response includes immune responses indirectly mediated by T cell activation, e.g., antibody production (humoral response) and cytokine-responsive cells, e.g., macrophage activation.

[0277] Immune checkpoint molecules include not only proteins on the cell surface of immune cells such as CD4+ and / or CD8+ T cells, dendritic cells, NK cells, and macrophages, but also proteins on certain tumor cells that modulate the immune response. It will be understood by those skilled in the art that PD-1 is an inhibitory immune checkpoint molecule.

[0278] Blocking or neutralizing immune checkpoint molecules such as PD-1 can block or neutralize inhibitory signaling, thereby upregulating the immune response to more effectively treat cancer. Examples of agents useful for blocking inhibitory immune checkpoints include antibodies, small molecules, peptides, peptide mimes, natural ligands, and derivatives of natural ligands, or fragments thereof, that can bind to and / or inactivate or inhibit inhibitory immune checkpoint proteins; as well as RNA interference, antisense, nucleic acid aptamers, or fragments thereof, that can downregulate the expression and / or activity of inhibitory immune checkpoint nucleic acids. Examples of agents for upregulating the immune response include antibodies against one or more inhibitory immune checkpoint proteins that block the interaction between the protein and its innate receptor(s); inactive forms of one or more immune checkpoint inhibitor proteins (e.g., dominant-negative polypeptides); small molecules or peptides that block the interaction between one or more inhibitory immune checkpoint proteins and their innate receptor(s); fusion proteins that bind to their innate receptor(s) (e.g., extracellular components of immune checkpoint inhibitor proteins fused to the Fc portion of an antibody or immunoglobulin); and nucleic acid molecules that block the transcription or translation of inhibitory immune checkpoint nucleic acid. Such agents can directly block the interaction between one or more inhibitory immune checkpoints and their innate receptor(s) (e.g., antibodies), thereby blocking inhibitory signaling and upregulating the immune response. Alternatively, agents can indirectly block the interaction between one or more inhibitory immune checkpoint proteins and their innate receptor(s), thereby blocking inhibitory signaling and upregulating the immune response. For example, soluble forms of immune checkpoint protein ligands, such as stabilized extracellular domains, can bind to their receptors and indirectly reduce the effective concentration of the receptor required for binding to the appropriate ligand. In one embodiment, an anti-PD-1 antibody and / or an anti-PD-L1 antibody are used alone or in combination to inhibit the immune checkpoint.

[0279] Therefore, in one embodiment, a PD-1 inhibitor binds to an inhibitory immune checkpoint molecule and inhibits its function.

[0280] A "PD-1 inhibitor" (or "PD-1 pathway inhibitor") contains elements that can inhibit the PD-1 pathway.

[0281] PD-1, when engaged with its ligand PD-L1 or PD-L2, functions as a negative regulator of T cell activation. PD-L1 is expressed by many solid tumors, particularly melanoma. Therefore, these tumors can downregulate immune-mediated antitumor effects through the activation of inhibitory PD-1 receptors on T cells. Blocking the interaction between PD-1 and PD-L1 can remove a checkpoint in the immune response, leading to an enhancement of the antitumor T cell response. This interaction can be blocked by antibodies specific to PD-1 or PD-L1, or any other suitable agent. Such antibodies and agents may generally be referred to as PD-1 inhibitors.

[0282] Therefore, PD-1 inhibitors block the interaction between PD-1 (programmed cell death protein 1) and its ligand PD-L1 (programmed cell death ligand 1). Thus, such PD-1 inhibitors can act on either PD-L1 or PD-1, or both. Therefore, the term PD-1 inhibitor includes both PD-1 and PD-L1 inhibitors. PD-1 inhibitors block the activity of PD-1 and PD-L1 immune checkpoint proteins.

[0283] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody or its antigen-binding fragment. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody or its antigen-binding fragment.

[0284] "PD-1" specifically refers to the human PD-1 protein, such as the one described in GenBank accession number NP_005009.2 (whose sequence is described in sequence number 470 below). PD-1 is also known in scientific literature as PD1, CD279, PDCD1, and SLEB2. MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL [Sequence ID 470]

[0285] "PD-L1" specifically refers to the human PD-L1 protein, such as the one described in GenBank accession number AAI13735.1 (whose sequence is described in sequence number 471 below). PD-L1 is also known in scientific literature as CD274, B7-H1, B7-H, PDCD1L1, and PDCD1LG1. MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET [Sequence ID 471]

[0286] Accordingly, the combination therapy or pharmaceutical composition of the present invention comprises a PD-1 inhibitor that specifically binds to PD-1 or PD-L1, i.e., has specificity for PD-1 or PD-L1. "Specificity" means that the inhibitor can bind to PD-1 or PD-L1 in vivo, i.e., under the physiological conditions in which PD-1 or PD-L1 is present in the human body. Preferably, the PD-1 inhibitor does not bind to any other protein (other than PD-1 or PD-L1) in vivo. Such binding specificity can be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blotting, and flow cytometry, using transfected cells expressing PD-1 or PD-L1.

[0287] PD-1 inhibitors that specifically bind to PD-1 or PD-L1 are preferably 10 × 10 -9 Less than M or 7×10 -9 Less than M, more preferably 4, or 2 × 10 -9 Less than M, most preferably 1.2 × 10 -9PD-1 inhibitors bind to human PD-1 or PD-L1 with a Kd value of less than M. Advantageously, PD-1 inhibitors can selectively bind to PD-1 or PD-L1, i.e., they bind at least 10 times more strongly to PD-1 or PD-L1 than to any other protein. Preferably, PD-1 inhibitors bind specifically to PD-1 or PD-L1, i.e., they bind to PD-1 or PD-L1 but not to other molecules (e.g., OX40 and / or CD40, etc.), or bind with lower affinity (e.g., 1 / 10th lower affinity). Thus, they bind to PD-1 or PD-L1 with higher binding affinity than they would to other molecules. Therefore, typically, the Kd of an antibody against human PD-1 or PD-L1 will be twice, preferably five times, and more preferably less than ten times, the Kd of an antibody against mouse PD-1 or PD-L1, other immune checkpoint molecules, or any other unrelated substances or accompanying substances in the environment. More preferably, Kd would be less than 50 times, even more preferably less than 100 times, and even more preferably less than 200 times.

[0288] The overall affinity (K) of interactions (such as interactions between antibodies and ligands) D ) and on speed (k a Methods for measuring the flow rate (kd) and off-speed are well known in the art. Exemplary in vitro methods are described in the attached examples. Methods based on flow cytometry may also be used (Sklar et al., Annu Rev Biophys Biomol Struct, (31), 97-119, 2002).

[0289] As used herein, the terms PD-1 and PD-L1 typically refer to human PD-1 and PD-L1. Inhibitors may have some binding affinity to PD-1 and PD-L1 from other mammals, such as PD-1 and PD-L1 from non-human primates, e.g., Macaca fascicularis (cynomolgus monkeys). Antibodies preferably do not bind to mouse PD-1 or PD-L1 and / or other immune checkpoint molecules.

[0290] In one embodiment, a PD-1 inhibitor that specifically binds to PD-1 or PD-L1 may have affinity for its native PD-1 or PD-L1, for example, PD-1 or PD-L1 localized on the surface of a cell. In one embodiment, the PD-1 inhibitor blocks the PD-1 PD-L1 interaction. For example, the PD-1 inhibitor may bind to PD-1 or PD-L1 in a manner that inhibits the ability of PD-L1 to bind to PD-1, thereby blocking the PD-1 / PD-L1 interaction.

[0291] "Localizing on the cell surface" means that PD-1 or PD-L1 associates with a cell such that one or more regions of PD-1 are present on the outer surface of the cell. For example, PD-1 can be inserted into the cell plasma membrane with one or more regions presented on the extracellular surface (i.e., oriented as a transmembrane protein). This can occur during the process of PD-1 expression by the cell. Therefore, in one embodiment, "localizing on the cell surface" may mean "expression on the cell surface." Alternatively, PD-1 may be present outside the cell by covalent and / or ionic interactions, causing it to localize to specific regions or multiple regions on the cell surface.

[0292] In one embodiment, the PD-1 inhibitors described herein can induce antitumor immunity via immune checkpoint blockade. The PD-1 inhibitors bind to PD-1 or PD-L1 in a manner that inhibits PD-L1 from binding to PD-1, i.e., they block the PD-1 / PD-L1 interaction. The PD-1 inhibitors can enhance the T cell response, for example, by enhancing or restoring T cell effector function. In one embodiment, the PD-1 inhibitors can promote the infiltration of tumor-reactive CD8+ T cells into an established tumor.

[0293] Therefore, PD-1 inhibitors can modulate the activity of cells expressing PD-1 or PD-L1, and this modulation is either an increase or decrease in the activity of such cells. These cells are typically T cells. Inhibitors can increase the activity of CD4+ or CD8+ effector cells, decrease the activity of regulatory T cells (T reg), or even deplete such cells. In any case, the net effect of the antibody is an increase in the activity of effector T cells, particularly CD4+, CD8+, or NK effector T cells. Methods for determining changes in effector T cell activity are well known and have been described previously.

[0294] PD-1 inhibitors preferably induce an increase in activity in T cells (preferably CD8+ T cells) in vitro, and optionally, this increase in activity is an increase in T cell proliferation, IFN-γ production, and / or IL-2 production. The increase is preferably at least 2-fold, more preferably at least 10-fold, and even more preferably at least 25-fold higher than the change in activity caused by isotype control antibodies measured in the same assay.

[0295] In one embodiment, a PD-1 inhibitor can improve the effectiveness of another immunotherapy.

[0296] In one embodiment, a PD-1 inhibitor blocks the programmed death-1 (PD-1) receptor from binding to its ligand PD-L1, which is expressed on the surface of cells within tumors (Ribas and Wolchok 2018). PD-1 is an immune checkpoint, and its inhibitory function is mediated by the tyrosine phosphatase SHP-2, which dephosphorylates signaling molecules downstream of T cell receptor (TCR) signaling molecules. In a preferred embodiment, a PD-1 inhibitor reactivates PD-1 expressing T cells, preferably by blocking inhibitory signaling mediated by the tyrosine phosphatase SHP-2 (which dephosphorylates signaling molecules downstream of T cell receptor (TCR) signaling molecules).

[0297] PD-1 inhibitors can be anti-PD-1 antibodies or their antigen-binding fragments that can inhibit PD-1 function (e.g., pembrolizumab (also known as lambrolizumab), nivolumab, pizilizumab, semiprimab, AMP-224, PDR-001, MEDI-0680 (also known as AMP-514), JTX-4014 (pimivarimab), spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, INCMGA00012 (retifanlimab), and acrixolimab (YBL006).

[0298] Alternatively, PD-1 inhibitors may include or consist of anti-PD-L1 antibodies or their antigen-binding fragments that can inhibit PD-1 function (e.g., atezolizumab (Tecentriq®, MPDL3280A), durvalumab (MEDI-4736), avelumab, MDX-1105, KN035 (emvafolimab), and CK-301 (kosivelimab)).

[0299] Alternatively, the PD-1 inhibitor may be a small molecule or peptide inhibitor of PD-1 or PD-L1. For example, the PD-1 inhibitor may be a small molecule inhibitor of PD-L1 such as CA-170. Alternatively, the PD-1 inhibitor may be a peptide inhibitor of PD-L1 such as AUNP12 or BMS-986189.

[0300] PD-1 inhibitors are formulated for parenteral delivery. Parenteral delivery includes any parenteral delivery means. In some preferred embodiments, PD-1 inhibitors are formulated for intravenous, subcutaneous, or intratumoral delivery. In some embodiments, PD-1 inhibitors are formulated for intravenous delivery.

[0301] In one embodiment, the PD-1 inhibitor binds to an epitope that blocks the PD-1 PD-L1 interaction.

[0302] "Pembrolizumab" refers to an intact IgG antibody containing heavy and light chains having amino acid sequences of SEQ ID NOs. 440 and 441, respectively. Pembrolizumab heavy chain sequence (SEQ ID NO: 440): QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK Pembrolizumab light chain sequence (SEQ ID NO: 441) EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

[0303] "Nivolumab" refers to an intact IgG antibody containing heavy and light chains having amino acid sequences of SEQ ID NOs. 442 and 443, respectively. Nivolumab heavy chain sequence (SEQ ID NO: 442): QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK Nivolumab light chain sequence (SEQ ID NO: 443) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Pizilizumab heavy chain sequence (SEQ ID NO: 444) QVQLVQSGSE LKKPGASVKI SCKASGYTFT NYGMNWVRQA PGQGLQWMGW INTDSGESTY AEEFKGRFVF SLDTSVNTAY LQITSLTAED TGMYFCVRVG YDALDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK Pizilizumab light chain sequence (SEQ ID NO: 445) EIVLTQSPSS LSASVGDRVT ITCSARSSVS YMHWFQQKPG KAPKLWIYRT SNLASGVPSR FSGSGSGTSY CLTINSLQPE DFATYYCQQR SSFPLTFGGG TKLEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC Semiprimab heavy chain sequence (SEQ ID NO: 446) EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA PGKGLEWVSG ISGGGRDTYF ADSVKGRFTI SRDNSKNTLY LQMNSLKGED TAVYYCVKWG NIYFDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK Semiprimab light chain sequence (SEQ ID NO: 447) DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP GKAPNLIYA ASSLHGGVPS RFSGSGSGTD FTLTIRTLQP EDFATYYCQQ SSNTPFTFGP GTVVDFRRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Spartalizumab heavy chain sequence (SEQ ID NO: 448) EVQLVQSGAE VKKPGESLRI SCKGSGYTFT TYWMHWVRQA TGQGLEWMGN IYPGTGGSNF DEKFKNRVTI TADKSTSTAY MELSSLRSED TAVYYCTRWT TGTGAYWGQG TTVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG Spartalizumab light chain sequence (SEQ ID NO: 449) EIVLTQSPAT LSLSPGERAT LSCKSSQSLL DSGNQKNFLT WYQQKPGQAP RLLIYWASTR ESGVPSRFSG SGSGTDFT ISSLEAEDAA TYYCQNDYSY PYTFGQGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC Camrelizumab heavy chain sequence (SEQ ID NO: 450) EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYMMSWVRQA PGKGLEWVAT ISGGGANTYY PDSVKGRFTIS RDNAKNSLYL QMNSLRAEDT AVYYCARQLY YFDYWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL GK Camrelizumab light chain sequence (SEQ ID NO: 451) DIQMTQSPSS LSASVGDRVT ITCLASQTIG TWLTWYQQKP GKAPKLLIYT ATSLADGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ VYSIPWTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC tislerizumab heavy chain sequence (SEQ ID NO: 452) QVQLQESGPG LVKPSETLSL TCTVSGFSLT SYGVHWIRQP PGKGLEWIGV IYADGSTNYN PSLKSRVTIS KDTSKNQVSL KLSSVTAADT AVYYCARAYG NYWYIDVWGQ GTTVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT CNVDHKPSNT KVDKRVESKY GPPCPPCPAP PVAGGPSVFL FPPKPKDTLM ISRTPEVTCV VVAVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVVHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK Light chain sequence of tislerizumab (SEQ ID NO: 453) DIVMTQSPDS LAVSLGERAT INCKSSESVS NDVAWYQQKP GQPPKLLINY AFHRFTGVPD RFSGSGYGTD FTLTISSLQA EDVAVYYCHQ AYSSPYTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Tripalimab heavy chain sequence (SEQ ID NO: 454) QGQLVQSGAE VKKPGASVKV SCKASGYTFT DYEMHWVRQA PIHGLEWIGV IESETGGTAY NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCAREG ITTVATTYYW YFDVWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL GK Light chain sequence of tripalimab (SEQ ID NO: 455) DVVMTQSPLS LPVTLGQPAS ISCRSSQSIV HSNGNTYLEW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCFQGSHVP LTFGQGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC dostallimab heavy chain sequence (SEQ ID NO: 456) EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYDMSWVRQA PGKGLEWVST ISGGGSYTYY QDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASPY YAMDYWGQGT TVTVSSASTK GPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTKTYTCN VDHKPSNTKV DKRVESKYGP PCPPCPAPEF LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAKGQPR EPQVYTLPPS QEEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSRLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS LGK dostallimab light chain sequence (SEQ ID NO: 457) DIQLTQSPSF LSAYVGDRVT ITCKASQDVG TAVAWYQQKP GKAPKLLIYW ASTLHTGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCQH YSSYPWTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Heavy chain sequence of INCMGA00012 (SEQ ID NO: 458) QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK YGPPCPPCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLG Light chain sequence of INCMGA00012 (SEQ ID NO: 459) EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

[0304] "Atezolizumab" refers to an intact IgG antibody containing heavy and light chains having amino acid sequences of SEQ ID NOs. 460 and 461, respectively. Atezolizumab heavy chain sequence (SEQ ID NO: 460) EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK Atezolizumab light chain sequence (SEQ ID NO: 461) DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Durvalumab heavy chain sequence (SEQ ID NO: 462) EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVAN IKQDGSEKYY VDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PCPPAPEFEG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPASIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K Durvalumab light chain sequence (SEQ ID NO: 463) EIVLTQSPGT LSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIY DASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC Avelumab heavy chain sequence (SEQ ID NO: 464) EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSS IYPSGGITFY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK Avelumab light chain sequence (SEQ ID NO: 465) QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVL GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS Heavy chain sequence of CK-301 (kosiberimab) (SEQ ID NO: 466) EVQLVQSGAE VKKPGSSVKV SCKASGGTFS RSAISWVRQA PGQGLEWMGV IIPAFGEANY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARGR QMFGAGIDFW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK Light chain sequence of CK-301 (kosiberimab) (SEQ ID NO: 467) NFMLTQPHSV SESPGKTVTI SCTRSSGSID SNYVQWYQQR PGSAPTTVIY EDNQRPSGVP DRFSGSIDSS SNSASLTISG LKTEDEADYY CQSYDSNNRH VIFGGGTKLT VLGQPKAAPS VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADSSP VKAGVETTTP SKQSNNKYAA SSYLSLTPEQ WKSHRSYSCQ VTHEGSTVEK TVAPTECS Heavy chain sequence of JTX-4014 (SEQ ID NO: 468) QVQLVQSGAE VKKPGASVKV SCKASGYTFP SYYMHWVRQA PGQGLEWMGI INPEGGSTAY AQKFQGRVTM TRDTSTSTVY MELSSLRSED TAVYYCARGG TYYDYTYWGQ GTLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK Light chain sequence of JTX-4014 (SEQ ID NO: 469) DIQMTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GKAPKLLIYE ASSLESGVPS RFSGSGTE FTLTISSLQP DDFATYYCQQ YNSFPPTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[0305] Acrixolimab (formerly known as YBL006) is an anti-PD1 monoclonal antibody with PubChem SID number 461454357 and CAS number 2506324-29-2.

[0306] Such PD-1 inhibitors are also described in US8354509B2 and US8779105B2, and the PD-1 inhibitors (in particular anti-PD-1 antibodies) of US8354509B2 and US8779105B2 are incorporated herein by reference.

[0307] For parenteral administration to human patients, the daily dose level of PD-1 inhibitors (e.g., anti-PD-1 and / or anti-PD-L1 antibody molecules) is typically 1 mg / kg patient body weight to 20 mg / kg, or in some cases up to 100 mg / kg, and is administered as a single dose or in divided doses. In some preferred embodiments, the dose is 10 mg / kg. In special circumstances, lower doses may also be used, for example, in combination with long-term administration. In any case, the physician will determine the most suitable actual dose for any individual patient, which will vary depending on the age, weight, and response of the particular patient. The above doses are examples of average cases. Of course, there may be individual cases where higher or lower dose ranges are required, and such cases are within the scope of the present invention.

[0308] Typically, the combination therapies or pharmaceutical compositions described herein contain a PD-1 inhibitor (e.g., an anti-PD-1 and / or anti-PD-L1 antibody molecule) at a concentration of about 2 mg / ml to 150 mg / ml, or about 2 mg / ml to 200 mg / ml. In some embodiments, the pharmaceutical composition contains an anti-PD-1 and / or anti-PD-L1 antibody molecule at a concentration of 10 mg / ml to 25 mg / ml.

[0309] In some embodiments, the PD-1 inhibitor (e.g., anti-PD-1 and / or anti-PD-L1 antibody or its antigen-binding fragment) is administered in doses of 10 mg to 1500 mg. For example, the dose may be 100 mg to 200 mg, or 200 mg to 500 mg.

[0310] In some embodiments, when the PD-1 inhibitor is the anti-PD-1 antibody pembrolizumab, the antibody is used at a dose of approximately 25 mg / ml. In some other embodiments, pembrolizumab is used at a dose of 200 mg(iv) every 3 weeks, or at a dose of 400 mg(iv) every 6 weeks.

[0311] In some embodiments, when the anti-PD-1 antibody is nivolumab, the antibody is used at a dose of approximately 10 mg / ml. In some embodiments, nivolumab is used at a dose of 240 mg(iv) every two weeks, or at a dose of 480 mg(iv) every four weeks. In some embodiments, nivolumab may be used in combination with the anti-CTLA-4 antibody ipilimumab, in which case nivolumab is used at a dose of 1 mg / kg every three weeks for up to four cycles, or at a dose of 3 mg / kg every two or three weeks.

[0312] In some embodiments, when the anti-PD-L1 antibody is atezolizumab, the antibody is used at a dose of approximately 60 mg / ml. In some other embodiments, atezolizumab is used at a dose of 840 mg(iv) every two weeks, or 1200 mg(iv) every three weeks, or 1680 mg(iv) every four weeks.

[0313] In one embodiment, the daily dose level of the PD-1 inhibitor is: (a) Body weight of 1 mg / kg to body weight of 100 mg / kg, (b) Whether the body weight is between 1 mg / kg and 20 mg / kg, (c) 1 mg / kg, 10 mg / kg, 20 mg / kg, 100 mg / kg, subject body weight, preferably 10 mg / kg body weight.

[0314] In one embodiment, the concentration of the PD-1 inhibitor is approximately 2 mg / ml to 150 mg / ml, or approximately 2 mg / ml to 200 mg / ml, and preferably, the concentration of the PD-1 inhibitor is 10 mg / ml to 25 mg / ml.

[0315] In one embodiment, the dose of the PD-1 inhibitor is 10 mg to 1500 mg, and optionally, the dose is 100 mg to 200 mg or 200 mg to 500 mg.

[0316] In one embodiment, (a) The PD-1 inhibitor is pembrolizumab, administered at a dose of approximately 25 mg / ml, 200 mg (intravenously) every 3 weeks, or 400 mg (intravenously) every 6 weeks. (b) The PD-1 inhibitor is nivolumab, administered at a dose of approximately 10 mg / ml, 250 mg (intravenously) every two weeks, or 480 mg (intravenously) every four weeks, or (c) The PD-1 inhibitor is atezolizumab, administered at a dose of approximately 60 mg / ml every two weeks (intravenously) or 840 mg every four weeks (intravenously).

[0317] In some embodiments, administration of the combination therapies described herein results in the activation of exhausted T cells in a subject. "Exhausted T cells" includes a state in which T cells (CD4 or CD8) lose the ability to kill cells (e.g., cancer cells). Activation of exhausted T cells results in the reactivation of these T cells. Advantageously, this results in enhanced immune-mediated killing of cancer cells. As shown in the examples herein, the combination therapies described herein result in a synergistic effect on CD4 and CD8 T cell activity, as measured by IFN-γ levels. In some embodiments, the increase in exhausted T cell activation observed for the combination therapy is a synergistic effect compared to the PD-1 inhibitor alone or the CD40×CEA bispecific antibody alone. This means that the effect of the combination therapy on T cell activation (e.g., as measured by IFN-γ levels) is greater than the additive effect of the PD-1 inhibitor alone or the CD40×CEA bispecific antibody alone.

[0318] In some embodiments, administration of the CD40×CEA bispecific antibody described herein results in upregulation of PD-1 and / or PD-L1 gene expression. In particular, the CD40×CEA bispecific antibody described herein has been shown to result in upregulation of PD-L1 on the surface of tumor-infiltrating immune cells. In some embodiments, administration of the CD40×CEA bispecific antibody described herein results in upregulation of PD-L1 on the surface of macrophages and / or dendritic cells.

[0319] In some embodiments, administration of the combination therapies described herein results in an improved reduction in tumor volume compared to administration of a control, a PD-1 inhibitor alone, or a CD40×CEA bispecific antibody alone. In some embodiments, the improved reduction in tumor volume for the combination therapy is a synergistic effect compared to the PD-1 inhibitor alone or the CD40×CEA bispecific antibody alone. This means that the effect of the combination therapy on tumor volume is greater than the additive effect of the PD-1 inhibitor alone or the CD40×CEA bispecific antibody alone.

[0320] In some embodiments, administration of the combination therapies described herein results in increased survival rates compared to administration of a control, a PD-1 inhibitor alone, or a CD40×CEA bispecific antibody alone. In some embodiments, the increase in survival rates for the combination therapy is a synergistic effect compared to the PD-1 inhibitor alone or the CD40×CEA bispecific antibody alone. This means that the effect of the combination therapy on survival rates is greater than the additive effect of the PD-1 inhibitor alone or the CD40×CEA bispecific antibody alone.

[0321] In one embodiment, the PD-1 inhibitor is an antibody or its antigen-binding fragment, which specifically binds to PD-1 or PD-L1, and the antibody or antigen-binding fragment contained in the combination therapy or pharmaceutical composition of the present invention includes an antibody Fc region. Those skilled in the art will understand that the Fc region may be derived from an IgG antibody or from a different class of antibody (such as IgM, IgA, IgD, or IgE). In one embodiment, the Fc region is derived from an IgG1, IgG2, IgG3, or IgG4 antibody. However, advantageously, the Fc region is derived from an IgG4 antibody.

[0322] Fc regions can be naturally occurring (e.g., as part of an endogenously produced antibody) or artificial (e.g., involving one or more point mutations in a naturally occurring Fc region). Variants of the Fc region typically bind to Fc receptors such as FcγR and / or neonatal Fc receptors (FcRn) with modified affinity, improving the function and / or half-life of the polypeptide. The biological function and / or half-life may be either prolonged or decreased compared to the half-life of the polypeptide containing the natural Fc region. Examples of such biological functions that can be modulated by the presence of a variant Fc region include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), and / or apoptosis.

[0323] Therefore, the Fc region can be naturally occurring (e.g., as part of an endogenously produced human antibody) or it can be artificial (e.g., containing one or more point mutations in a naturally occurring human Fc region).

[0324] As is well known in the art, the Fc region of an antibody mediates its serum half-life and effector functions such as complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP).

[0325] The Fc region can be manipulated as described above with respect to the bispecific antibody of the combination therapy of the present invention.

[0326] Polynucleotides, vectors, and cells Further aspects of the present invention include a first isolated nucleic acid molecule encoding a bispecific polypeptide comprising a first binding domain, referred to as B1, which can specifically bind to CD40, and a second binding domain, referred to as B2, which can specifically bind to carcinoembryonic antigen (CEA), and (i) PD-1 inhibitors, and / or (ii) The kit comprises an antibody that specifically binds to PD-1 or PD-L1, or an antigen-binding fragment thereof, or a second isolated nucleic acid molecule encoding a constituent polypeptide chain thereof.

[0327] In one embodiment, the first isolated nucleic acid molecule encodes a bispecific polypeptide, or its constituent polypeptide chain, as described above in relation to any other aspect of the present invention.

[0328] For example, a nucleic acid molecule may contain any of the nucleotide sequences provided in Tables A and B.

[0329] Therefore, the first polynucleotide may encode any polypeptide described herein, or all or part of B1, or all or part of B2. The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention may be provided in isolated or substantially isolated forms. Substantially isolated means that the polypeptide may be substantially, but not completely, isolated from any surrounding medium. Polynucleotides may be mixed with a carrier or diluent that does not interfere with their intended use and are still considered substantially isolated.

[0330] A nucleic acid sequence that "codes" a selected polypeptide is a nucleic acid molecule that, under the control of appropriate regulatory sequences, is transcribed in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. In this specification, such nucleic acid sequences may include, but are not limited to, cDNA from viruses, mRNA from prokaryotes or eukaryotes, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence is typically located at 3' of the coding sequence.

[0331] Representative polynucleotides encoding examples of heavy or light chain amino acid sequences of antibodies may include or consist of any one of the nucleotide sequences disclosed herein, for example, the sequences shown in Tables A and B.

[0332] A preferred polynucleotide sequence may, alternatively, be a variant of one of these specific polynucleotide sequences. For example, the variant may be a substitution, deletion, or addition variant of any of the nucleic acid sequences described above. A variant polynucleotide may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions from the sequences shown in the sequence listing.

[0333] A suitable variant may be at least 70% homologous to any one of the nucleic acid sequences disclosed herein, preferably at least 80% or 90%, and more preferably at least 95%, 97%, or 99% homologous. Preferably, homology and identity at these levels exist with respect to at least the coding region of the polynucleotide. Methods for measuring homology are well known in the art, and in this context, it will be understood by those skilled in the art that homology is calculated based on nucleic acid identity. Such homology may exist over regions of at least 15, preferably at least 30, for example, at least 40, 60, 100, 200 or more consecutive nucleotides. Such homology may exist over the entire length of the unmodified polynucleotide sequence.

[0334] Methods for measuring polynucleotide homology or identity are known in the art. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology (for example, in its default settings) (Devereux et al., 1984, the disclosure of which is incorporated herein by reference).

[0335] The PILEUP and BLAST algorithms can also be used to compute homology or (typically with their default settings) align sequences, as described, for example, in Altschul, 1993 and Altschul et al., 1990 (their disclosures are incorporated herein by reference).

[0336] Software for performing BLAST analysis is available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm identifies high-scoring sequence pairs (HSPs) by first identifying short words of length W. Query sequences are obtained that, when aligned with words of the same length in the database sequences, match or satisfy a positive threshold score T. T is referred to as the neighbor word score threshold (see Altschul et al., above). These initial neighbor word hits serve as seeds to initiate the search for HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The extension of word hits in each direction stops when the cumulative alignment score becomes zero or less due to the accumulation of one or more negative scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses, by default, word length (W) 11, alignment (B) 50 of the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1992, the disclosure of which is incorporated herein by reference), expected value (E) 10, M=5, N=4, and comparison of both strands.

[0337] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin & Altschul, 1993, the disclosure of which is incorporated herein by reference. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum sum probability in the comparison between a first sequence and a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the sequences are considered similar to each other.

[0338] Homogenies can differ in the sequence within the associated polynucleotide and by 3, 5, 10, 15, or 20 or more mutations (each of which may be a substitution, deletion, or insertion). These mutations can be measured over at least 30, for example, at least 40, 60, or 100 or more consecutive nucleotide regions of the homologue.

[0339] In one embodiment, a variant sequence may vary from a particular sequence shown in a sequence listing due to the duplication of the gene code. The DNA code has four primary nucleic acid residues (A, T, C, and G) which are used to "spell" three letter codons that represent the amino acids of the proteins encoded by the genes of an organism. The linear sequence of codons along the DNA molecule is translated into a linear sequence of amino acids in the protein(s) encoded by those genes. The code is highly degenerate, with 61 codons that code for 20 native amino acids and 3 codons that represent a "stop" signal. Thus, most amino acids are coded by two or more codons, and in fact, some are coded by four or more different codons. Therefore, a variant polynucleotide of the present invention may code for the same polypeptide sequence as another polynucleotide of the present invention, but may have a different nucleic acid sequence to use different codons to code for the same amino acids.

[0340] Therefore, polypeptides can be produced from or delivered in the form of polynucleotides that encode and can be expressed.

[0341] Polynucleotides can be synthesized according to methods well known in the art, as exemplified in Green & Sambrook (2012, Molecular Cloning - a laboratory manual, 4th edition; Cold Spring Harbor Press, its disclosure incorporated herein by reference).

[0342] A further aspect of the present invention is a vector (such as an expression vector) comprising a first isolated nucleic acid molecule encoding a bispecific polypeptide having a first binding domain called B1 that can specifically bind to CD40, and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), and (i) PD-1 inhibitors, and / or (ii) A kit comprising an antibody that specifically binds to PD-1 or PD-L1, or an antigen-binding fragment thereof, or a second isolated nucleic acid molecule encoding a constituent polypeptide chain thereof.

[0343] In one embodiment, the first isolated nucleic acid is as described in relation to the previous embodiment.

[0344] Nucleic acid molecules may be provided in the form of expression cassettes containing control sequences operably linked to an insert sequence, thus enabling in vivo expression of the polypeptide of the present invention. These expression cassettes are then typically provided within a vector (e.g., a plasmid or recombinant viral vector). Such expression cassettes may be administered directly to a host target. Alternatively, a vector containing the polynucleotide of the present invention may be administered to a host target. Preferably, the polynucleotide is prepared and / or administered using a gene vector. A suitable vector may be any vector capable of carrying a sufficient amount of genetic information and enabling the expression of the polypeptide of the present invention.

[0345] The kit of the present invention may include an expression vector comprising a first isolated nucleic acid. Such an expression vector is conventionally constructed in the field of molecular biology and may include, for example, the use of plasmid DNA and other elements such as appropriate initiators, promoters, enhancers, and, if necessary, polyadenylation signals, to enable the expression of the peptide of the present invention, and these are positioned in the correct orientation. Other suitable vectors will be obvious to those skilled in the art (see Green & Sambrook above).

[0346] Further aspects of the present invention include recombinant host cells (mammalian cells, e.g., human cells, or Chinese hamster ovary cells, e.g., CHOK1SV cells) comprising a first nucleic acid molecule encoding a bispecific polypeptide having a first binding domain, called B1, that can specifically bind to CD40, and a second binding domain, called B2, that can specifically bind to carcinoembryonic antigen (CEA), and (i) PD-1 inhibitors, and / or (ii) A kit comprising an antibody that specifically binds to PD-1 or PD-L1, or an antigen-binding fragment thereof, or a second isolated nucleic acid molecule encoding a constituent polypeptide chain thereof.

[0347] In one embodiment, the first isolated nucleic acid is as described in relation to the previous embodiment.

[0348] Recombinant host cells modified to express a bispecific polypeptide or a portion of its components. Such cells include transient, or preferably stable, higher eukaryotic cell lines, e.g., mammalian or insect cells; lower eukaryotic cells, e.g., yeast or prokaryotic cells; e.g., bacterial cells. Specific examples of cells that can be modified by insertion of a vector or expression cassette encoding the polypeptide of the present invention include mammalian human fetal kidney (HEK) (e.g., HEK293T), CHO, HeLa, NS0, and COS cells. Preferably, the selected cell lines are not only stable but also allow for the maturation glycosylation and cell surface expression of the polypeptide.

[0349] Such cell lines may be cultured using routine methods to produce bispecific polypeptides, or they may be used therapeutically or prophylactically to deliver antibodies to a target. Alternatively, the polynucleotides, expression cassettes, or vectors of the present invention may be administered ex vivo to cells derived from the target, and the cells may then be returned to the target body.

[0350] In one embodiment, the first isolated nucleic acid molecule encodes an antibody heavy chain or its variable region.

[0351] In one embodiment, the first isolated nucleic acid molecule encodes an antibody light chain or its variable region.

[0352] "Nucleic acid molecules" include DNA (e.g., genomic DNA or complementary DNA) and mRNA molecules, which can be single-stranded or double-stranded. "Isolated" means that the nucleic acid molecule is not located within a cell or is not otherwise supplied into a cell.

[0353] In one embodiment, it is a nucleic acid molecule or a cDNA molecule.

[0354] Those skilled in the art will understand that nucleic acid molecules can be codon-optimized for the expression of antibody polypeptides in specific host cells, for example, for expression in human cells (see, for example, Angov, 2011, the disclosure of which is incorporated herein by reference).

[0355] Production method As described above, a second aspect of the present invention is an effective amount of: (a) A bispecific polypeptide comprising a first binding domain called B1 that can specifically bind to CD40, and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), (b) The present invention relates to a pharmaceutical composition comprising a PD-1 inhibitor which is formulated for parenteral delivery.

[0356] In one embodiment, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

[0357] Those skilled in the art will understand that the pharmaceutical composition may also include additional compounds, such as chelating agents including EDTA, citrate, EGTA, or glutathione.

[0358] Pharmaceutical compositions can be prepared in ways known in the art, having sufficient storage stability and being suitable for administration to humans and animals. For example, pharmaceutical compositions can be freeze-dried by lyophilization, spray-drying, spray-cooling, or by using particle formation from supercritical particle formation.

[0359] "Pharmacologically acceptable" means a non-toxic material that does not impair the effectiveness of the CD40 and CEA binding activity of a bispecific polypeptide, or / or the effectiveness of the PD-1 or PD-L1 binding activity of a PD-1 inhibitor.

[0360] Such pharmaceutically acceptable buffers, carriers, or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000) (the disclosure thereof is incorporated herein by reference)).

[0361] The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH. Examples of buffers include Trizma, Bicine, Tricin, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0362] The term “diluent” is intended to mean an aqueous or non-aqueous solution intended to dilute polypeptides in a pharmaceutical preparation. The diluent may be one or more of the following: physiological saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0363] The term “adjuvant” is intended to mean any compound added to a formulation to enhance the biological effect of a bispecific polypeptide and / or PD-1 inhibitor. Adjuvants may be, but are not limited to, one or more zinc, copper, or silver salts having different anions, such as fluorides, chlorides, bromides, iodides, thiocyanates, sulfites, hydroxides, phosphates, carbonates, lactates, glycolates, citrates, borates, tartrates, and acetates with different acyl compositions. Adjuvants may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, and cationic dendrimers, cationic synthetic polymers such as poly(vinylimidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0364] Excipients may be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrin, which are added to compositions, for example, to facilitate lyophilization. Examples of polymers include starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginates, carrageenan, hyaluronic acid and their derivatives, polyacrylic acid, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate of different degrees of hydrolysis, and polyvinylpyrrolidone, all of which have different molecular weights, and are added to formulations, for example, for viscosity control, to achieve bioadhesion, or to protect lipids from chemical and proteolytic degradation. Examples of lipids include fatty acids, phospholipids, mono, di, and triglycerides, ceramides, sphingolipids and glycolipids, all with different acyl chain lengths and saturations, egg lecithin, soy lecithin, hydrogenated egg, and soy lecithin, which are added to compositions for similar reasons as those for polymers. Examples of minerals include talc, magnesium oxide, zinc oxide, and titanium dioxide, which are added to compositions to obtain advantages such as reduced liquid accumulation or favorable pigment properties.

[0365] Bispecific polypeptides and / or PD-1 inhibitors can be formulated into any type of pharmaceutical composition known in the art to be suitable for their delivery. PD-1 inhibitors are formulated for parenteral administration.

[0366] In one embodiment, the pharmaceutical composition of the present invention may be in the form of liposomes, in which a bispecific polypeptide and / or PD-1 inhibitor is combined with an amphiphilic agent, such as a lipid, which exists in aggregate form as a micelle, an insoluble monolayer, or a liquid crystal, in addition to other pharmaceutically acceptable carriers. Suitable lipids for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponins, and bile acids. Also suitable lipids include the above lipids modified with poly(ethylene glycol) at a polar head group to extend blood flow circulation time. Preparation of such liposomal formulations can be found, for example, in US4,235,871, the disclosure of which is incorporated herein by reference.

[0367] The pharmaceutical compositions of the present invention may also be in the form of biodegradable microparticles. Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA), or poly(caprolactone) (PCL), as well as polyanhydrides, are widely used as biodegradable polymers in the production of microparticles. Preparations of such microparticles can be found in US5,851,451 and EP0213303, their disclosures incorporated herein by reference.

[0368] In further embodiments, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, and polymers such as starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, ethyl hydroxyethylcellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polyvinylimidazole, polysulfonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate of different degrees of hydrolysis, and polyvinylpyrrolidone are used to thicken the solution containing the drug. The polymers may also include gelatin or collagen.

[0369] Alternatively, polypeptides can simply be dissolved in physiological saline, water, polyethylene glycol, propylene glycol, ethanol or oil (e.g., safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and / or various buffers.

[0370] It should be understood that the pharmaceutical compositions of the present invention may include ions and a defined pH for enhancing the action of the active polypeptide. In addition, the compositions may be subjected to conventional pharmaceutical operations such as sterilization and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, and fillers.

[0371] The pharmaceutical compositions according to the present invention can be administered by any preferred route known to those skilled in the art. Therefore, possible routes of administration include parenteral (intravenous, subcutaneous, intratumoral, and intramuscular), topical, ocular, nasal, lung, cheek, oral, parenteral, vaginal, and rectal. Administration via implant is also possible. Preferably, the route of administration is parenteral. Most preferably, the route of administration is intravenous, subcutaneous, or intratumoral.

[0372] In a preferred embodiment, the pharmaceutical composition is administered parenterally, for example, intravenously, intrarebroventricularly, intraarticularly, intraarterially, intraperitoneally, intraarachnoidally, intraventricularly, intrathoracically, intracranially, intramuscularly, or subcutaneously, or they may be administered by infusion techniques. For example, it is conveniently used in the form of a sterile aqueous solution that can contain sufficient salts or other substances such as glucose to be isotonic with blood. The aqueous solution should be preferably buffered as needed (preferably to a pH of 3 to 9). Preparation of a suitable parenteral formulation under sterile conditions is readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0373] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Formulations may be supplied in unit or multi-dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from the aforementioned types of sterile powders, granules, and tablets.

[0374] Therefore, the pharmaceutical composition of the present invention is particularly suitable for parenteral administration, such as intravenous administration.

[0375] Alternatively, the pharmaceutical composition may be administered intranasally or by inhalation (e.g., in the form of an aerosol spray) from a pressurized container, pump, spray, or nebulizer using a suitable propellant, for example, by hydrofluoroalkanes such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane (HFA134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA227EA3)), carbon dioxide, or other suitable gases. In the case of a pressurized aerosol, the dose unit may be determined by providing a valve for delivering a measured amount. The pressurized container, pump, spray, or nebulizer may contain a solution or suspension of the active polypeptide, for example, using a mixture of ethanol and a propellant as a solvent, and may additionally contain a lubricant, for example, sorbitan trioleate. Capsules and cartridges used in inhalers or air inhalers (for example, made from gelatin) can be formulated to contain a powder mixture of the compounds of the present invention and a suitable powder base such as lactose or starch.

[0376] The pharmaceutical composition will be administered to the patient in a pharmaceutically effective dose. As used herein, “therapeutic effective dose,” “effective dose,” or “therapeutably effective” refers to the amount that provides a therapeutic effect for a given condition and administration regimen. This is a predetermined amount of active material calculated to produce the desired therapeutic effect in relation to the necessary additives and diluents, i.e., the carrier or administration vehicle. Furthermore, it is intended to mean an amount sufficient to mitigate, and most preferably prevent, clinically significant deficits in the host’s activity, function, and response. Alternatively, a therapeutic effective dose is an amount sufficient to produce an improvement in a clinically important condition in the host. As will be understood by those skilled in the art, the amount of a compound may vary depending on its particular activity. A suitable dose may contain a predetermined amount of the active composition calculated to produce the desired therapeutic effect in relation to the necessary diluents. In the method of producing and using the compositions of the present invention, a therapeutic effective dose of the active ingredient is provided. The therapeutic effective dose can be determined by a normally skilled medical or veterinary professional based on patient characteristics such as age, weight, sex, condition, complications, and other diseases, as is well known in the art. The administration of a pharmaceutically effective dose can be carried out both as a single dose in the form of individual dose units or several smaller dose units, and as multiple doses of subdivided doses at specific intervals. Alternatively, the dose may be provided as a series of infusions over a long period.

[0377] Particularly preferred compositions are formulated for systemic administration.

[0378] The composition may preferably be formulated for sustained release over a period of time. Therefore, the composition may be provided in or as part of a matrix that facilitates sustained release. Preferred sustained-release matrices may include montanide or γ-polyglutamic acid (PGA) nanoparticles.

[0379] Bispecific polypeptides can be formulated at various concentrations depending on the potency / toxicity of the polypeptide used. For example, a formulation may contain the active polypeptide at concentrations of 0.1 μM to 1 mM, more preferably 1 μM to 500 μM, 500 μM to 1 mM, 300 μM to 700 μM, 1 μM to 100 μM, 100 μM to 200 μM, 200 μM to 300 μM, 300 μM to 400 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 800 μM to 900 μM, or 900 μM to 1 mM. Typically, a formulation contains the active polypeptide at a concentration of 300 μM to 700 μM.

[0380] Typically, therapeutic doses of bispecific polypeptides (with or without the therapeutic portion) in human patients range from 100 μg to 700 mg per dose (based on a body weight of 70 kg). For example, the maximum therapeutic dose may range from 0.1 to 20 mg / kg per dose, e.g., 0.1 to 5 mg / kg or 1 to 5 mg / kg or 0.1 to 2 mg / kg. Such doses may be administered at different intervals, as determined by the oncologist / physician; for example, doses may be administered daily, twice a week, weekly, every other week, or monthly.

[0381] For example, the combination therapy or pharmaceutical composition of the present invention may be administered in combination with a further immunotherapeutic agent that conjugates to a target selected from the group consisting of VGFR, EGFR, HER2, CTLA-4, CD137, OX40, GITR, LAG3, TIM3, CD27, VISTA, and KIR.

[0382] Accordingly, the present invention encompasses combination therapies or pharmaceutical compositions comprising the bispecific polypeptide and PD-1 inhibitor of the present invention together with further immunotherapeutic agents that specifically bind to immune checkpoint molecules and are effective in the treatment of cancer and / or tumors. It will be understood that the therapeutic benefits of the further immunotherapeutic agents may be mediated by attenuating the function of inhibitory immune checkpoint molecules and / or activating the function of stimulating immune checkpoints or co-stimulatory molecules.

[0383] In one embodiment, further immunotherapeutic agents are (a) Immunotherapy agents that inhibit the function of CTLA-4, (b) Immunotherapy agents that activate the function of CD137, (c) Immunotherapy agents that bind to and activate the function of OX40, (d) Immunotherapy agents that inhibit the function of LAG3, (e) Immunotherapy agents that inhibit the function of TIM3, (f) Immunotherapy agents that inhibit the function of VISTA, (g) Immunotherapy agents that inhibit the function of VGFR, (h) Immunotherapy agents that inhibit EGFR function, and (i) selected from the group consisting of immunotherapies that inhibit the function of HER2.

[0384] In another embodiment, further immunotherapeutic agents include CTLA-4 inhibitors such as anti-CTLA-4 antibodies or their antigen-binding moieties.

[0385] In further embodiments, additional immunotherapeutic agents activate CD137, such as an agonist anti-CD137 antibody or its antigen-binding moiety.

[0386] In further embodiments, additional immunotherapeutic agents activate OX40, such as an agonist anti-OX40 antibody or its antigen-binding moiety.

[0387] In further embodiments, additional immunotherapeutic agents inhibit the function of LAG3, TIM3, or VISTA (Lines et al. 2014).

[0388] In another embodiment, further immunotherapeutic agents are VGFR inhibitors, such as anti-VGFR antibodies or their antigen-binding moieties.

[0389] In further embodiments, the additional immunotherapeutic agent activates EGFR, such as an agonist anti-EGFR antibody or its antigen-binding moiety.

[0390] In further embodiments, the additional immunotherapeutic agent activates HER2, such as an agonist anti-HER2 antibody or its antigen-binding moiety.

[0391] Those skilled in the art will understand that the presence of additional immunotherapeutic agents (detailed above) may provide synergistic benefits in the treatment of tumors in the subject. "Synergistic" means that the therapeutic effect of the combination of agents (determined, for example, by reference to tumor growth rate or size) is greater than the additive therapeutic effect of the individual agents administered alone. Such synergies can be identified by testing the activators individually and in combination in relevant cell line models of solid tumors.

[0392] Medical use and methods The combination therapies and pharmaceutical compositions according to the present invention may be used in treatment or prevention. In therapeutic use, the polypeptide or composition is administered to a subject already suffering from a disorder or condition in an amount sufficient to cure, alleviate, or partially suppress one or more of the condition or its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of asymptomatic periods. An amount sufficient to achieve this is defined as the “therapeutic effective dose.” In preventive use, the polypeptide or composition is administered to a subject who has not yet shown symptoms of the disorder or condition in an amount sufficient to prevent or delay the onset of symptoms. Such an amount is defined as the “preventive effective dose.” A subject may be identified as being at risk of developing the disease or condition by any preferred means.

[0393] A fourth aspect of the present invention provides a combination therapy or pharmaceutical composition according to the first or second aspect of the present invention for use in pharmaceuticals.

[0394] Further embodiments provide combination therapies or pharmaceutical compositions according to the first or second embodiment for use in treating cancer and / or tumors. Cancer and / or tumors may be referred to as neobiotic disorders.

[0395] In one embodiment, the combination therapy or pharmaceutical composition is intended for use in combination with one or more additional immunotherapeutic agents.

[0396] In one embodiment, one or more additional therapeutic agents are immunotherapeutic agents that bind to a target selected from the group consisting of VGFR, EGFR, HER2, CTLA-4, CD137, OX40, GITR, LAG3, TIM3, CD27, VISTA, and KIR.

[0397] In one embodiment, the bispecific polypeptide is intended for parenteral or systemic administration.

[0398] As described above, a fifth aspect of the present invention provides a method for treating cancer and / or tumors in a subject, comprising: (a) administering to the subject an effective amount of a bispecific polypeptide comprising a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA); and (b) administering to the subject an effective amount of a PD-1 inhibitor, wherein the PD-1 inhibitor is administered parenterally.

[0399] Bispecific polypeptides and / or PD-1 inhibitors may be as described in relation to the prior embodiments of the present invention.

[0400] In one embodiment, the bispecific polypeptide and the PD-1 inhibitor are administered simultaneously or within 24 hours of each other.

[0401] In one embodiment, the bispecific polypeptide and / or PD-1 inhibitor is administered parenterally.

[0402] In one embodiment, administration is intravenous, subcutaneous, or intratumor.

[0403] In one embodiment, the dose of the PD-1 inhibitor is: (i) The patient's body weight is approximately 1 to 100 mg / kg, and the dose of the PD-1 inhibitor is optional, either 1 mg / kg, 10 mg / kg, 20 mg / kg, or 100 mg / kg, and / or (ii) The dosage is approximately 2 mg / ml to 200 mg / ml, and the dosage is optional, being 2 mg / ml, 10 mg / ml, 25 mg / ml, 150 mg / ml, or 200 mg / ml. PD-1 inhibitors are administered as a single dose or in divided doses, on an optional basis.

[0404] In one embodiment, the method includes administering a bispecific polypeptide systemically.

[0405] In one embodiment, the method further includes administering one or more additional therapeutic agents to the subject.

[0406] "Treatment" includes both therapeutic and prophylactic treatment of a patient. The term "prophylactic" is used to encompass the use of any of the agents or formulations described herein that either prevent or reduce the potential for neoplasms in a patient or subject, or the spread, dissemination, or metastasis of cancer cells. The term "prophylactic" also encompasses the use of any of the agents or formulations described herein to prevent the recurrence of neoplasms in patients who have been previously treated for cancer and / or tumors.

[0407] Preferably, cancer and / or tumor is cancer and / or tumor associated with CEA, for example, CEA expression. "Associated with CEA" includes CEA being cancer and / or the tumor being caused by CEA and / or CEA being a marker of cancer and / or tumor.

[0408] In one embodiment, cancer and / or tumors include target cells that express CEA.

[0409] Preferably, CEA expression is at an intermediate level or a high level.

[0410] In one embodiment, intermediate levels of CEA expression are approximately 10,000 or more CEA receptors per target cell, for example, approximately 11,000 or more, approximately 12,000 or more, approximately 13,000 or more, approximately 14,000 or more, approximately 15,000 or more, approximately 16,000 or more, approximately 17,000 or more, approximately 18,000 or more, approximately 19,000 or more, approximately 20,000 or more, approximately 2 The target cells are characterized by expressing 5,000 or more, approximately 30,000 or more, approximately 35,000 or more, approximately 40,000 or more, approximately 50,000 or more, approximately 60,000 or more, approximately 70,000 or more, approximately 80,000 or more, approximately 90,000 or more, approximately 100,000 or more, approximately 125,000 or more, approximately 150,000 or more, or approximately 175,000 or more CEA receptors. In another embodiment, intermediate levels of CEA expression are characterized by target cells expressing approximately 10,000 to 200,000 CEA receptors per target cell, for example, approximately 20,000 to 175,000 CEA receptors per target cell, or approximately 20,000 to 200,000 CEA receptors per target cell, or approximately 50,000 to 175,000 CEA receptors per target cell, or approximately 50,000 to 200,000 CEA receptors per target cell. Preferably, the CEA receptor is the CEACAM5 receptor.

[0411] In one embodiment, high levels of CEA expression are defined as approximately 200,000 or more CEA receptors per target cell, for example, approximately 225,000 or more, approximately 250,000 or more, approximately 275,000 or more, approximately 300,000 or more, approximately 325,000 or more, approximately 350,000 or more, approximately 375,000 or more, approximately 400,000 or more, approximately 425,0 The target cells are characterized by expressing 00 or more, approximately 450,000 or more, approximately 475,000 or more, approximately 500,000 or more, approximately 600,000 or more, approximately 700,000 or more, approximately 800,000 or more, approximately 900,000 or more, or approximately 1,000,000 CEA receptors, preferably more than approximately 300,000 CEA receptors per target cell. In another embodiment, high levels of CEA expression are characterized by target cells expressing approximately 200,000 to approximately 1,000,000 CEA receptors per target cell, for example, approximately 200,000 to approximately 500,000 CEA receptors per target cell, or approximately 300,000 to approximately 500,000 CEA receptors per target cell. Preferably, the CEA receptor is the CEACAM5 receptor.

[0412] In one embodiment, cancer and / or tumors do not include cells that have no CEA expression or have low levels of CEA expression. In one embodiment, low levels of CEA expression are characterized by cells expressing about 10,000 or fewer CEA receptors per target cell, for example, about 9,000 or fewer, about 8,000 or fewer, about 7,000 or fewer, about 6,000 or fewer, about 5,000 or fewer, about 4,000 or fewer, about 3,000 or fewer, about 2,000 or fewer, or about 1,000 or fewer CEA receptors per cell.

[0413] In one embodiment, the CEA is tumor-related CEA.

[0414] Preferably, CEA is CEACAM.

[0415] In one embodiment, CEACAM is one or more selected from the enumeration consisting of CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21. The aforementioned references to CEACAM molecules will be understood to include splice variants.

[0416] Preferably, CEACAM is one or more selected from the enumeration consisting of CEACAM1, CEACAM5, and CEACAM6. Preferably, CEACAM is CEACAM1. Most preferably, CEACAM is CEACAM5.

[0417] In a preferred embodiment, B2 can specifically bind to CEACAM5 but cannot bind to other CEACAMs, particularly CEACAM1.

[0418] In one embodiment, cancer and / or tumor is one or more cancers and / or tumors selected from the list consisting of prostate cancer and / or prostate tumors, breast cancer and / or breast tumors, lung cancer and / or lung tumors, colorectal cancer and / or colorectal tumors, melanoma, bladder cancer and / or bladder tumors, brain / CNS cancer and / or brain / CNS tumors, cervical cancer and / or cervical tumors, esophageal cancer and / or esophageal tumors, stomach cancer and / or stomach tumors, head and neck cancer and / or head and neck tumors, kidney cancer and / or kidney tumors, liver cancer and / or liver tumors, carcinoma, leukemia, lymphoma, ovarian cancer and / or ovarian tumors, pancreatic cancer and / or pancreatic tumors, tonsil cancer and / or tonsil tumors, and sarcoma. Preferably, carcinoma.

[0419] Preferably, one or more cancers and / or tumors are selected from a list consisting of breast cancer and / or breast tumors, lung cancer and / or lung tumors, colorectal cancer and / or colorectal tumors, stomach cancer and / or stomach tumors, and / or pancreatic cancer and / or pancreatic tumors.

[0420] In a preferred embodiment, the cancer and / or tumor is colorectal cancer and / or colorectal tumor. In a preferred embodiment, the cancer is gastric cancer and / or gastric tumor.

[0421] In a preferred embodiment, the cancer and / or tumor is tonsil cancer and / or tonsil tumor.

[0422] Preferably, the carcinoma is one or more carcinomas selected from the list consisting of gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, bile duct cancer, and medullary thyroid carcinoma.

[0423] In a preferred embodiment, the cancerous tumor is colorectal cancer.

[0424] Preferably, the tumor is a solid tumor.

[0425] In one embodiment, the non-cancerous state is a non-cancerous state that is associated with CEA, for example, CEA expression. "Associated with CEA" includes the fact that CEA is a non-cancerous state caused by CEA and / or that CEA is a marker of the non-cancerous state.

[0426] In one embodiment, the non-cancerous state includes target cells that express CEA.

[0427] Preferably, CEA expression is at an intermediate level or a high level, as discussed herein.

[0428] Preferably, one or more non-cancerous conditions are selected from the list consisting of ulcerative colitis, pancreatitis, cirrhosis, COPD, Crohn's disease, and / or hypothyroidism.

[0429] In one embodiment, the subject is a human being.

[0430] Optimized RUBY (trademark) format The bispecific polypeptide may include an optimized RUBY® format, and preferably, the bispecific polypeptide has specificity for a first antigen and a second antigen.

[0431] In particular, bispecific polypeptides are (a) an immunoglobulin molecule having specificity for a first antigen, comprising a first heavy chain polypeptide and a first light chain polypeptide, and (b) A Fab fragment comprising at least one Fab fragment having specificity for a second antigen, comprising a second heavy chain polypeptide and a second light chain polypeptide, The second light chain polypeptide is fused to the C-terminus of the first heavy chain polypeptide. The bispecific antibody contains one or more mutations considered in relation to the optimized RUBY® format, which facilitate the association of polypeptides, in particular, the association of a first heavy chain polypeptide with a first light chain polypeptide and / or the association of a second heavy chain polypeptide with a second light chain polypeptide.

[0432] The optimized RUBY® format has the structure shown in Figure 23, which has further optimized mutations compared to the RUBY® format. As will be understood by those skilled in the art, the technology relating to the antibody format has broad applicability to a wide range of different target antigens.

[0433] While bispecific polypeptides in the "RUBY™ format" can be produced with excellent purity and reproducibility, bispecific polypeptides in the "Optimized RUBY™ format" can be produced with even higher purity and reproducibility. Furthermore, bispecific polypeptides in the "Optimized RUBY™ format" are engineered to have a reduced risk of inducing immunogenic responses directed towards the bispecific polypeptide itself.

[0434] In one embodiment, the bispecific polypeptide comprises an immunoglobulin arranged as an antibody having two arms, thus two binding sites for a first antigen, and two of the Fab fragments, each providing a binding site for a second antigen. Thus, there are two binding sites for the first antigen and two binding sites for the second antigen. The first antigen and / or the second antigen are not CD40 and / or CEA. In a more preferred embodiment, the first antigen and / or the second antigen are proteins and / or peptides that are not CD40 and / or CEA.

[0435] In one embodiment, one or more Fab fragments are linked to the C-terminus of an immunoglobulin via a linker.

[0436] In one embodiment, the bispecific polypeptide is tetravalent and can bind to each of the two antigens in a divalent state.

[0437] The optimized mutations are described below as including “Optimized Mutation Set 1” and “Optimized Mutation Set 2” – “Set 2a” and / or “Set 2b”. Those skilled in the art will understand that various combinations of these optimized mutations can be used in bispecific polypeptides and in combination with any of the “RUBY™ format” mutations described above. It will also be understood that variations of these mutations described herein are functional. All mutations within variable domains (VH or VL) are numbered according to the IMGT numbering system, and all mutations within constant domains are numbered according to the EU numbering system.

[0438] Mutation Set 1 - Mutations in Variable Domain Weight (VH): T65E, T65A, T65I.

[0439] Mutation Set 2 - Any individual and / or any combination of mutations listed in Sets 2a and 2b. Set 2a - Mutations in CH1: Y180A, Y180G, Y180I, Y180N, Y180S, Y180T, Y180V, or Y180W, and / or S183N or S183T, and / or V188G, preferably Y180T. Set 2b - Mutations in the C kappa domain: A111R, A111T, A111W, or A111V, and / or T109P, preferably T109P and / or A111V, and / or mutations in the variable domain light (VL): I126A, I126G, I126H, I126N, I126P, I126Q, I126S, or I126T.

[0440] In one embodiment, the mutation is (a) T65 position in VH (according to the IMGT numbering system), and / or (b) One or more of the following positions in CH1: Y180, S183, and V188, preferably Y180 (according to the EU numbering system), and / or (c) C. The following locations in the Kappa Domain: one or more of A111 and T109 (according to the EU or Kabat numbering system), and / or (d) It is located in a position selected from the group consisting of I126th position in the VL (according to the IMGT numbering system).

[0441] In certain embodiments, the mutation is located at position T65 in the variable domain weight (VH) (according to the IMGT numbering system).

[0442] In certain embodiments, the mutation is one or more of the following positions in CH1: Y180, S183, and V188, preferably Y180 (according to the EU numbering system).

[0443] In certain embodiments, the mutation is one or more of the following positions in the C kappa domain: A111 and T109 (according to the EU or Kabat numbering system), and / or position I126 in the VL (according to the IMGT numbering system).

[0444] In one embodiment, the mutation is (a) X65E / A / I in VH (according to the IMGT numbering system), and / or (b) One or more of the following mutations in CH1: X180A / G / I / N / S / T / V / W, X183N / T, and X188G, preferably X180T (according to the EU numbering system), and / or (c) The following mutations in the C kappa domain: X111R / T / W / V and X109P, preferably one or more of X111V and X109P (according to the EU or Kabat numbering system), and / or (d) Selected from the group consisting of X126A / G / H / N / P / Q / S / T in VL (according to the IMGT numbering system). *X refers to any amino acid.

[0445] In certain embodiments, the mutation is X65E / A / I in the VH chain (according to the IMGT numbering system). *X refers to any amino acid.

[0446] In certain embodiments, the mutation is one or more of the following mutations in CH1: X180A / G / I / N / S / T / V / W, X183N / T, and X188G, preferably X180T (according to the EU numbering system). *X refers to any amino acid.

[0447] In certain embodiments, the mutation is one or more of the following mutations in the C kappa domain: X111R / T / W / V and X109P, preferably X111V and X109P (according to the IMGT numbering system), and / or the mutation is X126A / G / H / N / P / Q / S / T (according to the IMGT numbering system) in the VL. *X refers to any amino acid.

[0448] For example, mutations are (a) The following mutations in VH: one or more of T65E, T65A, and T65I (according to the IMGT numbering system), and / or (b) One or more of the following mutations in CH1: Y180A, Y180G, Y180I, Y180N, Y180S, Y180T, Y180V, Y180W, S183N, S183T, V188G, preferably Y180T (according to the EU numbering system), and / or (c) The following mutations in the C kappa domain: A111R, A111T, A111W, A111V, and T109P, preferably one or more of T109P and A111V (according to the EU numbering system), and / or (d) The following mutations in the VL may be selected from the group consisting of one or more of I126A, I126G, I126H, I126N, I126P, I126Q, I126S, and I126T (according to the IMGT numbering system).

[0449] In certain cases, the mutation is one or more of the following mutations in VH: T65E, T65A, and T65I (according to the IMGT numbering system).

[0450] In certain cases, the mutation is one or more of the following mutations in CH1: Y180A, Y180G, Y180I, Y180N, Y180S, Y180T, Y180V, Y180W, S183N, S183T, V188G, preferably Y180T (according to the EU numbering system).

[0451] In certain cases, the mutations are one or more of the following mutations in the C-kappa domain: A111R, A111T, A111W, A111V, and T109P, preferably T109P and A111V (according to the EU or Kabat numbering system), and / or one or more of the following mutations in the VL: I126A, I126G, I126H, I126N, I126P, I126Q, I126S, and I126T (according to the IMGT numbering system).

[0452] As discussed above, any combination of the "RUBY(trademark) format" mutation and the "optimized RUBY(trademark) format" mutation can be used with the same bispecific antibody, such as one or more of the following "RUBY(trademark) format" mutations in (a) to (d), or variations described herein, and can be combined with one or more of the following "optimized RUBY(trademark) format" mutations in (e) to (g), or variations described herein: (a) One or more of the following mutations in the CH1 domain: H168A, F170G, and / or T187E (according to the EU numbering system) (b) One or more of the following mutations in the C kappa domain: L135Y, S176W, S114A, and / or N137K (according to the EU or Kabat numbering system), and / or one or more of the following mutations in the C lambda domain: L135Y, S176W, T114A, and / or S137K (according to the Kabat numbering system), (c)Variant mutations in VL: Q44R or Q44E (according to the IMGT numbering system), ad (d) Variations in VH: Q44E or Q44R (according to the IMGT numbering system), (e) Variations in VH: T65E, T65A, or T65I (according to the IMGT numbering system), (f) Mutations in CH1: Y180T (according to the EU numbering system), and / or (g) Variations in C. kappa: T109P and / or A111V (according to the EU numbering system).

[0453] Therefore, in certain embodiments, a bispecific antibody having a combined "RUBY(trademark) format" mutation and an "optimized RUBY(trademark) format" mutation is defined as having the following mutations: ● One or more of the following mutations in the CH1 domain: H168A, F170G, Y180T, and / or T187E (according to the EU numbering system) ●One or more of the following mutations in the C kappa domain: T109P, A111V, L135Y, S176W, S114A, and / or N137K (according to the EU or Kabat numbering system), and / or one or more of the following mutations in the C lambda domain: L135Y, S176W, T114A, and / or S137K (according to the Kabat numbering system), ●Vulnerabilities in VL: Q44R or Q44E (according to the IMGT numbering system), and / or ●The following mutations in VH may include: Q44E or Q44R, and / or one or more of T65E, T65A, or T65I (according to the IMGT numbering system).

[0454] Any listing or discussion of previously published literature in this specification should not necessarily be construed as an endorsement that such literature is part of cutting-edge technology or common general knowledge.

[0455] When the words “a” or “an” are used in the claims and / or specification with the term “including,” they may mean “one,” but may also be synonymous with “one or more,” “at least one,” and “one or more than one.”

[0456] These and other embodiments of the present invention will be better recognized and understood when considered in conjunction with the above description and accompanying drawings. However, it should be understood that the above description, while illustrating various embodiments of the present invention and numerous specific details thereof, is provided for illustrative purposes only and is not limiting. Many substitutions, modifications, additions, and / or reorganizations can be made within the scope of the present invention without departing from the spirit of the invention, and the present invention includes all such substitutions, modifications, additions, and / or reorganizations.

[0457] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

[0458] Preferred non-limiting embodiments that embody specific aspects of the present invention will be described with reference to the following drawings. [Brief explanation of the drawing]

[0459] [Figure 1] ELISA analysis of 22 CEA antibodies isolated using phages displayed in combination with next-generation sequencing. IgG1-format conjugates were analyzed for binding to human CEACAM5 (abbreviated as CAM5) (slash bar) or human CEACAM1 (abbreviated as CAM1) (back bar). [Figure 2] The ability of CD40 and CEACAM5-targeted RUBY® bsAb to simultaneously bind to both antigens, as measured by dual-target ELISA. Panel A shows the binding curves for Multi34, Multi35, Multi37, and Multi38. Panel B shows the binding curves for Multi39, Multi40, Multi41, and Multi42, and Panel C shows the binding curves for Multi44, Multi45, Multi46, and Multi47. Finally, Panel D shows the binding curves for Multi48, Multi4, and AC_05339. [Figure 3] Cross-reactivity of RUBY™ bsAb (Multi34, Multi35, Multi37-Multi42, Multi44-Multi49) with CEA protein family members CEACAM1, 5, 6, and 8, as evaluated by ELISA. [Figure 4] Reaction kinetics in Octet of the interaction between captured CD40 CEACAM5-targeted bispecific antibodies (Multi34, Multi42, Multi46, and AC_5339) and soluble monomeric human CEACAM5 (various concentrations ranging from 100 to 1.6 nM). Association was measured for 100 seconds, followed by dissociation in 1× rate buffer for 100 seconds. [Figure 5] Reaction kinetics in Octet of bispecific antibodies in solution (various concentrations ranging from 50 to 0.8 nM) interacting with captured human CEACAM5-biotin. Association was measured for 100 seconds, followed by dissociation in reaction rate buffer for 100 seconds. [Figure 6] Binding of CD40-CEACAM5 bispecific antibodies to CEACAM5-transfected CHO cells. Binding of CD40-CEA bispecific antibodies was detected by flow cytometry using fluorochrome-conjugated anti-human IgG. Panel A shows binding curves for Multi34 and Multi35. Panel B shows binding curves for Multi41, Multi42, Multi44, and ffAC_5339. Panel C shows binding curves for Multi46, Multi47, Multi48, and Multi49. Panel D shows binding curves for ffAC_5337 and AC_5339. Panel E shows binding curves for AC_05355 and AC_05339. [Figure 7] Binding of CD40-CEACAM5 bispecific antibody to CEACAM1-transfected CHO cells (A-D) and CHO wt cells (F). Binding of CD40-CEACAM5 bispecific antibody was detected by flow cytometry using fluorochrome-conjugated anti-human IgG. [Figure 8]Binding of CD40-CEACAM5 bispecific antibodies to CEACAM5-expressing tumor cells. Tumor cell lines MKN45 (A-C) expressing high levels of CEACAM5, LS174T (D-E) expressing intermediate levels of CEACAM5, and Lovo (G-J) expressing low levels of CEACAM5. Binding of CD40-CEACAM5 bispecific antibodies was detected by flow cytometry using fluorochrome-conjugated anti-human IgG. [Figure 9] Effect of a CD40-CEACAM5 bispecific antibody on cultured CD40 reporter cells using a dose-controlled antibody, in the presence or absence of CEACAM5 expressed in CHO cells. Responses were calculated as induction factors relative to background. [Figure 10] Effect of CD40-CEACAM5 bispecific antibodies on CD40 reporter cells when co-cultured with tumor cells having different CEACAM5 receptor densities. CD40 clone G12 containing CD40-CEACAM5 (A-D), CD40 clone G12 containing CEACAM5-CD40 (E-H), and CD40 clone 1132 containing CD40-CEACAM5 (I-J). Response was calculated as an induction factor relative to the background. [Figure 11] Effect of a CD40-CEACAM5 bispecific antibody on CD40 reporter cells co-cultured with CEACAM5-expressing CHO cells and dose-set antibodies in the presence or absence of soluble CEACAM5. Responses were calculated as induction factors relative to background. [Figure 12] Effect of CD40-CEACAM5 bispecific antibody on B cell activation. Primary human B cells were cultured with dose-controlled antibodies in the presence or absence of CEACAM5 expressed in CHO cells. After 2 days, CD86 expression in B cells was analyzed by FACS. [Figure 13]Effect of the CD40-cCEACAM5 bispecific antibody AC_05355 on B cell activation in the presence of human or cynomolgus monkey CEACAM5 transfected cells. Primary human B cells were cultured with dose-controlled antibody in the presence of human or cynomolgus monkey CEACAM5 expressed in CHO cells. After 2 days, CD86 expression in B cells was analyzed by FACS. The graph shows results pooled from 6 donors. [Figure 14] HEK Blue CD40L™ reporter cells were activated in the presence of Raji cells after incubation in the Transwell system for 20 hours with BsAb AC_05339 (0.02 nM) that does not crosslink with CHO-CEACAM5 or CHO-wt cells. Activation was monitored as SEAP release into culture medium, measured using QuantiBlue™. Mean + SD of triple measurements. [Figure 15] BsAb Multi34 (3-0.004 nM, single sample) cross-linked to CHO-CEACAM5 cells activates HEK Blue CD40L™ reporter cells, which do not have Raji sync cells in the Transwell system. Activation was monitored as the release of SEAP into the culture medium after 20 hours of culture, measured using QuantiBlue™. [Figure 16] BsAb Multi42 (3-0.004 nM, single sample) cross-linked to CHO-CEACAM5 cells activates HEK Blue CD40L™ reporter cells, which do not have Raji sync cells in the Transwell system. Activation was monitored as the release of SEAP into the culture medium after 20 hours of culture, measured using QuantiBlue™. [Figure 17] BsAb Multi46 (100~0.1nM, single sample) cross-linked to CHO-CEACAM5 cells activates HEK Blue CD40L™ reporter cells, which do not have Raji sync cells in the Transwell system. Activation was monitored as the release of SEAP into the culture medium after 20 hours of culture, measured using QuantiBlue™. [Figure 18] Co-localization of tumor debris and Raji cells. Raji cells were incubated with CEACAM5 expressing MKN45 tumor debris and control antibody 1132 (referred to herein as 1132.m2) having AC_05339 or silenced Fc, more specifically IgG1 isotype and L234A and L235A mutations. Images were captured with a Cytation5 live imaging system, and the number of tumor debris co-localized with Raji cells was analyzed after 4 hours using Gen5 software. [Figure 19] MC38-CEACAM5 tumor growth and MC38-wt reloading. hCD40tg mice inoculated with MC38-CEACAM5 tumors were administered the prescribed treatments on days 7, 10, and 13 post-inoculation. Tumors were frequently measured until the first mouse in either treatment group reached a tumor volume exceeding ethical limits. Statistical analysis of tumor volume was performed on day 38 using the Mann-Whitney test (n=10, *p<0.05). Naive control hCD40tg mice or mice cured of MC38-CEACAM5 tumors by treatment with CD40-CEACAM5 bsAb (complete responders) were inoculated (reloaded) with MC38-wt tumors. Tumors were frequently measured until the first mouse in either treatment group reached a tumor volume exceeding ethical limits. [Figure 20] Effect of CD40-CEACAM5 bispecific antibody AC_05355 on B cell activation in the presence of cynomolgus monkey CEACAM5 (cCEACAM5) transfected CHO cells. Primary cynomolgus monkey B cells were cultured with dose-controlled antibody in the presence of cCEACAM5 expressed in CHO cells. After 2 days, CD86 expression in B cells was analyzed by FACS. The graph shows pooled data from two donors. [Figure 21]MC38-CEACAM5 tumor growth. hCD40tg mice inoculated with MC38-CEACAM5 tumors were administered the prescribed treatment on days 10, 13, and 16 post-inoculation. Tumors were frequently measured until the first mouse in either treatment group reached a tumor volume exceeding ethical limits. Statistical analysis of tumor volume was performed on day 17 using the Mann-Whitney test (n=10, *p<0.05). [Figure 22] This shows a schematic diagram of the structure of an exemplary format of a bispecific antibody. In each format, the constant region is shown as a solid gray, the variable heavy chain region VH1 as a black and white grid, the variable light chain region VL1 as an open white, the variable heavy chain region VH2 as a solid black, and the variable light chain region VL2 as a white background with diagonal lines. The CD40 binding domain (binding domain 1) is typically represented as a pair of a black and white grid domain and a solid white domain (VH1 / VL1), and the CEA binding domain (binding domain 2) is typically represented as a pair of a solid black domain and a white domain with diagonal lines (VH2 / VL2). However, it should be understood that in all the formats shown, binding domains 1 and 2 can be interchanged. That is, the CD40 binding domain can occur in the position shown for the CEA binding domain in this diagram, and vice versa. [Figure 23] This shows an exemplary configuration of a bispecific antibody construct in the RUBY® format. The bispecific antibody in Figure 21 is assembled from three types of polypeptide chains: (1) an IgG heavy chain (white) fused to a Fab light chain (lattice) via a polypeptide linker; (2) an IgG light chain (brick-like); and (3) a Fab heavy chain (black). Mutations are introduced at the interface between the heavy and light chains. [Figure 24] CD40×TAA bsAb mediates the localization of tumor debris to antigen-presenting cells. The number of CEA+ tumor debris clusters with CD40+ cells was quantified using live cell imaging software 8 hours after culture. The graph shows the mean (+SD) of overlapping wells in one of four representative experiments (CEA). [Figure 25] Dissociated cells from human colorectal cancer tumors were analyzed for (left) their CEA expression (gated in whole-cells), (center) their ability to provide crosslinking to CD40×CEA Neo-X-Prime bsAb in a CD40 reporter assay, and (right) CD83 upregulation after stimulation of tumor-infiltrating immune cells (CD45+CD3-CD56-gated in whole-cells) using CD40×CEA Neo-X-Prime bsAb or isotype×CD40 bsAb (data from one of three representative experiments). [Figure 26] Co-binding of CD40 and CEA by CD40×CEA bsAb mediates the activation of tonsillar APCs in vitro. Human CD45+ HLA-DR+ CD3- cells from tonsillar biopsies were co-cultured with UV-irradiated CHO cells transfected with human CEA in the presence of CD40×CEA bsAb, CD40 mAb, or isotype control. After 13 hours of culture, cells were collected and the frequency of CD86+CD40+ cells was examined using flow cytometry of CD19+CD20+ B cells, CD14+ macrophages, CD1c+cDC2, and XCR1+cDC1. [Figure 27] This study describes the accumulation of CD40×CEA bsAb in CEA-expressing tumors, but not the corresponding accumulation of CD40 mAb. Human CD40 transgenic mice were sc-inoculated with MC38-hCEA tumor cells (MC-38-CEA-2, Kerafast), and on days 10 and 13, 100 μg of anti-CD40 antibody or molar equivalent dose (167 μg) of CD40×CEA bsAb or isotyped bsAb was administered via ip (intravenous injection). On day 14, the tumors were dissected. Frozen tumor sections were stained for human IgG to assess the accumulation of the administered antibody, and stained for CEA to assess the CEA expression pattern in the tumors. (A) Representative images of IgG staining and (B) representative images of CEA staining are shown. The staining pattern obtained after treatment with CD40×CEA was significantly stronger than that of the control, and the staining pattern was consistent with the CEA staining of the tumors. [Figure 28]Tumors cryopreserved from human CD40 transgenic mice (B16.F10-hCD40+#6, 7, and 9 used as controls, hereafter referred to as B16 and MB49#2, 4, and 5) were analyzed. 8 μm frozen sections were prepared and stained. Mouse spleens were used as a positive control. Sections were analyzed using a Leica DMRX-e microscope, and representative images were taken. A) Representative image from MB49 tumors. B) Immunohistochemical staining was determined as follows: negative (0), few positive cells (1+), moderate number of positive cells (2+), many positive cells (3+), or very many positive cells (4+). The analysis shows a significantly higher degree of invasive T cells in MB49 tumors compared to the B16 tumors used as controls. Antibodies used for staining: CD4: Rat IgG, Affymetrix, 14-0042 1:200, CD8: Rat IgG, Affymetrix, 14-0081 1:200, CD3: Rabbit, Dako, A0452 1:100, CD45: Rat IgG, Biodesign 1:100. C) Frequency of immune cell populations in MB49-EpCAM tumors with in vivo proliferation and immune infiltration similar to MB49 tumors. Human CD40 transgenic mice were sc-injected with MB49-hEpCAM cells (0.25 × 10⁶) into the right flank in 100 μl of PBS. Twelve days after tumor inoculation, the tumors were dissected, isolated, and stained for flow cytometry analysis of immune cell content. The frequencies of NK cells (CD45+, CD11b-, CD19-MHC II-, TCR beta-, NK1.1+), T cells (CD45+, CD11b-, CD19-MHC II-, TCR beta+, NK1.1-), B cells (CD45+, Ly6G-, CD3-, NK1.1-, CD19+), monocytes / macrophages (CD45+, Ly6G-, CD3-, NK1.1-, CD64+), and DCs (CD45+, Ly6G-, CD3-, NK1.1-, CD64-, CD11c+, MHC II+) were evaluated within the overall survivor CD45+ population. [Figure 29]Data from the NHP study. A) B cell activation of cynoCEA×CD40 RUBY® in cynomolgus monkey and human B cells in the presence of CEA-transfected cells (macaque CEA, NP_001040590.1). Primary cynomolgus monkey B cells were cultured with dose-set antibodies in the presence of CEA expressed in CHO cells. After 2 days, CD86 expression in B cells was analyzed by FACS. The graph shows pooled data from two cynomolgus monkeys and four human donors. The data show that CEA×CD40 bsAb in RUBY® format induces similar levels of upregulation of CD86 in cynomolgus monkey and human B cells. CEA-conditional activation of CD40 in cynomolgus monkey B cells and human B cells is similar to that observed with human CEA×CD40 bsAb in RUBY® used in the in vitro assay. The cynoCEA×CD40 bsAb binds to human and cynomolgus monkey CEA with similar affinity (hCEA vs. cCEA, right panel). In B) and C), key data from toxicological evaluations in cynomolgus monkeys are presented. The cynoCEA×CD40 bispecific antibody was administered to cynomolgus monkeys once weekly via intravenous infusion at two different dose levels (10 mg / kg and 37.5 mg / kg) for two weeks. One female and one male were evaluated at each dose level. B) Data on L-aspartate aminotransferase (ASAT) and L-alanine aminotransferase (ALAT), and C) Levels of IL-6 and TNF-α over time. In addition, plasma levels of the following cytokines: IL-2, IL-6, IL-8, IL-10, MCP-1, IFN-γ, and TNF-α were measured by a bead-based multiplex immunoassay. The conclusion from the study was that there were no findings associated with cyoCEA × CD40 bsAb at the evaluated dose levels. [Figure 30]Structure of RUBY® bsAb and its binding to various antigenic targets as measured by ELISA. (A) Chain 1 consists of an IgG heavy chain, a short polypeptide linker, and an additional Fab fragment light chain; Chain 2 is a light chain that binds to the VH and CH1 domains of the IgG moiety; and Chain 3 is a short heavy chain that binds to the light chain attached to IgG. (B) Dual ELISA showing the co-binding of CD40×EpCAM RUBY® bsAb to each antigenic target. ELISA plates were coated with human CD40, bsAb was added, and then detected using biotinylated EpCAM. (C) Dual ELISA showing the co-binding of CD40×CEA RUBY® bsAb to each antigenic target. ELISA plates were coated with human CEACAM5, bsAb was added, and then detected using biotinylated CD40. (D) MonoELISA showing the binding of GFP×EpCAM-controlled RUBY® bsAb to human EpCAM. ELISA plates were coated with human EpCAM, bsAb was added, and then detected using goat anti-human kappa light chain-HRP. (E) MonoELISA showing the binding of cynoCEA×CD40 RUBY® bsAb to human CD40. ELISA plates were coated with human CD40, cynoCEA×CD40 RUBY® bsAb was added, and then detected using goat anti-human kappa light chain-HRP. (F) MonoELISA showing the binding of cynoCEA×CD40 RUBY® bsAb to human CEACAM5. ELISA plates were coated with human CEA, cynoCEA×CD40 RUBY® bsAb was added, and then detected using goat anti-human kappa light chain-HRP. In summary, bispecific antibodies were well generated in the RUBY® format, and the generated bsAbs showed good binding to their respective antigen targets, as illustrated by ELISA binding evaluation. [Figure 31]MC38-CEACAM5 2 tumor growth and survival. hCD40tg mice inoculated with MC38-CEACAM5 2 tumors were administered the prescribed treatments on days 7, 10, and 13 post-inoculation. Tumors were measured frequently, and the graphs show the mean tumor volume (+SD) in each group until the first mouse in either treatment group reached a tumor volume exceeding ethical limits, and the survival percentage of mice in each treatment group. [Figure 32] Dissociated cells from human gastric cancer tumors were analyzed for (left) their CEA expression (gated in whole-cells), (center) their ability to provide crosslinking to CD40×CEA Neo-X-Prime bsAb (ffAC_05337) in a CD40 reporter assay, and (right, 1nM CD40×CEA) CD40×CEA Neo-X-Prime bsAb or isotype × CD40 bsAb, followed by CD83 upregulation after stimulation of tumor-infiltrating immune cells (CD45+CD3-CD56-gated in whole-cells) (data from one of four representative experiments). [Figure 33] Effect of the bispecific antibody ffAC_05337 on CD40 reporter cells when co-cultured with tumor cells having different CEA receptor densities in the presence and absence of soluble CEA. MKN45, high CEA expression cells (A), LS174T, moderate CEA expression cells (B), HT29, and LOVO, low CEA expression cells (C-D). Response was calculated as an induction factor relative to the background. [Figure 34] Effect of the bispecific antibody ffAC_05337 on CD40 reporter cells co-cultured with CEA-expressing CHO cells and dose-controlled antibody in the presence or absence of soluble CEA. Response was calculated as an induction factor relative to the background. [Figure 35] This shows T cell activation in a mixed lymphocyte reaction (MLR) assay using exhausted CD4 T cells, induced by the CD40 x CEA bispecific antibody (ffAC_05337) combined with the PD-1 inhibitor (nivolumab). The additive effects of individual monotherapies are shown by the dotted line. [Figure 36]This shows T cell activation in a mixed lymphocyte reaction (MLR) assay using exhausted CD8 T cells, induced by the CD40 x CEA bispecific antibody (ffAC_05337) combined with the PD-1 inhibitor (nivolumab). The additive effects of individual monotherapies are shown by dotted lines in Figures 36B and 36D. [Figure 37] This shows T cell activation in a mixed lymphocyte response (MLR) assay using exhausted CD4 or CD8 T cells, induced by a CD40 × CEA bispecific antibody (ffAC_05337) combined with a PD-L1 inhibitor (atezolizumab). The additive effects of individual monotherapies are shown by dotted lines in Figures 37B and C. [Figure 38] The effects of treatment with CD40×CEA bispecific antibody (ffAC_05337) on PD-1 and PD-L1 gene expression compared to a vehicle control are shown. Figure 38A shows the gene expression of CD274 (PD-L1) in selected immune cells (CD45+Ly6G-) after treatment. Figure 38B shows the gene expression of Pdcd1 (PD-1) in selected immune cells (CD45+Ly6G-) after treatment. Figure 38C shows the gene expression of CD274 (PD-L1) in the entire tumor after treatment. Figure 38D shows the gene expression of Pdcd1 (PD-1) in the entire tumor after treatment. [Figure 39] This graph shows the number of PD-L1-expressing tumor-infiltrating macrophages and dendritic cells per mg of tumor after treatment with CD40 × CEA bispecific antibody (ffAC_05337). Each dot represents one mouse, and the bars represent the average. [Figure 40][Figure 40A] Survival curves of MC38-CEACAM5-vaccinated F1 hCD40tg×C57BL / 6 mice treated with vehicle, CD40×CEA bispecific antibody (ffAC_05337), aPD-1, or a combination of CD40×CEA bispecific antibody (ffAC_05337) and aPD-1. N=9~10 / group. [Figure 40B] Mean tumor growth curves in mice treated with CD40×CEA bispecific antibody (ffAC_05337), aPD-1 mAb, or a combination of CD40×CEA bispecific antibody (ffAC_05337) and aPD-1 mAb. Each line represents the mean, and the bars represent SEM. Mice euthanized by the human endpoint are also included in the graph after the endpoint and plotted as the same size as on the day of euthanasia until there are no more mice remaining in the group. N=9~10 / group. [Figure 41] This study demonstrates the effects of 5-day stimulation of dissociated cells from human gastric cancer tumors with a CD40×CEA bispecific antibody (ffAC_05337) and / or anti-PD1 (nivolumab). Cells were analyzed by flow cytometry for CD25 upregulation on CD8+ T cells. The percentages shown are CD25+CD8 T cells (after subtracting untreated samples), mean ± SEM from two patient samples.

[0460] Sequence List [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 5-1] Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 5-14 Table 5-15 Table 5-16 Table 5-17 Table 5-18 [Table 5-19] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

[0461] Control antibody sequence 3174 VH aa (Sequence ID 422) EVQLLEQSGAELVRPGTSVKISKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSS 3174 VH nt (Sequence ID 423) GAGGTGCAGCTGCTGGAACAGTCTGGCGCCGAACTCGTTAGACCTGGCACAAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCACAAATTATTGGCTCGGCTGGGTCAAACAGAGGCCAGGACACGGACTGGAATGGATCGGCGATATCTTCCCCGGCAGCGGCAACATCCAC TACAACGAGAAGTTCAAGGGCAAAGCCACACTGACCGCCGACAAGAGCAGCAGCACAGCCTATATGCAGCTGAGCAGCCTGACCTTCGAGGACAGCGCCGTGTACTTCTGCGCCAGGCTGAGAAACTGGGACGAGCCTATGGATTACTGGGGCCAGGGCACCACAGTGACAGTGTCTAGC 3174 VL aa (Sequence ID 424) ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIK 3174 VL nt (Accession No. 425) GAACTGGTTATGACACAGAGCCCTAGCAGCCTGACAGTGACAGCCGGCGAGAAAGTGACAATGAGCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTATCAGCAGAAGCCCGGACAGCCTCCTAAGCTGCTGATCTATTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACAGGCAGCGGCAGCGGAACCGACTTTACCCTGACAATTAGCAGCGTGCAGGCCGAGGACCTGGCCGTGTATTATTGTCAGAACGACTACAGCTACCCTCTGACCTTCGGAGCCGGCACCAAGCTGGAAATCAAG 1210 VH aa (Accession No. 426) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS 1210 VH nt (Accession No. 427) GAGGTGCAGCTGCTCGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 1210 VL aa (SEQ ID NO: 428) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK 1210 VL nt (SEQ ID NO: 429) GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAA...

Claims

1. It is a combination therapy, (a) A bispecific polypeptide comprising a first binding domain called B1 that can specifically bind to CD40, and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), (b) A combination therapy comprising a PD-1 inhibitor, which is formulated for parenteral delivery.

2. The combination therapy according to claim 1, wherein the parenteral delivery includes intravenous administration, subcutaneous administration, or intratumor administration.

3. The combination therapy according to claim 1 or 2, wherein the first and / or second binding domain of the bispecific polypeptide is selected from the group consisting of an antibody and its antigen-binding fragment, and a CD40 ligand.

4. The combination therapy according to claim 3, wherein the antigen-binding fragment is selected from the group consisting of Fv fragments (such as single-chain Fv fragments or disulfide-bonded Fv fragments), Fab-like fragments (such as Fab fragments, Fab' fragments, or F(ab)2 fragments), and domain antibodies.

5. The combination therapy according to any one of claims 1 to 4, wherein the bispecific polypeptide is a bispecific antibody.

6. The aforementioned bispecific polypeptide (a) The binding domain B1 and / or binding domain B2 is an intact IgG antibody, (b) The binding domain B1 and / or binding domain B2 is the Fv fragment, (c) The binding domain B1 and / or the binding domain B2 is a Fab fragment and / or (d) The binding domain B1 and / or binding domain B2 is a single-domain antibody. The combination therapy according to any one of claims 1 to 5, wherein the combination therapy is as follows:

7. The combination therapy according to claim 6, wherein the bispecific polypeptide comprises a human Fc region or a variant of the region, and the region is an IgG1, IgG2, IgG3, or IgG4 region, preferably an IgG1 or IgG4 region.

8. The combination therapy according to claim 7, wherein the bispecific polypeptide comprises Fc that does not exhibit affinity for FcγR or exhibits very low affinity for FcγR.

9. The Fc region is located at the following position: The combination therapy according to claim 8, wherein the human IgG1 Fc region variant comprises a mutation in one or more of L234, L235, P239, D265, N297, and / or P329.

10. The combination therapy according to claim 9, wherein alanine is present at the mutated position(s) mentioned above.

11. The combination therapy according to claim 10, wherein the Fc region is a variant of the human IgG1 Fc region containing the double mutations L234A and L235A.

12. The aforementioned bispecific polypeptide (a) A bivalent bispecific antibody such as an IgG-scFv bispecific antibody (for example, B1 is intact IgG and B2 is scFv bound to B1 at the N-terminus of the light chain and / or the C-terminus of the light chain and / or the N-terminus of the heavy chain and / or the C-terminus of the heavy chain of the IgG, or vice versa) (b) Monovalent bispecific antibodies such as “nob-in-hole” bispecific antibodies (e.g., scFv-KIH, scFv-KIHr, BiTE-KIH, or BiTE-KIHr), (c) scFv2-Fc bispecific antibody, (d) BiTE / scFv2 bispecific antibody, (e) DVD-Ig bispecific antibody, (f) DART-based bispecific antibody (e.g., DART2-Fc or DART), (g) DNL-Fab3 bispecific antibody, and (h) scFv-HSA-scFv bispecific antibody A combination therapy according to any one of claims 5 to 11, selected from the group consisting of the following.

13. The combination therapy according to claim 12, wherein the bispecific polypeptide is an IgG-scFv bispecific antibody.

14. The combination therapy according to any one of claims 1 to 13, wherein binding domain B1 and binding domain B2 are directly fused to each other.

15. The combination therapy according to any one of claims 1 to 14, wherein binding domain B1 and binding domain B2 are linked via a polypeptide linker.

16. The combination therapy according to claim 15, wherein the linker is selected from the group consisting of amino acid sequences SGGGGGGGGGS (SEQ ID NO: 337), SGGGGGGGGGGSAP (SEQ ID NO: 338), NFSQP (SEQ ID NO: 339), KRTVA (SEQ ID NO: 340), GGGGGGGGG (SEQ ID NO: 341), GGGGGGGGGGS (SEQ ID NO: 342), GGGGGGGGGGGGGGS (SEQ ID NO: 343), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 344), THTCPPCPEPKSSDK (SEQ ID NO: 345), GGGS (SEQ ID NO: 346), EAAKEAAKGGGGGS (SEQ ID NO: 347), EAAKEAAK (SEQ ID NO: 348), or (SG)m, where m = 1 to 7.

17. The combination therapy according to any one of claims 1 to 16, wherein one of B1 or B2 is an immunoglobulin molecule, and one of B1 or B2 is a Fab fragment, wherein the Fab fragment is fused to the C-terminus of the heavy chain of the immunoglobulin via the light chain of the Fab fragment.

18. The combination therapy according to any one of claims 1 to 17, wherein the bispecific polypeptide comprises one or more mutations that promote the association of the immunoglobulin heavy chain polypeptide with the immunoglobulin light chain polypeptide and / or promote the association of the Fab heavy chain polypeptide with the Fab light chain polypeptide.

19. The combination therapy according to claim 18, wherein one or more of the mutations prevent the formation of aggregates and Fab byproducts.

20. The combination therapy according to claim 18 or 19, wherein the mutation prevents the formation of aggregates and Fab byproducts by generating steric hindrance and / or charge mismatch.

21. The combination therapy according to any one of claims 18 to 20, wherein the bispecific polypeptide comprises one or more mutant pairs, each containing two functionally compatible mutations.

22. The combination therapy according to any one of claims 1 to 21, wherein the bispecific polypeptide can regulate the activity of myelocytes and / or activate myelocytes.

23. The combination therapy according to any one of claims 1 to 22, wherein the bispecific polypeptide cannot induce antibody-dependent cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

24. The combination therapy according to any one of claims 1 to 23, wherein the bispecific polypeptide can induce tumor immunity.

25. The aforementioned bispecific polypeptide (a) Tumor-specific immune activation, and / or (b) Activation of dendritic cells, and / or (c) Internalization of related tumor debris and / or extracellular vesicles containing CEA antigen as well as tumor neoantigen, and / or (d) Cross-presentation of peptides derived from endogenous tumor antigens in MHC, and / or (e) Priming and activation of effector T cells, and / or (f) Direct tumor-killing effects selected from the list consisting of apoptosis, necroptosis, antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). The combination therapy according to any one of claims 1 to 24, which can induce the following.

26. The aforementioned bispecific polypeptide (a) Activation of B cells in the presence of CEA, and / or (b) Activation of dendritic cells in the presence of the CEA, and / or (c) Ability to cross-present dendritic cells with increased neoantigens, and / or (d) Inducing the proliferation of neoantigen-specific T cells The combination therapy according to any one of claims 1 to 25, which is possible.

27. The combination therapy according to any one of claims 1 to 26, wherein the bispecific polypeptide promotes the uptake of tumor-derived material derived from tumor cells that overexpress CEA.

28. The combination therapy according to claim 26, wherein the B cell activation is characterized by CD86 upregulation.

29. The binding domain B1 is less than 2×10 －７ M, or less than 1.5×10 －７ M, or less than 8.5×10 －８ M, or less than 8×10 －８ M, or less than 7.5×10 －８ M, or less than 7×10 －８ M, or less than 9×10 －８ M, or less than 9×10 －９ M, or less than 5×10 －１０ M, or less than 3×10 －１０ M, preferably less than 8.5×10 －８ M, more preferably less than 5×10 －１０ M, or less than 3×10 －１０ M of K Ｄ The combination therapy according to any one of claims 1 to 28, which binds to human CD40.

30. The combination therapy according to any one of claims 1 to 29, wherein the binding domain B1 comprises one or more heavy chain CDR sequences selected from those in Table C(1), and / or the binding domain B1 comprises one or more light chain CDR sequences selected from those in Table C(2).

31. The combination therapy according to any one of claims 1 to 30, wherein the binding domain B1 comprises one, two, or three light chain CDR sequences from a specific row for an individual antibody reference in Table C(2), and / or one, two, or three heavy chain CDR sequences from a corresponding row for an antibody having the same reference in Table C(1).

32. The combination therapy according to any one of claims 1 to 31, wherein the binding domain B1 contains all three heavy chain CDR sequences of a specific antibody reference shown in Table C(1) and / or all three light chain CDR sequences of an antibody reference shown in Table C(2), or the binding domain B1 contains a heavy chain VH sequence and / or a light chain VL sequence shown in Table A.

33. B1, independently, is as follows: (a) Heavy chain CDR1 sequence consisting of the sequence "G, F, T, F, S, S, Y, A", (b) A heavy chain CDR2 sequence having a length of 8 amino acids and containing the consensus sequence: "I, G / S, S / G, Y / S, G / S, G / S, G / Y / S, T", (c) A heavy chain CDR3 sequence having a length of 9 to 12 amino acids and containing the consensus sequence "A, R, Y / R / G, Y / P / V / -, N / S / V, F / Y / W, G / H / S, - / S, - / V, M / F, D, Y", (d) Sequence: Light chain CDR1 sequence consisting of "Q, S, I, S, S, Y", (e) Sequence: Light chain CDR2 sequence consisting of "A, A, S", (f) A light chain CDR3 sequence having a length of 9 amino acids and containing the consensus sequence: "Q, Q, Y / S, G / Y, R / S / V, N / A / Y / T, P, P / F / Y, T" A combination therapy according to any one of claims 1 to 32, comprising one, two, three, four, five, or all of the features selected from the above.

34. The binding domain B1 is (a) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1132 / 1133 (SEQ ID NOs. 73, 74, and 75, and / or 90, 91, and 92), or (b) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1107 / 1108 (SEQ ID NOs. 73, 78, and 80, and / or SEQ ID NOs. 90, 91, and 95), or (c) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1150 / 1151 (SEQ ID NOs. 73, 76, and 77, and / or SEQ ID NOs. 90, 91, and 93), or (d) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 1140 / 1135 (SEQ ID NOs. 73, 78, and 79, and / or SEQ ID NOs. 90, 91, and 94), or (e) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody G12 or G12_mut or ffAC_05337 (SEQ ID NOs: 81, 82, and 83, and / or SEQ ID NOs: 96, 97, and 98), or (f) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody APX005 (SEQ ID NOs: 84, 85, and 86, and / or SEQ ID NOs: 99, 100, and 101), or (g) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody 21.4.1 (SEQ ID NOs: 87, 88, and 89, and / or SEQ ID NOs: 102, 103, and 104) A combination therapy according to any one of claims 1 to 33, including the combination therapy described in any one of claims 1 to 33.

35. The binding domain B1 is (a) The heavy chain variable region and / or light chain variable region of antibody 1132 / 1133 (SEQ ID NOs. 3 and 1), or (b) Heavy chain variable region and / or light chain variable region of antibody 1107 / 1108 (SEQ ID NOs. 15 and 13), or (c) Heavy chain variable region and / or light chain variable region of antibody 1150 / 1151 (SEQ ID NOs. 7 and 5), or (d) Heavy chain variable region and / or light chain variable region of antibody 1140 / 1135 (SEQ ID NOs: 11 and 9), or (e) The heavy chain variable region and / or light chain variable region of antibody G12 (SEQ ID NOs: 19 and 17), or (f) The heavy chain variable region and / or light chain variable region of antibody APX005 (SEQ ID NOs. 23 and 21), or (g) Heavy chain variable region and / or light chain variable region of antibody 21.4.1 (SEQ ID NOs. 27 and 25), or (h) The heavy chain variable region and / or light chain variable region of antibody G12_mut (SEQ ID NOs. 29 and 17), or (i) Heavy chain variable region and / or light chain variable region of antibody ffAC_05337 (SEQ ID NOs: 431 and 430) A combination therapy according to any one of claims 1 to 34, including the combination therapy described in any one of claims 1 to 34.

36. The combination therapy according to any one of claims 1 to 35, wherein the binding domain B1 comprises the light chain of antibody 1132 / 1133 (SEQ ID NO: 372 or 379) and / or the heavy chain of antibody 1132 / 1133 (SEQ ID NO: 371 or 378).

37. The combination therapy according to any one of claims 1 to 36, wherein the binding domain B1 comprises the light chain of antibody G12 (SEQ ID NO: 381) and / or the heavy chain of antibody G12 (SEQ ID NO: 380), or the light chain of antibody G12_mut (SEQ ID NO: 383) and / or the heavy chain of antibody G12_mut (SEQ ID NO: 382).

38. The combination therapy according to any one of claims 1 to 37, wherein the binding domain B1 comprises the light chain of antibody ffAC_05337 (SEQ ID NO: 430) and / or the heavy chain of antibody ffAC_05337 (SEQ ID NO: 431).

39. The combination therapy according to any one of claims 1 to 38, wherein the CEA is a tumor-related CEA.

40. The combination therapy according to any one of claims 1 to 39, wherein the CEA is a carcinoembryonic antigen-associated cell adhesion molecule (CEACAM).

41. The combination therapy according to claim 40, wherein the CEACAM is one or more selected from the enumerated list consisting of CEACAM1, CEACAM6, and CEACAM5.

42. The combination therapy according to claim 41, wherein the CEACAM is CEACAM5.

43. The combination therapy according to any one of claims 1 to 42, wherein B2 can specifically bind to CEA on target cells.

44. The combination therapy according to claim 43, wherein the target cells are cancer cells and / or tumor cells.

45. The combination therapy according to claim 44, wherein the CEA on the target cells is at an intermediate level or a high level.

46. The combination therapy according to claim 45, characterized in that the intermediate level of CEA is a target cell expressing about 10,000 or more CEA receptors per target cell, preferably about 50,000 or more CEA receptors per target cell.

47. The combination therapy according to claim 45, characterized in that the high level of CEA is a target cell expressing approximately 200,000 more CEA receptors per target cell, preferably approximately 300,000 more CEA receptors per target cell.

48. The binding domain B2 is 2 × 10 －６ Less than M, or 1.5 × 10 －８ Less than M, or 2.5 × 10 －９ Less than M, or 2 x 10 －９ Less than M, or 1.5 × 10 －１２ Less than M, or 1 x 10 －１２ Less than M, preferably 1.5 × 10 －８ Less than M, or 2.5 × 10 －９ Less than M, or 1.5 × 10 －１２ K less than M Ｄ The combination therapy according to any one of claims 1 to 47, wherein human CEA is bound.

49. The combination therapy according to any one of claims 1 to 48, wherein the binding domain B2 preferentially binds to CEA on cells rather than soluble CEA.

50. The combination therapy according to any one of claims 1 to 49, wherein the binding domain B2 comprises one or more heavy chain CDR sequences selected from Table D(1a) and / or Table D(1b), and / or the binding domain B2 comprises one or more light chain CDR sequences selected from Table D(2).

51. The combination therapy according to any one of claims 1 to 50, wherein the binding domain B2 comprises one, two, or three light chain CDR sequences from a specific row for an individual antibody reference in Table D(2), and / or one, two, or three heavy chain CDR sequences from the corresponding row for an antibody having the same reference in Table D(1a) and / or Table D(1b).

52. The combination therapy according to any one of claims 1 to 51, wherein the binding domain B2 comprises all three heavy chain CDR sequences of a specific antibody reference shown in Table D(1a) and / or Table D(1b), and / or all three light chain CDR sequences of an antibody reference shown in Table D(2), or the binding domain B1 comprises a heavy chain VH sequence and / or a light chain VL sequence shown in Table B.

53. B2, independently, is as follows: (a) Sequence: A heavy chain CDR1 sequence consisting of "G, F, T, F, S, S, S, Y" or containing the consensus sequence "G, F, T, F, G / S, S, Y, Y / A", (b) Sequence: A heavy chain CDR2 sequence consisting of "I, G, S, G, S, Y, S, T" or containing the consensus sequence "I, S, G, Y / S, G, Y / G, S, T", (c) A heavy chain CDR3 sequence containing the consensus sequence "A, R, Y, P, S, V, P / L, F, P, Q, S, P / H / L, H / P / L, L / F / V / W, D, Y" or the consensus sequence "A, R, H / Y, G, Y, G / S / T, V / H, L / F, D, Y", (d) Sequence: Light chain CDR1 sequence consisting of "Q, S, I, S, S, Y" or containing the consensus sequence "Q, S, I, R / S, S, Y", (e) Sequence: Light chain CDR2 sequence consisting of "A, A, S", (f) A light chain CDR3 sequence consisting of "Q, Q, A, G, N, P, H, T" or containing the consensus sequence "Q, Q, G / Y, T / P / A, W / -, Y / -, F / V, P, F / Y, T". A combination therapy according to any one of claims 1 to 52, comprising one, two, three, four, five, or all of the features selected from the above.

54. The binding domain B2 is (a) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05059 (SEQ ID NOs. 216, 217, and 218, or 280, 281, and 218, and / or SEQ ID NOs. 90, 91, and 311) (b) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05060 (SEQ ID NOs. 219, 220, and 221, or 282, 283, and 221, and / or SEQ ID NOs. 312, 91, and 313) (c) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05061 (SEQ ID NOs: 222, 223, and 224, or 284, 285, and 224, and / or SEQ ID NOs: 90, 91, and 314) (d) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05062 (SEQ ID NOs. 222, 223, and 225, or 284, 285, and 225, and / or SEQ ID NOs. 315, 316, and 94) (e) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05064 (SEQ ID NOs. 222, 223, and 226, or 284, 285, and 226, and / or SEQ ID NOs. 90, 91, and 317) (f) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05079 (SEQ ID NOs. 216, 217, and 227, or 280, 281, and 227, and / or SEQ ID NOs. 90, 91, and 311) (g) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05080 (SEQ ID NOs. 216, 217, and 228, or 280, 281, and 228, and / or SEQ ID NOs. 90, 91, and 311) (h) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05081 (SEQ ID NOs. 216, 217, and 229, or 280, 281, and 229, and / or SEQ ID NOs. 90, 91, and 311) (i) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05082 (SEQ ID NOs: 222, 223, and 230, or 284, 285, and 230, and / or SEQ ID NOs: 90, 91, and 311) (j) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05083 (SEQ ID NOs: 222, 223, and 231, or 284, 285, and 231, and / or SEQ ID NOs: 318, 91, and 319) (k) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05084 (SEQ ID NOs: 222, 223, and 232, or 284, 285, and 232, and / or SEQ ID NOs: 90, 91, and 320) (l) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05085 (SEQ ID NOs. 219, 233, and 234, or 286, 287, and 234, and / or SEQ ID NOs. 90, 91, and 311) (m) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05086 (SEQ ID NOs: 216, 217, and 235, or 280, 281, and 235, and / or SEQ ID NOs: 90, 91, and 311) (n) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05087 (SEQ ID NOs. 216, 217, and 236, or 280, 281, and 236, and / or SEQ ID NOs. 90, 91, and 311) (o) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05088 (SEQ ID NOs: 216, 217, and 237, or 280, 281, and 237, and / or SEQ ID NOs: 90, 91, and 311) (p) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05089 (SEQ ID NOs. 216, 217, and 238, or 280, 281, and 238, and / or SEQ ID NOs. 90, 91, and 311) (q) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05090 or ffAC_05337 (SEQ ID NOs. 216, 217, and 239, or 280, 281, and 239, and / or SEQ ID NOs. 90, 91, and 311) (r) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05091 (SEQ ID NOs: 216, 217, and 240, or 280, 281, and 240, and / or SEQ ID NOs: 90, 91, and 311) (s) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05092 (SEQ ID NOs. 216, 217, and 218, or 280, 281, and 218, and / or SEQ ID NOs. 321, 91, and 311) (t) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05093 (SEQ ID NOs. 216, 217, and 241 or 280, 281, and 241, and / or SEQ ID NOs. 90, 91, and 311) (u) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05094 (SEQ ID NOs. 216, 217, and 242, or 280, 281, and 242, and / or SEQ ID NOs. 90, 91, and 311) (v) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05095 (SEQ ID NOs. 216, 217, and 243, or 280, 281, and 243, and / or SEQ ID NOs. 90, 91, and 311) (w) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05096 (SEQ ID NOs. 216, 217, and 244, or 280, 281, and 244, and / or SEQ ID NOs. 90, 91, and 311) (x) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05097 (SEQ ID NOs: 217, 216, and 245, or 280, 281, and 245, and / or SEQ ID NOs: 90, 91, and 311) (y) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05098 (SEQ ID NOs. 219, 220, and 246, or 282, 283, and 246, and / or SEQ ID NOs. 312, 91, and 313) (z) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05099 (SEQ ID NOs: 222, 223, and 224, or 288, 285, and 224, and / or SEQ ID NOs: 90, 91, and 311) (aa) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody AC_05100 (SEQ ID NOs: 222, 223, and 247, or 288, 285, and 247, and / or SEQ ID NOs: 90, 91, and 311) (ab) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab1 (SEQ ID NOs: 248, 249, and 250, or 289, 290, and 250, and / or SEQ ID NOs: 90, 91, and 322) (ac) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab2 (SEQ ID NOs: 251, 252, and 253, or 291, 292, and 253, and / or SEQ ID NOs: 90, 91, and 323) (ad) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab3 (SEQ ID NOs: 254, 255, and 256, or 293, 294, and 256, and / or SEQ ID NOs: 324, 325, and 326) (ae) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab4 (SEQ ID NOs: 257, 258, and 259, or 295, 296, and 259, and / or SEQ ID NOs: 90, 91, and 327) (af) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab5 (SEQ ID NOs: 260, 261, and 262, or 297, 298, and 262, and / or SEQ ID NOs: 324, 325, and 328) (ag) three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab6 (SEQ ID NOs: 263, 264, and 265, or 299, 300, and 265, and / or SEQ ID NOs: 324, 325, and 329) (ah) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab7 (SEQ ID NOs: 266, 267, and 268, or 301, 302, and 268, and / or SEQ ID NOs: 90, 91, and 330) (ai) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab8 (SEQ ID NOs: 269, 270, and 271, or 303, 304, and 271, and / or SEQ ID NOs: 90, 91, and 331) (aj) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab9 (SEQ ID NOs: 272, 335, and 273, or 305, 306, and 273, and / or SEQ ID NOs: 90, 91, and 332) (ak) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab10 (SEQ ID NOs: 274, 275, and 276, or 307, 308, and 276, and / or SEQ ID NOs: 90, 91, and 333) and / or (al) Three CDRs of the heavy chain and / or three CDRs of the light chain of antibody Fab11 (SEQ ID NOs: 277, 278, and 279, or 309, 310, and 279, and / or SEQ ID NOs: 324, 325, and 334) The combination therapy according to any one of claims 1 to 53, including

55. The binding domain B2 is (a) Heavy chain variable region and / or light chain variable region of antibody AC_05059 (SEQ ID NO: 33 and / or SEQ ID NO: 31) (b) Heavy chain variable region and / or light chain variable region of antibody AC_05060 (SEQ ID NO: 37 and / or SEQ ID NO: 35) (c) Heavy chain variable region and / or light chain variable region of antibody AC_05061 (SEQ ID NO: 41 and / or SEQ ID NO: 39) (d) Heavy chain variable region and / or light chain variable region of antibody AC_05062 (SEQ ID NO: 45 and / or SEQ ID NO: 43) (e) Heavy chain variable region and / or light chain variable region of antibody AC_05064 (SEQ ID NO: 49 and / or SEQ ID NO: 47) (f) Heavy chain variable region and / or light chain variable region of antibody AC_05079 (SEQ ID NO: 53 and / or SEQ ID NO: 51) (g) Heavy chain variable region and / or light chain variable region of antibody AC_05080 (SEQ ID NO: 57 and / or SEQ ID NO: 55) (h) Heavy chain variable region and / or light chain variable region of antibody AC_05081 (SEQ ID NO: 61 and / or SEQ ID NO: 59) (i) Heavy chain variable region and / or light chain variable region of antibody AC_05082 (SEQ ID NO: 65 and / or SEQ ID NO: 63) (j) Heavy chain variable region and / or light chain variable region of antibody AC_05083 (SEQ ID NO: 69 and / or SEQ ID NO: 67) (k) Heavy chain variable region and / or light chain variable region of antibody AC_05084 (SEQ ID NO: 106 and / or SEQ ID NO: 71) (l) Heavy chain variable region and / or light chain variable region of antibody AC_05085 (SEQ ID NO: 110 and / or SEQ ID NO: 108) (m) Heavy chain variable region and / or light chain variable region of antibody AC_05086 (SEQ ID NO: 114 and / or SEQ ID NO: 112) (n) Heavy chain variable region and / or light chain variable region of antibody AC_05087 (SEQ ID NO: 118 and / or SEQ ID NO: 116) (o) Heavy chain variable region and / or light chain variable region of antibody AC_05088 (SEQ ID NO: 122 and / or SEQ ID NO: 120) (p) Heavy chain variable region and / or light chain variable region of antibody AC_05089 (SEQ ID NO: 126 and / or SEQ ID NO: 124) (q) Heavy chain variable region and / or light chain variable region of antibody AC_05090 (SEQ ID NO: 130 and / or SEQ ID NO: 128) (r) Heavy chain variable region and / or light chain variable region of antibody AC_05091 (SEQ ID NO: 134 and / or SEQ ID NO: 132) (s) Heavy chain variable region and / or light chain variable region of antibody AC_05092 (SEQ ID NO: 138 and / or SEQ ID NO: 136) (t) Antibody AC_05093 heavy chain variable region and / or light chain variable region (SEQ ID NO: 142 and / or SEQ ID NO: 140) (u) Heavy chain variable region and / or light chain variable region of antibody AC_05094 (SEQ ID NO: 146 and / or SEQ ID NO: 144) (v) Heavy chain variable region and / or light chain variable region of antibody AC_05095 (SEQ ID NO: 150 and / or SEQ ID NO: 148) (w) Heavy chain variable region and / or light chain variable region of antibody AC_05096 (SEQ ID NO: 154 and / or SEQ ID NO: 152) (x) Heavy chain variable region and / or light chain variable region of antibody AC_05097 (SEQ ID NO: 158 and / or SEQ ID NO: 156) (y) Heavy chain variable region and / or light chain variable region of antibody AC_05098 (SEQ ID NO: 162 and / or SEQ ID NO: 160) (z) Heavy chain variable region and / or light chain variable region of antibody AC_05099 (SEQ ID NO: 166 and / or SEQ ID NO: 164) (aa) Heavy chain variable region and / or light chain variable region of antibody AC_05100 (SEQ ID NO: 170 and / or SEQ ID NO: 168) (ab) Heavy chain variable region and / or light chain variable region of antibody Fab1 (SEQ ID NO: 174 and / or SEQ ID NO: 172) (ac) Heavy chain variable region and / or light chain variable region of antibody Fab2 (SEQ ID NO: 178 and / or SEQ ID NO: 176) (ad) Heavy chain variable region and / or light chain variable region of antibody Fab3 (SEQ ID NO: 182 and / or SEQ ID NO: 180) (ae) Heavy chain variable region and / or light chain variable region of antibody Fab4 (SEQ ID NO: 186 and / or SEQ ID NO: 184) (af) Heavy chain variable region and / or light chain variable region of antibody Fab5 (SEQ ID NO: 190 and / or SEQ ID NO: 188) (ag) Heavy chain variable region and / or light chain variable region of antibody Fab6 (SEQ ID NO: 194 and / or SEQ ID NO: 192) (ah) Heavy chain variable region and / or light chain variable region of antibody Fab7 (SEQ ID NO: 198 and / or SEQ ID NO: 196) (ai) Heavy chain variable region and / or light chain variable region of antibody Fab8 (SEQ ID NO: 202 and / or SEQ ID NO: 200) (aj) Heavy chain variable region and / or light chain variable region of antibody Fab9 (SEQ ID NO: 206 and / or SEQ ID NO: 204) (ak) Heavy chain variable region and / or light chain variable region of antibody Fab10 (SEQ ID NO: 210 and / or SEQ ID NO: 208) (al) Heavy chain variable region and / or light chain variable region of antibody Fab11 (SEQ ID NO: 214 and / or SEQ ID NO: 212) (am) The heavy chain variable region and / or light chain variable region of antibody mAb2 (SEQ ID NO: 387 and / or SEQ ID NO: 385) and / or (an) Heavy chain variable region and / or light chain variable region of antibody ffAC_05337 (SEQ ID NO: 433 and / or SEQ ID NO: 432) The combination therapy according to any one of claims 1 to 54, including

56. The binding domain B2 is (a) Light and / or heavy chains of antibody AC_05059 (SEQ ID NO: 388 and / or SEQ ID NO: 389) (b) Light and / or heavy chains of antibody AC_05060 (SEQ ID NO: 390 and / or SEQ ID NO: 391) (c) Light chain and / or heavy chain of antibody AC_05061 (SEQ ID NO: 392 and / or (SEQ ID NO: 393) (d) Light and / or heavy chains of antibody AC_05062 (SEQ ID NO: 394 and / or SEQ ID NO: 395) (e) Light and / or heavy chains of antibody AC_05064 (SEQ ID NO: 396 and / or SEQ ID NO: 397) (f) Light and / or heavy chains of antibody AC_05079 (SEQ ID NO: 398 and / or SEQ ID NO: 399) (g) Light and / or heavy chains of antibody AC_05081 (SEQ ID NO: 400 and / or SEQ ID NO: 401) (h) Light and / or heavy chains of antibody AC_05088 (SEQ ID NO: 402 and / or SEQ ID NO: 403) (i) Light and / or heavy chains of antibody AC_05089 (SEQ ID NO: 404 and / or SEQ ID NO: 405) (j) Light and / or heavy chains of antibody AC_05090 (SEQ ID NO: 406 and / or SEQ ID NO: 407) (k) Light and / or heavy chains of antibody AC_05091 (SEQ ID NO: 408 and / or SEQ ID NO: 409) (l) Light and / or heavy chains of antibody AC_05093 (SEQ ID NO: 410 and / or SEQ ID NO: 411) (m) Light and / or heavy chains of antibody AC_05094 (SEQ ID NO: 412 and / or SEQ ID NO: 413) (n) Light and / or heavy chains of antibody AC_05096 (SEQ ID NO: 414 and / or SEQ ID NO: 415) (o) Light and / or heavy chains of antibody AC_05097 (SEQ ID NO: 416 and / or SEQ ID NO: 417) (p) Light and / or heavy chains of antibody Fab1 (SEQ ID NO: 418 and / or SEQ ID NO: 419) (q) Light chain and / or heavy chain of antibody Fab3 (SEQ ID NO: 420 and / or (SEQ ID NO: 421) The combination therapy according to any one of claims 1 to 55, including the combination therapy described in any one of claims 1 to 55.

57. The aforementioned bispecific polypeptide ●Includes a chain H1 containing an array selected from the enumeration consisting of SEQ ID NO: 359, SEQ ID NO: 362, SEQ ID NO: 365, and / or SEQ ID NO: 367, and / or ●Includes a chain L1 containing an array selected from the enumeration consisting of SEQ ID NOs: 360, 363, 372, and / or 368, and / or ●The combination therapy according to any one of claims 1 to 56, comprising a chain H2 containing a sequence selected from the enumeration consisting of SEQ ID NO: 361, SEQ ID NO: 364, SEQ ID NO: 366, and / or SEQ ID NO:

369.

58. The combination therapy according to any one of claims 1 to 57, wherein the PD-1 inhibitor comprises or consists of an anti-PD-1 antibody or an antigen-binding fragment thereof capable of inhibiting PD-1 function.

59. The combination therapy according to claim 58, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, pizilizumab, semiprimab, AMP-224, PDR-001, MEDI-0680, JTX-4014 (pimivarimab), spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, INCMGA00012 (retifanlimab), and acrixolimab, and preferably the anti-PD-1 antibody is nivolumab.

60. The combination therapy according to any one of claims 1 to 59, wherein the PD-1 inhibitor comprises or consists of an anti-PD-L1 antibody or an antigen-binding fragment thereof capable of inhibiting PD-1 function.

61. The combination therapy according to claim 60, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab (MPDL3280A), durvalumab (MEDI 4736), avelumab, MDX-1105, KN035 (emvafolimab), and CK-301 (kosivelimab).

62. (a) The binding domain B1 comprises three heavy chain CDRs of sequence numbers 81, 82, and 83, and three light chain CDRs of sequence numbers 96, 97, and 98, (b) The binding domain B2 comprises three heavy chain CDRs of SEQ ID NOs. 216, 217, and 239, and three light chain CDRs of SEQ ID NOs. 90, 91, and 311, and (c) The combination therapy according to any one of claims 1 to 59, wherein the PD-1 inhibitor is nivolumab.

63. Effective amount (a) A bispecific polypeptide comprising a first binding domain called B1 that can specifically bind to CD40, and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), (b) A pharmaceutical composition comprising a PD-1 inhibitor which is formulated for parenteral delivery.

64. The pharmaceutical composition according to claim 63, wherein the parenteral delivery is intravenous, subcutaneous, or intratumor administration.

65. The pharmaceutical composition according to claim 63 or 64, wherein the bispecific polypeptide is as defined in any one of claims 1 to 57 or 62.

66. The pharmaceutical composition according to any one of claims 63 to 65, wherein the PD-1 inhibitor is as defined in any one of claims 58 to 62.

67. (a) The binding domain B1 comprises three heavy chain CDRs of sequence numbers 81, 82, and 83, and three light chain CDRs of sequence numbers 96, 97, and 98, (b) The binding domain B2 comprises three heavy chain CDRs of SEQ ID NOs. 216, 217, and 239, and three light chain CDRs of SEQ ID NOs. 90, 91, and 311, and (c) The pharmaceutical composition according to any one of claims 63 to 66, wherein the PD-1 inhibitor is nivolumab.

68. The daily dose level of the aforementioned PD-1 inhibitor is, (d) 1 mg / kg target body weight to 100 mg / kg target body weight (e) Whether the body weight is between 1 mg / kg and 20 mg / kg, (f) A combination therapy according to any one of claims 1 to 62, or a pharmaceutical composition according to any one of claims 63 to 67, wherein the dose is 1 mg / kg, 10 mg / kg, 20 mg / kg, or 100 mg / kg of the target body weight, preferably 10 mg / kg of body weight.

69. The combination therapy according to any one of claims 1 to 62 or 68, or the pharmaceutical composition according to any one of claims 63 to 68, wherein the concentration of the PD-1 inhibitor is about 2 mg / ml to 150 mg / ml, or about 2 mg / ml to 200 mg / ml, preferably 10 mg / ml to 25 mg / ml.

70. A combination therapy according to any one of claims 1 to 62, 68, or 69, wherein the dose of the PD-1 inhibitor is 10 mg to 1500 mg, and optionally the dose is 100 mg to 200 mg or 200 mg to 500 mg, or a pharmaceutical composition according to any one of claims 63 to 69.

71. (a) The PD-1 inhibitor is pembrolizumab, administered at a dose of approximately 25 mg / ml, 200 mg (intravenously) every three weeks, or 400 mg (intravenously) every six weeks. (b) The PD-1 inhibitor is nivolumab, administered at a dose of approximately 10 mg / ml, 250 mg (intravenously) every two weeks, or 480 mg (intravenously) every four weeks, or (c) The PD-1 inhibitor is atezolizumab, administered at a dose of approximately 60 mg / ml, 840 mg (intravenously) every two weeks, or 1200 mg (intravenously) every four weeks. A combination therapy according to any one of claims 1 to 62 or 68 to 70, or a pharmaceutical composition according to any one of claims 63 to 70.

72. A combination therapy according to any one of claims 1 to 62 or 68 to 71, or a pharmaceutical composition according to any one of claims 63 to 71, for use in pharmaceuticals.

73. The use of a combination therapy according to any one of claims 1 to 62 or 68 to 71, or a pharmaceutical composition according to any one of claims 63 to 71, in the preparation of a pharmaceutical product.

74. A method for treating cancer and / or tumors in a subject, comprising: (a) administering to the subject an effective amount of a bispecific polypeptide comprising a first binding domain called B1 that can specifically bind to CD40 and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA); and (b) administering to the subject an effective amount of a PD-1 inhibitor, wherein the PD-1 inhibitor is administered parenterally.

75. The method according to claim 74, wherein the bispecific polypeptide is as defined in any one of claims 1 to 57 or 62.

76. The method according to claim 74 or 75, wherein the PD-1 inhibitor is as defined in any one of claims 58 to 62.

77. The combination therapy or pharmaceutical composition according to claim 72, or the method according to any one of claims 74 to 76, wherein the bispecific polypeptide and the PD-1 inhibitor are administered simultaneously or to each other within 24 hours.

78. The combination therapy or pharmaceutical composition according to claim 72 or 77, or the method according to any one of claims 74 to 77, wherein the bispecific polypeptide and / or the PD-1 inhibitor is administered parenterally.

79. The combination therapy, pharmaceutical composition, or method according to claim 78, wherein the administration is intravenous, subcutaneous, or intratumor.

80. The dose of the PD-1 inhibitor is (j) Approximately 1 to 100 mg / kg of patient body weight, wherein the dose of the PD-1 inhibitor is optionally 1 mg / kg, 10 mg / kg, 20 mg / kg, or 100 mg / kg, and / or (iii) Approximately 2 mg / ml to 200 mg / ml, and the above dose can be optionally selected as 2 mg / ml, 10 mg / ml, 25 mg / ml, 150 mg / ml, or 200 mg / ml. The PD-1 inhibitor may be administered as a single dose or in divided doses, at the discretion of the patient. A combination therapy or pharmaceutical composition according to any one of claims 72, 77 to 79, or the method according to any one of claims 74 to 69.

81. A bispecific polypeptide for use in pharmaceuticals, comprising a first binding domain called B1 that can specifically bind to CD40, and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), A bispecific polypeptide, wherein the bispecific polypeptide is intended for use in combination with a PD-1 inhibitor, and the PD-1 inhibitor is formulated for parenteral administration.

82. A bispecific polypeptide for use in the treatment of cancer and / or tumors in a subject, comprising a first binding domain called B1 that can specifically bind to CD40, and a second binding domain called B2 that can specifically bind to carcinoembryonic antigen (CEA), A bispecific polypeptide, wherein the bispecific polypeptide is intended for use in combination with a PD-1 inhibitor, and the PD-1 inhibitor is formulated for parenteral administration.

83. The bispecific polypeptide for use according to claim 81 or 82, wherein the bispecific polypeptide is as defined in any one of claims 1 to 57 or 62.

84. A bispecific polypeptide for use according to any one of claims 81 to 83, wherein the PD-1 inhibitor is as defined in any one of claims 63 to 71.

85. The method according to any one of claims 74 to 80, or the bispecific polypeptide for use according to any one of claims 82 to 84, wherein the tumor is a solid tumor.

86. The combination therapy according to any one of claims 68 to 72, or the method according to any one of claims 74 to 85, wherein the subject is a human.

87. Combination therapies, pharmaceutical compositions, bispecific polypeptides, methods, or uses substantially described herein with reference to the specification and drawings.

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