Bispecific antibodies for immune cell activation

Abstract

To provide a bispecific antibody comprising an arm binding to a cancer associated antigen on a cancer cell and an arm binding to a costimulatory molecule.SOLUTION: The invention provides bispecific antibodies having one arm binding to a cancer associated antigen on a cancer cell, such as CD33, EGFR or PD-L1, and a second arm binding to a costimulatory molecule, such as OX40, CD40, GITR, ICOS or 4-1BB. Bridging by the bispecific antibody between cancer cells expressing the cancer associated antigen and immune cells expressing the costimulatory molecule results in clustering of the costimulatory molecules and selective activation of the immune cells at a location proximate to the cancer cells. Thus, the immune cells can exert an immunotherapeutic effect against the cancer cells with reduced toxicity to healthy tissue.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Cross-reference of related applications This application is the same as applications 62 / 760,328 filed on 13 November 2018 and 62 / 838,579 filed on 25 April 2019, each of which is incorporated by reference in whole for all purposes.

[0002] Inclusion by referencing the sequence list This application includes an array in a 438KB txt file 530874-ST25, which is incorporated by reference. [Background technology]

[0003] Antigen-specific immune responses are complex biological processes regulated by multiple positive and negative regulators. T cells are first stimulated via the T cell receptor (TCR) after recognizing allopeptide antigens presented by major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). Optimal T cell activation requires a second signal from co-stimulatory molecules belonging to the CD28 superfamily, such as CD28 and ICOS. Furthermore, the immune response is positively regulated by other co-stimulatory molecules expressed in a cell type and developmental stage-dependent manner, including CD40, OX40, GITR, CD27, HVEM, and 4-1BB belonging to the TNF receptor superfamily, and negatively regulated by checkpoint molecules such as PD-1, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD96, and CD112R. The function of checkpoint molecules is to prevent undesirable overreactions of the immune system. However, checkpoint molecules limit the immune system's ability to effectively fight cancer and infections. For reviews, see Pardoll, Nat. Rev. Cancer, 12:252-264, 2012; Mahoney et al., Nat. Rev. Drug Discov. 14:561-584, 2015; Shin et al., Curr. Opin. Immunol. 33:23-35, 2015; Marquez-Rodas et al. Ann. Transl. Med. 3:267, 2015; Mercier et al., Front. Immunol. 6:418, 2015; Topalian et al., Cancer Cell 27: 450-461, 2015; Baumeister et al., Annu. Rev. Immunol. 34:539-573, 2016; Ward-Kavanagh et al., Immunity 44:1005-1019, See Torphy et al., Int. J. Mol. Sci. 18:2642, 2017.

[0004] Blocking the function of checkpoint molecules with antagonistic monoclonal antibodies has been reported to be effective in enhancing immunity (Mercier et al., supra; Baumeister et al., supra). For example, the interaction between PD-1 (also known as CD279), which is expressed on the surface of T cells, and its ligand, PD-L1 (also known as CD274), which is expressed on antigen-presenting cells, suppresses the immune response in the body. The ligands of checkpoint molecules expressed on cancer cells interact with their respective receptors expressed on immune cells, weakening the immune response in the body. As a result, cancer cells are able to survive and proliferate. Inhibition of the interaction between PD-1 and PD-L1 by monoclonal antibodies has been shown to be effective in treating cancer. To date, three types of anti-PD-1 monoclonal IgG antibodies (nivolumab, pembrolizumab, and cemiprimab) and three types of anti-PD-L1 monoclonal IgG antibodies (durvalumab, atezolizumab, and avelumab) have been approved by the US FDA as cancer treatments. Furthermore, monoclonal IgG antibodies that inhibit the interaction of other checkpoint molecules such as TIGIT, TIM-3, LAG-3, and VISTA with their respective ligands are being investigated for their therapeutic effects in clinical trials (Marin-Acevedo et al., J. Hematol. Oncol. 11:39, 2018).

[0005] A monoclonal IgG antibody against CTLA-4 (ipilimumab) is used as a cancer treatment because it blocks the interaction between CTLA-4 on T cells and B7-1 and B7-2 (also known as CD80 and CD86, ligands for the costimulatory molecule CD28, respectively) on antigen-presenting cells, thereby stimulating CD28-mediated T cell activation. Furthermore, agonist IgG antibodies against costimulatory molecules such as ICOS, CD40, OX40, GITR, CD27, and 4-1BB also have the ability to activate immune cells (Peggs et al., Clin. Exp. Immunol. 157:9-19, 2009; Melero et al., Clin. Cancer Res. 19:1044-1053, 2013; Attanasio et al., Immunity 44:1053-1068, 2016; Strugill et al., Am. J. Hematol. Oncol. 13:4-15, 2017). Similar to ICOS, costimulatory molecules belonging to the TNF receptor superfamily require multimeric crosslinking on the surface of immune cells to initiate intracellular signaling in order to enhance the immune response (Watanabe et al., Int. Immunol. 17:269-278, 2005; Croft et al., Nat. Rev. Drug Discov. 12:147-168, 2013; Wikenheiser et al., Front. Immunol. 7: Article 304, 2016). Because IgG is divalent for antigen binding, unless IgG molecules bound to the cell surface are conjugated via binding to Fcγ receptors on other cells, they cannot efficiently trigger signaling through these costimulatory molecules. Anti-CD40 IgG2 antibodies with Fc mutations to enhance binding to FcγRIIB (also known as CD32B) showed superior antitumor activity compared to the parental IgG2 antibody in a mouse xenograft tumor model (Dahan et al., Cancer Cell 29:820-831, 2016).As another approach to promoting multivalent bonding of surface molecules, Tso et al. designed an IgG antibody by fusing the CH3 and CH4 regions of the human mu heavy chain to the C-terminus of the human gamma heavy chain (US Patent 10,053,517). This designed hexameric IgG antibody can efficiently crosslink costimulatory molecules on the cell surface and trigger intracellular signaling.

[0006] Bispecific antibodies are artificially created antibodies that can bind to two different antigens or two different epitopes on the same antigen. Various forms of bispecific antibodies have been produced (Lameris et al., Crit. Rev. Oncol. Hematol. 92:153-165, 2014; Kontermann et al., Drug Dicov. Today 20:838-847, 2015; Spiess et al., Mol. Immunol. 67:95-106, 2015; Brinkmann et al., MAbs 9:182-212, 2017; Sedykh et al., Drug Des. Dev. Ther. 12:195-208, 2018). Some bispecific antibodies, such as blinatumomab, which bind to CD3 and CD19, are designed to bring T cells closer to cancer cells that possess CD19, thereby inducing T cell-mediated cytotoxicity against cancer cells (Viardot et al., Cancer Treat. Rev. 65:87-95, 2018). [Overview of the project]

[0007] The present invention provides a bispecific antibody comprising a first binding site that specifically binds to CD33, and a second binding site that specifically binds to GITR, OX40, CD40, ICOS, or 4-1BB. Optionally, the bispecific antibody further comprises an IgG Fc region. Optionally, the second binding site agonizes OX40 or CD40. Optionally, the first binding site includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 94-96, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 97-99, and the second binding site includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 33-35, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 37-39, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 13-15, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 17-19, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 49-51, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 53-55, or a CDR of SEQ ID NOs. 109-111 It includes a mature heavy chain variable region containing H1, H2, and H3 respectively, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 113-115, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 128-130, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 132-134.Optionally, the first binding site includes the mature heavy chain variable region containing residues 20-135 of SEQ ID NO: 2 and the mature light chain variable region containing residues 20-131 of SEQ ID NO: 7, and the second binding site includes the mature heavy chain variable region containing residues 20-137 of SEQ ID NO: 32 and the mature light chain variable region containing residues 21-127 of SEQ ID NO: 36, or the mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 12 and the mature light chain variable region containing residues 21-127 of SEQ ID NO: 16. The variable chain region includes a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 48, and a mature light chain variable region containing residues 23-127 of SEQ ID NO: 52, or a mature heavy chain variable region containing residues 20-136 of SEQ ID NO: 108, and a mature light chain variable region containing residues 21-126 of SEQ ID NO: 112, or a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 127, and a mature light chain variable region containing residues 21-133 of SEQ ID NO: 131. Optionally, the first binding site includes a mature heavy chain variable region containing residues 20-135 of SEQ ID NO: 2, and a mature light chain variable region containing residues 20-131 of SEQ ID NO: 7, and the second binding site includes a single-stranded Fv fragment containing SEQ ID NOs: 22, 41, 57, 117, 122, 123, 124, 125, 136, or 139.

[0008] The present invention further provides a first binding site that specifically binds to EGFR, and a second binding site that specifically binds to GITR, OX40, CD40, ICOS, or 4-1BB. Optionally, the bispecific antibody further comprises an Fc region. Optionally, the first binding site antagonizes the binding of EGFR to EGF, and the second binding site agonizes GITR. Optionally, the first binding site includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 100-102, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 103-105, and the second binding site includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 33-35, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 37-39, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 13-15, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 17-19, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 49-51, and CDR of SEQ ID NOs. 53-55 It includes a mature light chain variable region containing L1, L2, and L3 respectively, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 109-111, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 113-115, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 128-130, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 132-134.Optionally, the first binding site includes the mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 29 and the mature light chain variable region containing residues 21-127 of SEQ ID NO: 30, and the second binding site includes the mature heavy chain variable region containing residues 20-137 of SEQ ID NO: 32 and the mature light chain variable region containing residues 21-127 of SEQ ID NO: 36, or the mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 12 and the mature light chain variable region containing residues 21-127 of SEQ ID NO: 16. The light chain variable region, or the mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 48, and the mature light chain variable region containing residues 23-127 of SEQ ID NO: 52, or the mature heavy chain variable region containing residues 20-136 of SEQ ID NO: 108, and the mature light chain variable region containing residues 21-126 of SEQ ID NO: 112, or the mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 127, and the mature light chain variable region containing residues 21-133 of SEQ ID NO: 131. Optionally, the first binding site includes the mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 29, and the mature light chain variable region containing residues 21-127 of SEQ ID NO: 30, and the second binding site includes a single-stranded Fv fragment containing SEQ ID NOs: 22, 41, 57, 117, 122, 123, 124, 125, 136, or 139.

[0009] The present invention further provides a monoclonal antibody comprising a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 13-15, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 17-19, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 49-51, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 53-55, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 109-111, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 113-115, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 128-130, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 132-134, respectively. Optionally, the monoclonal antibody includes a mature heavy chain variable region containing residues 20-137 of SEQ ID NO: 32 and a mature light chain variable region containing residues 21-127 of SEQ ID NO: 36, or a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region containing residues 21-127 of SEQ ID NO: 16, or a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 48 and a mature light chain variable region containing residues 23-127 of SEQ ID NO: 52, or a mature heavy chain variable region containing residues 20-136 of SEQ ID NO: 108 and a mature light chain variable region containing residues 21-126 of SEQ ID NO: 112, or a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 127 and a mature light chain variable region containing residues 21-133 of SEQ ID NO: 131.

[0010] The present invention further provides a bispecific antibody comprising a first binding site that specifically binds to PD-L1, and a second binding site that specifically binds to GITR, OX40, CD40, ICOS, or 4-1BB. Optionally, the bispecific antibody further comprises an Fc region. Optionally, the first binding site antagonizes the binding of PD-L1 to PD1, and the second binding site agonizes GITR, OX40, CD40, ICOS, or 4-1BB. Optionally, the first binding site includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 62-64, 75-77, or 85-87, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 66-68, 79-81, or 89-91, and the second binding site includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 33-35, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 37-39, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 13-15, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 17-19, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 49-51, and CDR Each includes a mature light chain variable region containing L1, L2, and L3 respectively, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 109-111, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 113-115, or a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 128-130, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 132-134.Optionally, the first binding site includes the mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 61, residues 20-138 of SEQ ID NO: 74, or residues 19-137 of SEQ ID NO: 84, and the mature light chain variable region containing residues 23-127 of SEQ ID NO: 65, residues 23-128 of SEQ ID NO: 78, or residues 23-128 of SEQ ID NO: 88, and the second binding site includes the mature heavy chain variable region containing residues 20-137 of SEQ ID NO: 32, and the mature light chain variable region containing residues 21-127 of SEQ ID NO: 36, or residues 20-12 of SEQ ID NO: 12 It includes a mature heavy chain variable region containing 38, and a mature light chain variable region containing residues 21-127 of SEQ ID NO: 16, or a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 48, and a mature light chain variable region containing residues 23-127 of SEQ ID NO: 52, or a mature heavy chain variable region containing residues 20-136 of SEQ ID NO: 108, and a mature light chain variable region containing residues 21-126 of SEQ ID NO: 112, or a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 127, and a mature light chain variable region containing residues 21-133 of SEQ ID NO: 131. Optionally, the first binding site includes a mature heavy chain variable region containing residues 20-138 of SEQ ID NO: 61, 20-138 of SEQ ID NO: 74, and 19-137 of SEQ ID NO: 84, and residues 23-128 of SEQ ID NO: 65, 23-128 of SEQ ID NO: 78, or 23-128 of SEQ ID NO: 88, and the second binding site includes a single-stranded Fv fragment containing SEQ ID NOs: 22, 41, 57, 117, 122, 123, 124, 125, 136, or 139.

[0011] Some of the bispecific antibodies described above include a first pair of heavy and light chain variable regions forming a first binding site, and a second pair of heavy and light chain variable regions forming a second binding site. Here, the C-terminuses of the first pair of heavy and light chain variable regions are fused to the N-terminuses of the heavy and light chain constant regions, and the heavy and light chain variable regions of the second pair are fused to the C-terminuses of the heavy chain constant region to form an scFv, or vice versa. In some bispecific antibodies, the C-terminuses of the first pair of heavy and light chain variable regions are fused to the N-terminuses of the heavy and light chain constant regions, and the heavy and light chain variable regions of the second pair are fused to the C-terminus of the heavy chain constant region to form an scFv. In some bispecific antibodies, the light chain variable region of the scFv is fused to the C-terminus of the heavy chain constant region. In some bispecific antibodies, the first and second binding sites are humanized, veneerized, or human, and the heavy and light chain constant regions are human. In some bispecific antibodies, the isotype of the heavy chain constant region is human IgG1, and the light chain constant region is kappa. Some bispecific antibodies contain two primary binding sites and two secondary binding sites. In some bispecific antibodies, the heavy chain constant region has at least one mutation that decreases or increases FcRγ binding. In some bispecific antibodies, the heavy chain constant region has at least one mutation that increases binding to FcRn.

[0012] The present invention further provides a bispecific antibody comprising a first binding site that specifically binds to an antigen on cancer cells, pathogen-infected cells, or immune cells, and a second binding site that specifically binds to a costimulatory molecule. Here, the heavy chain and light chain variable regions of a first pair form the first binding site, and the heavy chain and light chain variable regions of a second pair form the second binding site, where the C-terminuses of the heavy chain and light chain variable regions of the first pair are fused to the N-terminuses of the heavy chain and light chain constant regions, and the heavy chain and light chain variable regions of the second pair are fused to the C-terminus of the heavy chain constant region to form an scFv, or vice versa. Optionally, the first binding site is ligated to the N-terminuses of the heavy chain and light chain constant regions, and the second binding site is ligated to the C-terminus of the heavy chain constant region.

[0013] The present invention further provides a monoclonal antibody that specifically binds to PD-L1, comprising a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 62-64, 75-77, or 85-87, respectively, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 66-68, 79-81, or 89-91, respectively. Optionally, the monoclonal antibody comprises a mature heavy chain variable region containing residues 20-138 of SEQ ID NO. 61, residues 20-138 of SEQ ID NO. 74, or residues 19-137 of SEQ ID NO. 84, and a mature light chain variable region containing residues 23-127 of SEQ ID NO. 65, residues 23-128 of SEQ ID NO. 78, or residues 23-128 of SEQ ID NO. 88, respectively.

[0014] The present invention further provides a pharmaceutical composition comprising either a bispecific antibody or a monoclonal antibody, and a pharmaceutically acceptable carrier.

[0015] The present invention further provides a method for exerting a therapeutic or preventive effect against cancer, comprising the step of administering an effective regimen of the bispecific or monoclonal antibody defined above to a subject who has cancer or is at risk of developing cancer.

[0016] The present invention further provides a method for treating an infectious disease, comprising the step of administering an effective regimen of the bispecific antibody or monoclonal antibody defined above to a subject who has or is at risk of having an infectious disease. [Brief explanation of the drawing]

[0017] [Figure 1A-D] Schematic diagrams of the expression vectors pHuM195-IgG1 (A), pBS824 (B), pFCm331 (C), and pFCm267 (D). Relevant restriction enzyme sites are indicated. The diagrams are not drawn to scale.

[0018] [Figure 2] Schematic structure of the bispecific IgG antibody of the present invention.

[0019] [Figure 3]Analysis of luciferase activity in Jurkat Dual cells expressing OX40 (JD / OX40) using the bispecific antibody (BS824) that binds to CD33 and OX40. The mean relative light units (RLU) of triplicate analyses are shown with standard deviation bars.

[0020] [Figure 4] Analysis of luciferase activity in Jurkat Dual cells expressing GITR (JD / GITR) using the bispecific antibody (BS827) that binds to EGFR and GITR. The mean relative light units (RLU) of triplicate analyses are shown with standard deviation bars.

[0021] [Figure 5] FACS analysis of CD95 expression in Ramos cells using the bispecific antibody (BS828) that binds to CD33 and CD40. The X-axis and Y-axis represent the expression levels of CD20 and CD95, respectively. The numbers in the upper right and lower right indicate the percentage of CD20-positive cells in each quadrant.

[0022] [Figure 6] Activation of T cells by the bispecific antibody (BS809) that binds to PD-L1 and GITR. The mean expression level of IL-2 is shown with standard deviation bars.

[0023] [Figure 7] Analysis of luciferase activity in Jurkat Dual cells expressing OX40 (JD / OX40) using the bispecific antibody (BS813) that binds to PD-L1 and OX40. The mean relative light units (RLU) of triplicate analyses are shown with standard deviation bars.

[0024] [Figure 8]Analysis of luciferase activity in GITR-expressing Jurkat Dual cells (JD / GITR) using a bispecific antibody (BS809) that binds to PD-L1 and GITR. The mean relative luminescence units (RLU) of the triplicate analysis are shown along with the standard deviation bar.

[0025] [Figure 9] Activation of human T cells by bispecific antibodies (BS813, BS841, and BS839) that bind to PD-L1 and OX40. Mean IL-2 expression levels from triplicate analysis are shown with standard deviation bars.

[0026] [Figure 10] Activation of human peripheral blood mononuclear cells treated with SEB using bispecific antibodies (BS813, BS841, and BS839) that bind to PD-L1 and OX40. Mean IL-2 expression levels from triplicate analysis are shown with standard deviation bars.

[0027] [Figure 11] Activation of human T cells by a bispecific antibody (BS859) that binds to PD-L1 and ICOS. Average IL-2 and IL-10 expression levels are shown with standard deviation bars.

[0028] [Figure 12] Analysis of luciferase activity in GITR-expressing Jurkat Dual cells (JD / GITR) using a bispecific antibody (BS840) that binds to PD-L1 and GITR. The mean relative luminescence units (RLU) of the triplicate analysis are shown along with the standard deviation bar.

[0029] [Figure 13] Activation of Ramos cells using a bispecific antibody (BS846) that binds to PD-L1 and CD40.

[0030] definition The bispecific antibodies of the present invention are typically provided in isolated form. This means that the bispecific antibodies are typically at least 50% w / w pure with respect to interfering proteins and other contaminating substances resulting from their production or purification, but this does not preclude the possibility that the bispecific antibodies may be combined with excess pharmaceutically acceptable carriers or other vehicles intended to facilitate their use. The bispecific antibodies may be at least 60, 70, 80, 90, 95, or 99% w / w pure with respect to interfering proteins and contaminating substances resulting from their production or purification. Often, the bispecific antibody is the major high molecular weight species remaining after its purification.

[0031] The specific binding of bispecific antibodies to target antigens is at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 This refers to affinity. Affinity can differ for different targets. Specific binding is detected as being large enough to distinguish it from nonspecific binding that occurs for at least one unrelated target. Specific binding may result from binding between specific functional groups or the formation of a specific spatial fit (e.g., lock-and-key type), while nonspecific binding is typically the result of van der Waals forces. However, specific binding does not necessarily mean that a bispecific antibody with two different binding sites will only bind to the targets of those two binding sites.

[0032] The basic structural unit of an antibody is a tetramer of subunits. Each tetramer contains a pair of two identical polypeptide chains, each pair having one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region containing approximately 100-110 or more amino acids, primarily involved in antigen recognition. This variable region is initially expressed bound to a cleavable signal peptide. A variable region that does not contain a signal peptide is sometimes called a mature variable region. Therefore, for example, a light chain mature variable region means a light chain variable region that does not contain a light chain signal peptide. However, when referring to a variable region, a signal sequence is not necessarily present; in fact, the signal sequence is cleaved when the bispecific antibody of the present invention is expressed and secreted. The pair of variable regions of the heavy and light chains defines the antibody's binding region. The carboxyl-terminuses of the light and heavy chains define the light chain constant region and the heavy chain constant region. The heavy chain constant region primarily performs effector functions. In IgG antibodies, the heavy chain constant region is divided into CH1, hinge, CH2, and CH3 regions. In IgA, the heavy chain constant region is divided into CH1, CH2, and CH3. The CH1 region binds to the light chain constant region via disulfide and non-covalent bonds. The hinge region provides flexibility between the antibody's binding region and effector region, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions within the tetrameric subunit. The CH2 and CH3 regions are the main sites for effector function and FcRn binding.

[0033] The light chain is classified as kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, defining the antibody isotypes as IgG, IgM, IgA, IgD, and IgE, respectively. The variable and constant regions of the light and heavy chains are linked by a "J" segment containing approximately 12 or more amino acids, and the heavy chain also contains a "D" segment containing approximately 10 or more amino acids (see Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7) (the whole is incorporated by reference for all purposes).

[0034] The mature variable region of each light / heavy chain pair forms an antibody binding site. Therefore, an intact antibody has two binding sites, i.e., it is bivalent. In natural antibodies, the binding sites are identical. However, in bispecific antibodies, these binding sites may be identical or different depending on the format (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). All variable regions exhibit the same typical structure: a relatively conserved framework region (FR) to which three hypervariable regions, also called complementarity-determining regions (CDRs), are bound. The CDRs from the two strands of each pair are aligned by the framework region, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering convention (Kabat numbering) that assigns the same number to corresponding residues between different heavy chain variable regions or between different light chain variable regions. Kabat numbering can be used for antibody constant regions, but EU indexes (also called EU numbering) are more commonly used, as in this application.

[0035] The term "epitope" refers to the site on an antigen to which the arms of a bispecific antibody bind. Epitopes can be formed from consecutive amino acids or discontinuous amino acids juxtaposed by the three-dimensional folding of one or more proteins. Epitopes formed from consecutive amino acids (also called linear epitopes) are typically retained even when exposed to denaturing solvents, while epitopes formed by three-dimensional folding (also called conformational epitopes) are typically lost when treated with denaturing solvents. Some antibodies bind to terminal-specific epitopes; that is, antibodies selectively bind to polypeptides with free ends, resulting in the loss of free ends, compared to the same polypeptide fused to another polypeptide. Epitopes typically contain at least three, more commonly, at least five or eight to ten amino acids and have a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. For example, see Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0036] The terms "antigen" or "target antigen" refer to the target molecule to which a bispecific antibody binds. Antigens can be molecules such as proteins (natural, synthetic, or recombinant expression) of any length, nucleic acids, or carbohydrates. Antigens can include receptors, ligands, counterreceptors, and coat proteins.

[0037] Antibodies that recognize the same or overlapping epitopes can be identified by a simple immunoassay that demonstrates one antibody's ability to compete for the binding of another antibody to its target antigen. Antibody epitopes can also be defined by identifying contact residues through X-ray crystallography of the antibody bound to the antigen. Furthermore, if all amino acid mutations in an antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other, then the two antibodies have the same epitope. If several amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other, then the two antibodies have overlapping epitopes.

[0038] Antibody competition is determined by an assay in which the test antibody inhibits the specific binding of the reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). The test antibody competes with the reference antibody if an excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits the binding of the reference antibody by at least 50%, preferably 75%, 90%, or 99%, as measured by the competitive binding assay. Antibodies identified by the competitive assay (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody due to steric hindrance.

[0039] The term "subject" includes human and other mammalian subjects receiving preventive or therapeutic treatment. Other mammalian subjects include animal models of human conditions (e.g., rodents, non-human primates) and veterinary subjects.

[0040] For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions are substitutions between amino acids of the same class. Non-conservative substitutions are exchanges of one member of one class with a member of another class.

[0041] Percentage sequence identity is determined using antibody sequences that are maximally aligned according to Kabat numbering rules for variable regions and EU numbering rules for constant regions. After alignment, when comparing the target antibody region (e.g., the entire mature variable region of the heavy or light chain) with the same region of the reference antibody, the percentage of sequence identity between the target antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acid in both the target antibody region and the reference antibody region by the total number of aligned positions in these two regions (without considering gaps), and multiplying by 100 to convert it to a percentage.

[0042] A composition or method that "contains" one or more of the listed elements may also contain other elements not specifically listed. For example, a composition containing an antibody may contain only the antibody, or it may contain the antibody in combination with other components.

[0043] The term "antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a mechanism that induces cell death dependent on the interaction between antibody-coated target cells (i.e., cells to which antibodies are bound) and immune cells with lytic activity (also called effector cells). Examples of such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC occurs through the interaction between the Fc region of an antibody bound to a cell and Fcγ receptors (particularly FcγRI and FcγRIII) present on immune effector cells such as neutrophils, macrophages, and natural killer cells. The target cell is eliminated by phagocytosis or lysis, depending on the type of effector cell mediating the process. The death of the antibody-coated target cell occurs as a result of effector cell activity.

[0044] The term opsonization, also known as "antibody-dependent phagocytosis" or ADCP, refers to the process by which antibody-coated cells are whole or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc region.

[0045] The term "complement-dependent cell injury" or CDC (also known as CMC) refers to a mechanism in which the Fc effector domain of a target-binding antibody activates a series of enzymatic reactions, resulting in cell death through the formation of holes in the target cell membrane. Generally, an antigen-antibody complex (e.g., on an antibody-coated cell) binds to and activates complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation may also result in the deposition of complement components on the target cell surface, which promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0046] pH-dependent binding of antibodies to FcRn receptors means that antibodies bind to such receptors more strongly at pH 6.0 than at pH 7.5. Binding to FcRn at low pH in endosomes after internalization via iodine rescues IgG antibodies from catabolic degradation in lysosomes. The rescued IgG antibodies are then released from FcRn at neutral pH and reused in the bloodstream. This pH-dependent FcRn binding underlies the molecular mechanism of the long serum half-life of IgG antibodies (and the bispecific antibodies of the present invention) (Ghetie et al., Annu. Rev. Immunol. 18:739-766, 2000). For example, human IgG antibodies bind to human neonatal Fc receptors (FcRn) at pH 6.0, while binding only weakly at pH 7.5. The FcRn binding site in IgG antibodies is located at the junction of the CH2 and CH3 domains. Since muon heavy chains do not bind to FcRn at pH 6.0 or pH 7.5, natural IgM cannot rescue antibodies from degradation in leukocytes using the FcRn-mediated pathway, and therefore generally has a shorter half-life than natural IgG antibodies.

[0047] Protein A is a 40-60 kDa surface protein originally discovered in the cell wall of Staphylococcus aureus. Protein A specifically binds with high affinity to mouse IgG2a and IgG2b, as well as human IgG1, IgG2, and IgG4. Protein A does not bind to human IgG3, IgA, or IgM. Protein A is used in antibody affinity purification.

[0048] Protein G is a 65kDa (G148 protein G) and 58kDa (C40 protein G) cell surface protein of Streptococcus. Protein G contains a serum albumin-binding domain, but this is not required for IgG binding and is often removed. Protein G specifically binds to all isotypes of human IgG, but not to IgA and IgM. Protein G is also used in antibody purification. [Modes for carrying out the invention]

[0049] I. General Matters The present invention provides a bispecific antibody having a first arm that binds to cancer-related antigens such as CD33, EGFR, and PD-L1, and a second arm that binds to costimulatory molecules such as OX40, CD40, GITR, ICOS, and 4-1BB. While understanding the mechanism is not essential for implementing the present invention, it is thought that the bispecific antibody of the present invention bridges the gap between cancer cells expressing cancer-related antigens and immune cells expressing costimulatory molecules, causing the costimulatory molecules to cluster and selectively activating immune cells in close proximity to cancer cells. As a result, immune cells can exert immunotherapeutic effects against cancer cells while suppressing toxicity to healthy tissues. The bispecific antibody may have additional activity against cells expressing cancer-related antigens as a result of antagonistizing receptor-ligand interactions (e.g., interactions between EGFR and EGF, PD-L1 and PD1, or CD33 and sialic acid). Some bispecific antibodies of the present invention, which bind to cancer cells (or cells infected with pathogens) and costimulatory molecules, can create a bridge between these two types of cells, triggering an immune response through multivalent crosslinking of costimulatory molecules at the intercellular junction, thereby combating cancer and infectious diseases. Furthermore, some bispecific antibodies of the present invention can enhance the immune response by inhibiting the function of checkpoint molecules (e.g., PD-L1) and inducing signaling pathways through multivalent crosslinking of costimulatory molecules (e.g., GITR, OX40, CD40, ICOS, 4-1BB).

[0050] II. Target The bispecific antibodies of the present invention have at least two arms, each specifically binding to a different target antigen. One class of proteins that can be targeted are cancer-associated antigens. Such antigens are expressed by cancer and are typically expressed at higher levels (overexpression) than in normal control tissues. Examples of cancer-associated antigens include CD33, EGFR, and PD-L1. Exemplary human-type Swiss Prot numbers for these targets are P20138, P00533, and Q9NZQ7. CD33 binds to sialic acid and is overexpressed mainly in bone marrow-derived cancers such as acute myeloid leukemia. EGFR binds to EGF and is overexpressed mainly in gastric cancer, breast cancer, endometrial cancer, colorectal cancer, head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, and esophageal cancer. PD-L1 binds to PD1 and is overexpressed in cancers such as gastric cancer, hepatocyte carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer. Another class of proteins are antigens that are expressed on the surface of cells infected with pathogens.

[0051] Another class of proteins are co-stimulatory molecules such as CD40, OX40, GITR, ICOS, and 4-1BB. P25942, P23510, Q9Y5U5, Q9Y6W8, and Q07011 are exemplary Swiss Prot numbers for the human forms of these targets. Unless otherwise stated in context, references to specific targets should be understood as referring to the human form. However, non-human forms such as experimental animals (e.g., mice, rats), non-human primates, companion animals, or farm animals are also acceptable.

[0052] III. Exemplary antibodies against each target Bispecific antibodies are formed from a pair of heavy and light chain variable regions of component antibodies. Component antibodies can be, among other things, rodent, chimeric, veneerized, humanized, primateized, primate, or human. The component antibodies may be of the same type or different types. For example, one could be humanized and the other human.

[0053] The production of other non-human monoclonal antibodies against antigens, such as monoclonal antibodies from mice, guinea pigs, primates, rabbits, or rats, can be carried out, for example, by immunizing animals with the antigen or a fragment thereof, or with cells containing the antigen. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such antigens can be obtained from natural sources, by peptide synthesis, or by recombinant expression. Optionally, the antigen may be administered fused or complexed with a carrier protein. Optionally, the antigen may be administered with an adjuvant. Several types of adjuvants can be used, as follows: For immunization of experimental animals, it is preferable to use a complete Freund's adjuvant followed by an incomplete adjuvant.

[0054] Humanized antibodies are genetically engineered antibodies in which CDRs from non-human "donor" antibodies are transplanted into human "acceptor" antibody sequences (see, for example, Queen, US Pat. Nos. 5,530,101 and 5,585,089; Winter, US Pat. No. 5,225,539; Carter, US Pat. No. 6,407,213; Adair, US Pat. Nos. 5,859,205 and 6,881,557; Foote, US Pat. No. 6,881,557). The acceptor antibody sequence may be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, humanized antibodies are antibodies that have some or all of the CDRs derived entirely or substantially from the donor antibody, as well as constant region (if any) and variable region framework sequences derived entirely or substantially from the human antibody sequence. Similarly, the humanized heavy chain has at least one, two, and usually three CDRs derived entirely or substantially from the donor antibody heavy chain, as well as the heavy chain constant region (if present) and heavy chain variable region framework sequence, substantially derived from the human heavy chain variable region framework sequence and constant region sequence. Similarly, the humanized light chain has at least one, two, and usually three CDRs derived entirely or substantially from the donor antibody light chain, as well as the light chain constant region (if present) and light chain variable region framework sequence, substantially derived from the human light chain variable region framework sequence and constant region sequence. Except for nanobodies and dAbs, the humanized antibody contains the humanized heavy chain and humanized light chain. The CDRs in the humanized antibody are substantially derived from the corresponding CDRs in the non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical between the corresponding CDRs. The variable region framework sequence or the constant region of an antibody chain is substantially derived from the human variable region framework sequence or the human constant region, respectively, if at least 85%, 90%, 95%, or 100% of the corresponding residues, as defined by Kabato, are identical.

[0055] Humanized antibodies often incorporate all six CDRs (preferably according to Kabat's definition) derived from mouse antibodies, but they can also be produced with fewer than all CDRs (e.g., at least three, four, or five CDRs derived from mouse antibodies) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000).

[0056] Chimeric antibodies are antibodies in which the maturation variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) are combined with the constant light and heavy chain regions of a human antibody. Such antibodies substantially or completely retain the binding specificity of the mouse antibody, with approximately two-thirds of the sequence being human.

[0057] A beniated antibody is a type of humanized antibody that retains some, usually all, CDRs and non-human variable region framework residues of a non-human antibody, but in which other variable region framework residues that may contribute to B-cell or T-cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), are replaced with residues derived from the corresponding positions in the human antibody sequence. As a result, the CDRs are entirely or substantially derived from the non-human antibody, and the variable region framework of the non-human antibody is made human-like through substitution.

[0058] Human antibodies can be isolated from humans, but they can also be generated from the expression of human immunoglobulin genes (for example, in vitro or through genetically modified mice using phage presentation). Methods for producing human antibodies include the trioma method (Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, US Pat. No. 4,634,664; and Engleman et al., US Pat. No. 4,634,666), and the use of genetically modified mice containing human immunoglobulin genes (e.g., Lonberg et al., WO93 / 12227 (1993); US Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148). This includes methods such as 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991), and phage display methods (see, for example, Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047, US Pat. Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332).

[0059] Antibodies are screened for specific binding to antigens. Antibodies can be further screened for binding to specific regions of the antigen, competition with reference antibodies, and whether they are agonists or antagonists of cells containing the antigen. Non-human antibodies can be converted into chimeric, veneerized, or humanized forms as described above.

[0060] This specification discloses exemplary antibodies that specifically bind to CD33, EGFR, PD-L1, CD40, OX40, GITR, and ICOS. These antibodies are characterized by mature heavy and light chain variable region sequences and Kabat CDRs provided in the sequence listing, as shown in Table 1 below.

[0061] [Table 1]

[0062] This specification discloses exemplary single-chain Fv (scFv) antibodies that bind to CD40, OX40, GITR, ICOS, and 4-1BB. These scFv antibodies are shown in Table 2 below.

[0063] [Table 2]

[0064] Other antibodies with the same CDR as defined by Kabat or alternative definitions, e.g., Chothia, a combination of Chothia and Kabat, AbM, or Contact (see bioinf.org.uk / abs on the World Wide Web), or other antibodies that bind to the same epitope or compete for binding to any of these antibodies and their target proteins, may also be used. Alternatively, other means of binding to any of the above targets may be used instead of the antibodies mentioned above. Antibodies that bind to CD40, OX40, GITR, ICOS, and 4-1BB can agonize their receptors, thereby activating immune cells that express those receptors. Antibodies that bind to cancer-associated antigens may or may not antagonize the interaction between the cancer-associated antigen and its ligand or counterreceptor. Antagonism provides an additional mechanism of cytotoxicity against cancer cells, but is not essential for the activation of immune cells by the binding arm of bispecific antibodies that bind to costimulatory receptors.

[0065] Other antibodies against CD33 include gentuzumab, lintuzumab, and BI836858 (Heider Blood 2011;118(15):4159-4168). Antibodies against EGFR include the chimeric version of C225 marketed as cetuximab, panitumumab, matuzumab, and nesitumumab. Antibodies against PD-L1 include durvalumab, avelumab, atezolizumab, and MDX-1105 / BMS936559. Agonist antibodies against CD40 include CP-870, 893 (Pfizer and VLST), dacetuzumab (Seattle Genetics), Chi Lob 7 / 4 (University of Southampton), and lucatumab (Novartis) (Vonderheide et al., Clin Cancer Res 19, 1035-1043 (2013)). Agonist OX40 antibodies include MOXR0916 (Infante et al., Journal of Clinical Oncology 2016 34:15_suppl 101-101), PF-04518600 (Long et al., Journal of Clinical Oncology 2016 34:15_suppl, e14518-e14518), MEDI0562 (Glisson et al., Annals of Oncology, Volume 27, Issue suppl_6, 1 October 2016, 1052PD), and MEDI6469 (Bell et al., Clin Cancer Res 2017;23(23_Suppl):Abstract nr 37). Agonist antibodies against GITR include DTA-1 (Shimizu et al., Nat Immunol. 3, 135-142, 2002), INCAGN01876 (Gonzalez et al. Cancer Res. 2016 Volume 76, Issue 14, Abstract 3220), and 28F3, 19D3, 18E10, 3C3, 2G6, 8A6, 9G7, 14E3, 19H8, and 6G10 (WO2017087678).Agonist antibodies against ICOS include MEDI-570 (Medimmune; Nicholson et al. Reprod. Toxicol. 2017 74:116-133), GSK3359609 (GlaxoSmithKline; Angevin et al. 2017 Cancer Res. Volume 77, Issue 13 Supplement, Abstract CT039), BMS-986226 (Bristol-Myers Squib; Wang et al. 2019 BBA Rev. Cancer 1871:199-224), and JTX-2011 (Jounce Therapeutics; Michaelson et al. 2016 Cancer Res. Volume 76, Issue 14 Supplement, Abstract 573). Agonist antibodies against 4-1BB include urelumab (Bristol-Myers Squib), utomirumab (Pfizer), and AGEN2373 (Agenus; Galand et al. 2019 J. Clin. Oncol. 37, no. 15 suppl.e14005).

[0066] Any of these antibodies, or any other antibody having the same CDR as defined by Kabat or an alternative definition, such as Chothia, a combination of Chothia and Kabat, AbM, or Contact, or any other antibody that binds to the same epitope or competes for binding to any of these antibodies and its target protein, can be incorporated into the bispecific antibodies of the present invention. Alternatively, other means of binding to any of the above targets can be used instead of the antibodies.

[0067] The present invention also provides monoclonal antibodies that specifically bind to human PD-L1, including HuPRO1, HuPRO2, and HuPRO5, and other antibodies that share the same six CDRs (by any conventional definition) or the same pairs of mature heavy and light variable regions as one of the antibodies. Such antibodies can inhibit the interaction between PD-L1 and PD1. The present invention also provides monoclonal antibodies that specifically bind to human OX40, including HuOHX14DS, and other antibodies that share the same six CDRs (by any conventional definition) or the same pairs of mature heavy and light variable regions as the antibody. The present invention also provides monoclonal antibodies that specifically bind to human CD40, including HuACS2, and other antibodies that share the same six CDRs (by any conventional definition) or the same pairs of mature heavy and light variable regions. The present invention also provides monoclonal antibodies that specifically bind to human ICOS, including HuTAM14, and other antibodies that share the same six CDRs (by any conventional definition) or the same pairs of mature heavy and light variable regions. The present invention also provides monoclonal antibodies that specifically bind to human 4-1BB, including HuFOB5, and other antibodies that share the same six CDRs (as defined conventionally) or the same pairs of mature heavy and light chain variable regions.

[0068] IV. Format of bispecific antibodies For bispecific antibodies, more than 100 formats have been described (e.g., Kontermann et al., Drug Discovery Today 20, 838-847 (2015); Sedykh et al., Drug Des. Devel. Ther. 2, 195-209 (2018)). Such formats include at least one binding site for each of the two targets. Preferred formats include two or more binding sites for each target.

[0069] Some formats have a tetramer structure similar to that of a normal antibody, with two binding regions, one for each target. Each binding region is formed from the variable regions of a pair of heavy and light chains, which are linked to the constant regions of the heavy and light chains, respectively. Such bispecific antibodies differ from normal antibodies in that they have two different binding sites and the pairs of heavy and light chains that form them. Therefore, such antibodies require the association of two different pairs of heavy and light chains.

[0070] The "knob-into-hole" approach is employed to reduce homodimer formation and heavy chain mispairing by substituting a large amino acid in the CH3 domain of one antibody with a small amino acid ("knob") and doing the reverse in the other antibody ("hole") (Ridgway et al., Protein Eng 9:617-21, 1996; Atwell et al., J Mol Biol 270:26-35, 1997; and US Pat. No. 7,695,936). Light chain mispairing in such a format can be reduced by several strategies. One strategy is to use a common light chain variable region for two different heavy chain variable regions. However, this is only applicable to some antibodies. Another approach is to express the knob-containing half and the hole-containing half in separate bacteria. Another approach, called CrossMab, involves swapping one CH1 domain of the heavy chain with the corresponding constant CL domain of the light chain to induce the correct pairing between the designed heavy and light chains (Schaefer et al., Proc Natl Acad Sci USA 108:11187-92, 2011; WO 2009 / 080251; WO 2009 / 080252; WO 2009 / 080253). Another approach involves introducing additional mutations at the VH-VL and CH1-CL interfaces (Lewis et al., Nat. Biotechnol., 32 (2014), pp. 191-198). These mutations encourage the heavy chain to preferentially pair with the light chain. Another approach involves introducing a mutation that promotes protein A binding into one of the Fc regions and selecting heterodimer pairs with intermediate protein A binding from homodimers with higher or lower protein A binding by affinity chromatography (Tusdian et al, MAbs. 2016 May-Jun;8(4):828-38).

[0071] Other bispecific antibodies avoid the problem of mispairing by combining multiple binding specificities on the same heavy-light chain pair. One approach to doing this is called a dual variable domain, in which two different heavy-chain variable regions are tandem-linked to the heavy-chain constant region, and two different light-chain variable regions are tandem-linked to the light-chain constant region (Correia et al., MAbs. 2013 May 1; 5(3): 364-372). Such antibodies can be assembled as tetramers by the association of two identical pairs of heavy-light chains. The assembled antibody contains two different binding sites for each target.

[0072] Another approach followed in embodiments of the present invention is to incorporate a second binding specificity by ligating a single-chain Fv (scFv) to the C-terminus of the heavy chain constant region. Such a bispecific antibody, like a standard antibody, includes a first binding site formed by a heavy chain variable region and a light chain variable region bound to the N-terminus of the heavy chain constant region and the light chain constant region. The C-terminus of the heavy chain is bound to an scFv that provides a second binding site. The scFv is typically bound via a linker, which further links the heavy chain and light chain variable regions of the scFv. The scFv can bind to the Fc region via a linker from the end of its light chain variable region or heavy chain variable region. When assembled by the complexation of two identical pairs of heavy and light chains, such a bispecific antibody includes two binding sites in each of the two different specificities. The cancer-associated antigen or infected cell antigen and the costimulatory antigen binding arm of such a bispecific antibody can be attached in either direction. Arms that bind to the N-terminus of the constant regions of the heavy and light chains are provided separately as variable regions of the heavy and light chains, while arms that bind to the C-terminus are provided as scFv fragments. The advantage of this format is that the two distinct binding spaces are separated by the entire constant region of the heavy chain, which can facilitate intercellular bridging.

[0073] In another format, an scFv that specifically binds to the first target is ligated to the heavy chain constant region, and an scFv that specifically binds to the other target is ligated to the light chain constant region. Such antibodies are assembled as tetramers containing two copies of each binding site (Bs(scFv)4-IgG) (Zuo et al., Protein Eng 13: 361-367, 2000).

[0074] Other formats link scFv binding regions in a single strand without a constant region. For example, the BiTe format links two scFv fragments via a linker (see, e.g., Ross et al., PLoS ONE 12(8): e0183390, 2017). Such formats lack effector functionality and tend to have shorter half-lives, but may offer advantages in terms of accessibility and ease of production due to their smaller size.

[0075] Many of the above formats include a linker peptide between the heavy chain and the light chain variable region, or between the variable region and the constant region. The linker is a short peptide that provides flexibility and is often occupied primarily by Gly, Ala, and / or Ser. Some exemplary linkers are Gly-Gly-Ala-Ala, Gly-Gly-Gly-Gly-Ser, Leu-Ala-Ala-Ala-Ala, and their multimers.

[0076] V. Selection of the steady-state region Many bispecific antibody formats include at least a portion of the human constant region. The selection of the constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 possess complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. The light chain constant region can be lambda or kappa. Also, human IgG1 and IgG3 induce stronger cell-mediated effector function than human IgG2 and IgG4. Here, ADCC, ADCP, and CDC are useful for providing an additional mechanism of action against cancer or infected cells bound by one arm of the bispecific antibody, but are not useful for agonizing co-stimulatory molecules and activating immune cells by the other arm.

[0077] One or more amino acids at the amino or carboxyl termini of the light and / or heavy chains, for example, the C-terminal lysine of the heavy chain, may be deleted or derivatized in part or all of the molecule. Amino acid substitutions can be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For example, there are many known mutations in IgG Fc that increase FcRn binding. Exemplary substitutions include Gln at position 250 and / or Leu at position 428, Ser or Asn at position 434, Tyr at position 252, Thr at position 254, Glu at position 256, and Ala at position 434 (EU numbering). Increased FcRn binding is advantageous for the hybrid protein of the present invention to compete more strongly with endogenous IgG for binding to FcRn. Numerous mutations are also known to reduce ADCC, ADCP, or CDC. (See, for example, Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006). For example, substitution of any of the amino acid residues at positions 234, 235, 236, and / or 237 reduces affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., US 6,624,821). Optionally, substitution of the amino acid residues at positions 234, 236, and / or 237 of human IgG2 with Ala, and substitution of position 235 with Gln or Glu (see, e.g., US 5,624,821).Other substitutions that reduce the effector function include Ala at position 268, Gly or Ala at position 297, Leu at position 309, Ala at position 322, Gly at position 327, Ser at position 330, Ser at position 331, Ser at position 238, Ala at position 268, and Leu at position 309.

[0078] The human constant region exhibits allotypic and isoallotypic variations between different individuals. That is, the constant region can differ in one or more polymorphic sites within different individuals. Isoaltypes differ from allotypes in that the serum recognizing the isoallotype binds to the non-polymorphic regions of one or more other isotypes.

[0079] VI. Expression of recombinant antibodies Bispecific antibodies are typically produced by recombinant expression. Depending on the bispecificity format, the expression of one, two, or more antibody chains may be required. When multiple chains are expressed, they can be expressed from the same vector or different vectors. Recombinant polynucleotide constructs typically include expression control sequences operably ligated to the coding sequence of the antibody chain, including spontaneously occurring or heterologous expression control elements such as promoters. The expression control sequences may be promoter systems for vectors that can transform or transfect eukaryotic or prokaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high levels of nucleotide sequence expression and collection and purification of bispecific antibodies.

[0080] These expression vectors are typically replicable in the host organism, either as episomes or as an integral part of the host's chromosomal DNA. Typically, the expression vectors include a selection marker, such as ampicillin resistance or hygromycin resistance, to enable the detection of those cells transformed with the desired DNA sequence.

[0081] Escherichia coli (E. coli) is one of the prokaryotic hosts useful for antibody expression, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces is one of the yeast hosts that has suitable vectors with expression regulatory sequences, origins of replication, and stop sequences as desired. Typical promoters include glycosphagocytes, such as 3-phosphoglycerate kinase. Inducible yeast promoters include, in particular, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes that are responsible for the utilization of maltose and galactose.

[0082] Mammalian cells can be used to express nucleotides encoding immunoglobulins or their fragments. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). Numerous suitable host cell lines capable of secreting intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas containing Sp2 / 0 and NS0. The cells may also be non-human cells. Expression vectors used in these cells may include expression regulatory sequences such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Expression regulatory sequences may include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0083] Alternatively, the antibody coding sequence may be incorporated into a transgene and introduced into the genome of a transgenic animal, after which it may be expressed in the milk of the transgenic animal (see, for example, US Pat. No. 5,741,957; US Pat. No. 5,304,489; and US Pat. No. 5,849,992). Suitable transgenes include light chain and / or heavy chain coding sequences operably linked to promoters and enhancers derived from mammary gland-specific genes such as casein or beta-lactoglobulin.

[0084] Vectors containing the target DNA segment can be introduced into host cells using methods appropriate to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene guns, or virus-based transfection can be used for other cell hosts. Methods used for transforming mammalian cells include the use of polyblens, protoplast fusion, liposomes, electroporation, and microinjection. Transgenic animals can be created by microinjecting transgenes into oocytes or incorporating them into the genome of embryonic stem cells, and then transferring the nuclei of such cells into enucleated oocytes.

[0085] After introducing one or more vectors encoding the heavy and light chains of an antibody into a cell culture, the cell pool can be screened in serum-free medium for antibody productivity and quality. Next, the cell pool with the highest productivity can be subjected to FACS-based single-cell cloning to create a monoclonal line. Specific production volumes of over 50 pg / cell or over 100 pg / cell per day, corresponding to over 7.5 g per liter of medium, can be used. Antibodies produced by single-cell clones can be tested for turbidity, filtration characteristics, PAGE, IEF, UV scan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore. The selected clones are then stored in multiple vials and cryopreserved for later use.

[0086] After expression, the antibody can be purified according to standard methods in the art, including protein A capture, HPLC purification, column chromatography, and gel electrophoresis (see typically Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0087] Methodologies for the commercial production of antibodies can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single-cell cloning, cell banking, use of selection markers for copy number amplification, CHO terminators, or enhancement of protein titers (see, for example, US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; W02004 / 050884; W02008 / 012142; W02008 / 012142; W02005 / 019442; W02008 / 107388; W02009 / 027471; and US 5,888,809).

[0088] VII. Nucleic acids The present invention further provides nucleic acids encoding either the heavy chain or the light chain as described above. Optionally, such nucleic acids may further encode a signal peptide, which can be ligated to a constant region coding sequence of the nucleic acid for expression, and can be operably ligated to control sequences to ensure the expression of the coding sequence, such as promoters, enhancers, ribosome binding sites, and transcription termination signals. Nucleic acids encoding the heavy chain and light chain can be generated in isolation or cloned into one or more vectors. Nucleic acids can be synthesized, for example, by solid-phase synthesis or by PCR of overlapping oligonucleotides. Nucleic acids encoding the heavy chain and light chain can be bound together as a single continuous nucleic acid, for example, in an expression vector, or separately, for example, each can be cloned into its own expression vector.

[0089] VIII. Treatment methods and pharmaceutical compositions The bispecific antibodies of the present invention can be used in the treatment of cancer, including those in which one arm of the bispecific antibody binds to a target expressed or overexpressed in cancer, as disclosed above. The bispecific antibodies can be used in the treatment of solid tumors and hematological malignancies. Hematological malignancies include leukemia (e.g., T-cell large granular lymphocyte leukemia), lymphoma (Hodgkin or non-Hodgkin), or multiple myeloma. Solid tumors include skin (e.g., melanoma), ovaries, endometrium, kidneys, liver, pancreas, bladder, breast, prostate, rectum, colon, stomach, intestines, lungs, thymus, thyroid, kidneys, and brain.

[0090] The bispecific antibodies of the present invention may be used to treat pathogenic infections if the bispecific antibody has one arm that specifically binds to an antigen expressed in infected cells but not in corresponding uninfected cells. Such antigens may be encoded by a pathogen or expressed by cells in response to infection by a pathogen. Examples of antigens expressed in infected cells include the glycoproteins gp41 and gp120 of human immunodeficiency virus (HIV), the Env protein of human T-cell leukemia virus type 1 (HTLV-1), the glycoproteins gB and gH of herpes simplex virus (HSV), influenza hemagglutinin (HA) and neuraminidase (NA), and the F protein of respiratory syncytial virus (RSV). Examples of pathogenic infections treatable with bispecific antibodies include viral, bacterial, protozoan, and fungal infections. Examples of viral infections include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, Epstein-Barr virus), adenovirus, XMRV, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory cynthiavirus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, MLV-associated virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. Some examples of bacterial infections include Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Conococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum, Anthrax, Plague, Leptospirosis, Lymeymus bacillus, Streptococcus, or Neisseria. Examples of pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Examples of protozoa include Cryptosporidium, Giardia ramuria, and Plasmodium.

[0091] Bispecific antibodies are administered in an effective regimen that means a dosage, route of administration, and frequency of administration that delays the onset of the disease, reduces its severity, prevents further exacerbation, and / or improves at least one sign or condition of the disease. If the subject already has the disease, the regimen can be called a therapeutically effective regimen. If the subject is at higher risk of the disease than the general population but has not yet experienced symptoms, the regimen can be called a prophylactically effective regimen. In some cases, therapeutic or prophylactic effectiveness can be observed in individual subjects compared to past controls or past experience in the same subjects. In other cases, therapeutic or prophylactic effectiveness can be demonstrated in preclinical or clinical trials in the treated subject population compared to a control population of untreated subjects.

[0092] Preferably, the bispecific antibody exhibits at least additive activity, and more preferably synergistic activity, against cancer or infected cells when compared individually to its constituent antibodies. The synergistic effect is preferably evaluated quantitatively, as described in Tallarida, Genes Cancer. 2011 Nov; 2(11): 1003-1008. Also preferably, the bispecific antibody exhibits increased activity compared to a mixture of its constituent antibodies, each at equimolar concentration with respect to the bispecific antibody. Such activity can be measured, for example, as cytotoxicity or cytostatism against cancer or infected cells expressing an antigen specifically bound to one arm of the bispecific antibody, in the presence of immune cells expressing a costimulatory molecule specifically bound to the other arm of the bispecific antibody.

[0093] Exemplary doses of bispecific antibodies are 0.01–20 mg / kg body weight, or 0.5–5 mg / kg, or 0.01–1 mg / kg, or 0.01–0.5 mg / kg, or 0.05–0.5 mg / kg (e.g., 0.1, 0.5, 1, 2, 3, 4, or 5 mg / kg), or a fixed dose of 10–1500 mg. The dose depends on the patient's condition and response to prior treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0094] Administration can be carried out parenterally, intravenously, orally, subcutaneously, intra-arterially, intracranially, intrathecally, intraperitoneally, locally, intranasally, or intramuscularly. For systemic circulation, intravenous or subcutaneous administration is preferred. Intravenous administration can be carried out by infusion over a period of time, such as 30 to 90 minutes.

[0095] The frequency of administration depends, among other factors, particularly the half-life of the bispecific antibody in the circulatory system, the patient's condition, and the route of administration. Administration can be daily, weekly, monthly, quarterly, or irregularly depending on changes in the patient's condition or the progression of the patient's disorder. For intravenous administration, a frequency of once a week to once a quarter is exemplified during a continuous treatment period, but more or less frequent administration is also possible. For subcutaneous administration, an exemplary frequency is daily to monthly, but more or less frequent administration is also possible.

[0096] The number of administrations depends on whether the disorder is acute or chronic, and the disorder's response to treatment. In cases of acute disorder or acute exacerbation of chronic disorder, one to ten administrations are often sufficient. In cases of acute disease or acute exacerbation of chronic disease, a single bolus administration (optionally divided into multiple doses) may be sufficient. Treatment can be repeated for recurrences of acute disease or acute exacerbation. For chronic disease, bispecific antibodies can be administered regularly, for example, weekly, bi-weekly, monthly, quarterly, or every six months, for at least one year, five years, ten years, or throughout the patient's lifetime.

[0097] The pharmaceutical composition is preferably suitable for parenteral administration to humans (e.g., in accordance with FDA standards). The pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic and is produced under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., the dosage for a single administration). The pharmaceutical composition can be formulated using one or more pharmaceutically acceptable carriers, diluents, excipients or adjuvants. Pharmaceutically acceptable means suitable for administration to humans, e.g., approved by the FDA or approvable. The formulation varies depending on the selected route of administration. In the case of injection, the antibody can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, physiological saline or acetate buffer (to reduce discomfort at the injection site). This solution may contain modifiers such as suspending agents, stabilizers, dispersing agents, etc. Alternatively, the antibody may be in a lyophilized form to be constituted with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0098] The treatment with the bispecific antibody of the present invention can be combined with other treatments effective against the disorder to be treated. When used for the treatment of cancer, the bispecific antibody of the present invention can be combined with treatments with other biologics such as chemotherapy, radiation, stem cell therapy, surgery, or Herceptin TM (trastuzumab), Avastin TM (bevacizumab) against VEGF, or an antibody against the EGF receptor (e.g., (Arbitux TM , cetuximab), and Vectibix TM (panitumumab)). Chemotherapeutic agents include chlorambucil, cyclophosphamide or melphalan, carboplatinum, daunorubicin, doxorubicin, idarubicin, and mitoxantrone, methotrexate, fludarabine, and cytarabine, etoposide or topotecan, vincristine, vinblastine. For infectious diseases, antibiotics, antiviral agents, antifungal agents, antiparasitic agents, etc. can be used in combination for treatment.

[0099] IX. Other methods The bispecific antibodies of the present invention can also be used in diagnostic, prognostic, and experimental methods. They can be used to measure the level of antigen expressed by cancer, or the level of antigen in circulation in a patient with cancer, to determine whether the level is measurable or elevated, and thus to track and guide cancer treatment. This is because cancers associated with measurable or elevated levels of antigen are most sensitive to treatment with bispecific antibodies containing an arm that binds to cancer. Bispecific antibodies can be used in particular in ELISA assays, radioimmunoassays, or immunohistochemistry. Bispecific antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes and may be provided in the form of a kit containing all the reagents necessary to perform the assay.

[0100] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated by reference in whole to the same extent that each individual item is specifically and individually indicated to be incorporated by reference for all purposes. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number on the effective filing date of this application is meant. The effective filing date, where applicable, refers to the earlier of the actual filing date and the filing date of the priority application, with reference to the accession number. Similarly, if different versions of a publication, website, etc., are published at different times, unless otherwise indicated, the version most recently published on the effective filing date of this application is meant. Any feature, process, element, embodiment, or aspect of the present invention may be used in combination with others unless specifically indicated elsewhere. While the present invention has been described in some detail using examples and embodiments for clarity and understanding, it will be apparent that certain changes and modifications can be made within the scope of the appended claims. [Examples]

[0101] Example 1: General Method and Materials

[0102] Gene cloning, mutagenesis, plasmid construction, ELISA, and FACS were performed according to standard experimental techniques described in Green and Sambrook (Molecular Cloning, A Laboratory Manual, 4th ed., 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Greenfield (Antibodies, A Laboratory Manual, 2nd ed., 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556-565, 2001), Cole et al. (J. Immunol. 159:3613-3621, 1997), and Tsurushita et al. (Methods 36:69-83, 2005), as well as vendor protocols.

[0103] Mouse hybridomas producing monoclonal antibodies against desired antigens were constructed at JN Biosciences (Cosmo Bio, Carlsbad, CA) following standard cell fusion techniques using GenomONE CF EX Cell Fusion Reagent (Cosmo Bio, Carlsbad, CA). For immunization of mice, purified soluble recombinant antigens, typically fusions to the human IgG Fc region, or mouse cell lines expressing recombinant antigens on their surface were used. Mouse monoclonal antibodies secreted into the culture supernatant of hybridoma cells were subjected to a series of screenings to identify antibodies with the following characteristics: (1) specific binding to human antigens, (2) specific binding to cynomolgus monkey antigens, and (3) desired biological functions such as blocking the interaction between the antigen and its ligand, or stimulating cell activity by binding to antigens on the cell surface. Selected hybridoma cells were grown in serum-free media such as Hybridoma SFM (Thermo Fisher Scientific, Waltham, MA). Mouse monoclonal IgG antibodies were purified using a protein A affinity column (MabSelect SuRe, GE Healthcare Life Sciences, Marlborough, MA) according to the manufacturer's protocol. The buffer of the purified antibodies was replaced with phosphate-buffered saline (PBS) by dialysis. The antibody concentration was determined by measuring the absorbance at 280 nm (OD 1.4 = 1 mg / ml).

[0104] The expression level of mouse IgG antibody in the culture supernatant was measured by sandwich ELISA. In a typical experiment, an ELISA plate was coated with goat anti-mouse IgG Fc-specific polyclonal antibody (SouthenBiotech, Birmingham, AL), washed with Wash Buffer (PBS containing 0.05% Tween 20), and blocked with ELISA Buffer (PBS containing 2% skim milk and 0.05% Tween 20). After washing with Wash Buffer, the test sample, appropriately diluted with ELISA Buffer, was placed on the ELISA plate. An appropriate mouse IgG / kappa antibody was used as a standard. The ELISA plate was incubated at room temperature for 60 minutes, washed with Wash Buffer, and the conjugated mouse antibody was detected using HRP-conjugated goat anti-mouse kappa chain polyclonal antibody (Bethyl Laboratories, Montgomery, TX). The plate was incubated at room temperature for 30 minutes, washed with Wash Buffer, and then ABTS substrate (Sigma-Aldrich, St. Louis, MO) was added to initiate color development, which was stopped with 2% oxalic acid. The absorbance was read at 405 nm.

[0105] The expression of chimeric antibodies and humanized IgG / kappa antibodies in the culture supernatant was measured using the sandwich ELISA method described above, except that goat anti-human IgG Fc-specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA) was used to coat the ELISA plate, and HRP-conjugated goat anti-human kappa chain polyclonal antibody (Bethyl Laboratories) was used to detect the bound antibodies.

[0106] The heavy and light chain variable regions (VH and VL, respectively) of mouse monoclonal antibodies were sequenced using standard molecular biological techniques, such as those described by Tsurushita et al. (Methods 36: 69-83, 2005). Humanization of VH and VL was performed using the general procedure described by Tsurushita et al. (ibid.) as follows: First, a three-dimensional molecular model of the variable region of the mouse antibody to be humanized was constructed using appropriate software. Next, this molecular model was used to identify framework amino acid residues that are important for the formation of the CDR (complementarity-determining region) structure or that are necessary for antigen binding. In parallel, human VH and VL amino acid sequences derived from cDNA that have high homology to the amino acid sequences of mouse VH and VL were selected, respectively. Finally, the CDR sequence, along with the framework amino acid residues identified as important for antigen-binding site formation, was grafted from the mouse variable region onto the corresponding selected human framework sequence.

[0107] Stable transfection of the Chinese hamster ovary cell line CHO-K1 was performed by electroporation. Prior to transfection, the expression vector was linearized using appropriate restriction enzymes. In a typical experiment, approximately 10 7 CHO-K1 cells were transfected with 20 μg of linearized plasmid, suspended in SFM4CHO medium (GE Healthcare Life Sciences), and after appropriate cell dilution, plated in several 96-well plates at 100 μl / well. After 48 hours, 100 μl / well of SFM4CHO medium containing 20 μg / ml puromycin was added, and stable transfectants were isolated. Approximately 10 days after the start of selection, the culture supernatant of the transfectants was assayed for antibody production. CHO-K1 stable transfectants producing high levels of antibody were identified by ELISA as described above.

[0108] CHO-K1 stable transfectants, which highly produce recombinant antibodies such as the humanized IgG antibody and bispecific IgG antibody of the present invention, were cultured in SFM4CHO until the cell viability was less than 50%. After centrifugation and filtration, the culture supernatant was loaded onto a Protein A column (HiTrap MABSelect SuRe, GE Healthcare Life Sciences). After washing the column with PBS, the antibodies were eluted with 0.1M glycine-HCl (pH 3.0) containing 0.1M NaCl or 0.1M sodium acetate (pH 3.6). The buffer of the eluted antibodies was neutralized with 1M Tris-HCl (pH 8.0) and then converted to PBS by dialysis. The absorbance at 280 nm was measured to determine the antibody concentration (1 mg / ml = 1.4 OD).

[0109] Example 2: Construction of a bispecific antibody (BS824) that binds to CD33 and OX40.

[0110] The mammalian expression vector pHuM195-IgG1 (Figure 1A), designed for the production of a humanized anti-CD33 IgG1 / kappa antibody that binds to human CD33 (also known as Siglec-3; SEQ ID NO: 1) (Co et al., J. Immunol. 148:1149-1154, 1992), contains the following genetic elements. Starting clockwise from the SalI site in Figure 1A, the vector contains a heavy chain transcription unit that begins with the human cytomegalovirus (CMV) major early promoter and enhancer (CMV-P in the figure) to initiate transcription of the antibody heavy chain gene. Following the CMV promoter is a genome sequence containing an exon encoding humanized M195 VH (SEQ ID NO: 2) sandwiched between the SpeI and HindIII sites, exons encoding the constant regions of the human gamma-1 heavy chain CH1 (SEQ ID NO: 3), Hinge (SEQ ID NO: 4), CH2 (SEQ ID NO: 5), and CH3 (SEQ ID NO: 6), and intervening introns. After the EagI site is the polyadenylation site for the human gamma-1 heavy chain gene. Following the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter and enhancer (CMV-P), and includes a genome sequence containing an exon encoding humanized M195 (HuM195) VL (SEQ ID NO: 7) sandwiched between the NheI and EcoRI sites, an exon encoding the human kappa chain constant region exon (Ck) (SEQ ID NO: 8) preceded by a portion of an intron, and the polyadenylation site for the human kappa chain gene following the Ck exon. Following the light chain gene are the SV40 initial promoter (SV40-P), the puromycin N-acetyltransferase gene for puromycin resistance (puro), and a segment containing the SV40 polyadenylation site (SV40-A). Finally, pHuM195-IgG1 contains the bacterial origin of replication (pUC ori) and the β-lactamase gene (β-lactamase). The arrows in the figure indicate the direction of transcription. Humanized IgG1 / kappa monoclonal antibody (HuM195-IgG1) is expressed from pHuM195-IgG1 in mammalian cells.

[0111] Mouse hybridomas producing OHX14, an IgG / kappa monoclonal antibody that specifically binds to human and cynomolgus monkey OX40 (also known as TNFRSF4 and CD134), were isolated as described in Example 1. As an immunogen, mouse myeloma cell line NS0, which stably expresses recombinant human OX40 on its surface, was used. This recombinant human OX40 was constructed by fusing the extracellular domain of human OX40 to the FLAG polypeptide (SEQ ID NO: 9), and then fusing it to the glycosylphosphatidylinositol (GPI) anchorage signal (SEQ ID NO: 10) of human CD55 (OX40-FLAG-GPI; SEQ ID NO: 11). Sequencing and humanization of OHX14 VH and VL were performed as described in Tsurushita et al. (cited above).

[0112] The amino acid sequence of humanized OHX14 (HuOHX14DS)VH is MGRLTSSFLLLIVPAYVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS (SEQ ID NO: 12). According to Kabat et al.'s definition (Sequences of Proteins of Immunological Interests, Fifth edition, NIH Publication No. 91-3242, US Department of Health and Human Services, 1991), the amino acid sequences of CDR1, 2, and 3 of HuOHX14DS VH are SYIMH (SEQ ID NO: 13), YINPYNSGTKYNEKFKG (SEQ ID NO: 14), and YYGSTFTMDY (SEQ ID NO: 15), respectively. The gene encoding HuOHX14DS VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0113] The amino acid sequence of humanized OHX14 (HuOHX14DS)VL is MMSSAQFLGLLLLCFQGTRCDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 16). The amino acid sequences of CDR1, 2, and 3 of HuOHX14DS VL are RASQDIRTYLN (SEQ ID NO: 17), YTSRLHS (SEQ ID NO: 18), and QQGNTLPWT (SEQ ID NO: 19), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuOHX14DS VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0114] The expression vector pHuOHX14DS-IgG1.AA has the same structure as pHuM195-IgG1, except that (i) the HuOHX14DS VH exon is positioned between the SpeI and HindIII sites, (ii) the HuOHX14DS VL exon is positioned between the NheI and EcoRI sites, and (iii) two leucine residues at positions 234 and 235 of CH2 (Kabat et al.'s EU numbering, cited above) are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) in order to eliminate effector function (Hezareh et al., J. Virol. 75:12161-12168, 2001). The novel vector pHuOHX14DS-IgG1.AA expresses a humanized anti-OX40 IgG1 / kappa antibody (HuOHX14DS-IgG1.AA) in mammalian cells.

[0115] The mammalian expression vector pBS824 (Figure 1B), designed for the expression of a bispecific antibody that binds to both human CD33 (SEQ ID NO: 1) and human OX40 (SEQ ID NO: 21), was constructed by modifying pHuM195-IgG1 as follows: The VL and VH coding regions of the humanized anti-human OX40 monoclonal antibody pHuOHX14DS-IgG1.AA were converted to a single-stranded Fv (scFv) form from the N-terminus to the C-terminus in the order of VL, polypeptide linker, and VH (HuOHX14DS.scFv; SEQ ID NO: 22). The N-terminus of HuOHX14DS.scFv was fused to the second-to-last glycine residue of CH3 in pHuM195-IgG1, separated by a polypeptide linker (CH3-HuOHX14D.scFv; SEQ ID NO: 23). Furthermore, two leucine residues at positions 234 and 235 (EU numbering) in CH2 were replaced with alanine residues (L234A and L235A, respectively). The resulting vector pBS824 expresses a bispecific IgG antibody named BS824 that binds to both human CD33 and OX40.

[0116] The amino acid sequence of the mature heavy chain encoded in pBS824 is (SEQ ID NO: 24).

[0117] The amino acid sequence of the mature light chain encoded in pBS824 is DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25).

[0118] Figure 2 shows the schematic structure of the bispecific IgG antibody of the present invention, such as BS824.

[0119] Each of the three expression vectors, pHuM195-IgG1, pHuOHX14DS-IgG1.AA, and pBS824, was stably transfected into CHO-K1 cells as described above. HuM195-IgG1, HuOHX14DS-IgG1.AA, and BS824 were purified from the culture supernatant of the respective CHO-K1 stable transfectants using a protein A affinity column as described above. SDS-PAGE analysis under reducing conditions showed only two dominant bands for each of the three antibodies: a heavy chain of approximately 50 kD and a light chain of 25 kD (for HuM195-IgG1 and HuOHX14DS-IgG1.AA), or a heavy chain of approximately 75 kD and a light chain of 25 kD (for BS824).

[0120] Example 3: Biological activity of a bispecific antibody (BS824) that binds to CD33 and OX40

[0121] The biological activity of BS824, which binds to CD33 and OX40, was investigated using Jurkat Dual reporter cells (InvivoGen, San Diego, CA) (JD / OX40) expressing human OX40 on their surface and the mouse myeloma cell line NS0 (NS0 / CD33) expressing human CD33 on its surface. OX40 binding on the cell surface is known to trigger NF-κB signaling (Song et al., J. Immunol. 180:7240-7248, 2008). In Jurkat Dual cells, activation of the intracellular NF-κB signaling pathway leads to the expression and secretion of recombinant lutialciferase.

[0122] JD / OX40 was prepared by stably transfecting Jurkat Dual cells with the human OX40 expression vector pFCm331 via electroporation. The expression vector pFCm331 (Figure 1C) has the same structure as pHuM195-IgG1, except that (a) the SpeI-EagI fragment is replaced with a DNA fragment encoding human OX40 (SEQ ID NO: 21), and (b) the light chain gene is removed. Promycin-resistant Jurkat Dual cells expressing OX40 on their surface (JD / OX40) were maintained at 37°C in a 7.5% CO2 incubator in RPMI1640 medium containing 10% fetal bovine serum (FBS). In JD / OX40 cells, no CD33 expression was detected by flow cytometry using HuM195-IgG1 and a secondary PE-labeled goat anti-human IgG antibody.

[0123] NS0 / CD33 was prepared by stably transfecting NS0 cells with the expression vector pFCm267 for expressing human CD33 via electroporation. The expression vector pFCm267 (Figure 1D) has the same structure as pFCm331, except that (a) the puromycin N-acetyltransferase gene (puro) is replaced with E. coli xanthine-guanine-phosphoribosyltransferase (gpt), and (b) the SpeI-EagI fragment is replaced with a DNA fragment encoding recombinant human CD33, consisting of a synthetic signal peptide (SEQ ID NO: 26), the extracellular region of human CD33 (CD33 EC; SEQ ID NO: 27), a FLAG polypeptide (SEQ ID NO: 9), and a GPI anchorage signal (SEQ ID NO: 10) (CD33-FLAG-GPI) from the N-terminus to the C-terminus. (i) NS0 stable transfectants that survived in DME medium containing 10% FBS, 1 μg / ml mycophenolic acid, HT medium supplement (Sigma-Aldrich, St. Louis, MO), and 0.25 mg / ml xanthine, and (ii) expressed CD33-FLAG-GPI on their surface, were maintained in DME medium containing 10% FBS at 37°C in a 7.5% CO2 incubator.

[0124] OX40, a member of the TNF receptor superfamily of immunocostimulatory molecules, requires trimerization on the cell surface to induce the NF-κB pathway of intracellular signaling (Watts, Annu. Rev. Immunol. 23:23-68, 2005; Croft et al., Nat. Rev. Drug Discov. 12:147-168, 2013; Willoughby et al., Mol. Immunol. 83:13-22, 2017). In JD / OX40 cells, multimeric crosslinking of OX40 on the surface upregulates the expression and secretion of lutialciferase. Approximately 200,000 JD / OX40 cells were incubated in 200 μl of RPMI1640 medium containing 10% FBS, 0.5 μg / ml mouse anti-human CD3 IgG antibody OKT3 (BioLegend, San Diego, CA), and 5 μg / ml goat anti-mouse IgG antibody (human IgG-absorbed) (Jackson ImmunoResearch) in or without approximately 200,000 NS0 / CD33 cells, with or without (i) 1 μg / ml HuM195-IgG1 (anti-CD33), (ii) 1 μg / ml HuOHX14DS-IgG1.AA (anti-OX40), (iii) 1 μg / ml BS824, or (iv) no antibody, to provide a primary signal for activating T cells. Luciferase activity in the culture supernatant was measured in three separate steps using the QUANTI-Luc reagent (InvivoGen) according to the vendor's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek, Winooski, VT). The results are shown in Figure 3.

[0125] The mean relative luciferase units (RLUs) in JD / OX40 cells were 6,458 without antibody, 5,997 with HuM195-IgG1, 7,361 with HuOHX14DS-IgG1.AA, 5,009 with BS824, 6,918 with NS0 / CD33, 6,240 with HuM195-IgG1 and NS0 / CD33, 8,698 with HuOHX14DS-IgG1.AA and NS0 / CD33, and 21,684 with BS824 and NS0 / CD33. Luciferase activity in JD / OX40 cells was significantly increased only when both a bispecific antibody (BS824) that binds to CD33 and OX40 and CD33-expressing cells (NS0 / CD33) were present. These results indicate that only BS824, which can bridge JD / OX40 cells and NS0 / CD33 cells (unlike HuM195-IgG1 or HuOHX14DS-IgG1.AA), clusters OX40 on the surface of JD / OX40 cells in proximity to NS0 / CD33 cells, activating JD / OX40 cells and increasing luciferase expression.

[0126] Example 4: Construction of a bispecific antibody (BS827) that binds to EGFR and GITR

[0127] A mammalian expression vector, pCh225-IgG1, for expressing a chimeric IgG1 / kappa antibody that binds to the human epidermal growth factor receptor (EGFR; SEQ ID NO: 28), was constructed as follows: The gene encoding mouse 225(Ch225)VH (SEQ ID NO: 29) was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment. The gene encoding mouse 225(Ch225)VL (SEQ ID NO: 30) was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment. The VH and VL exons of Ch225 were cloned into the corresponding sites of the antibody expression vector. As a result, the plasmid pCh225-IgG1, used to express the anti-EGFR antibody Ch225-IgG1, had the same structure as pHuM195-IgG1 (Figure 1A), except that the VH and VL coding regions were replaced with Ch225 VH and VL, respectively.

[0128] Mouse hybridomas producing the IgG / kappa monoclonal antibody GAB11, which specifically binds to human and cynomolgus monkey GITR (also known as TNFRSF18 and CD357), were isolated as described in Example 1. As the immunogen, the extracellular region of human GITR fused to the Fc region of the human gamma-1 heavy chain (hGITR-Fc; SEQ ID NO: 31) was used. Sequencing and humanization of GAB11 VH and VL were performed as described by Tsurushita et al. (cited above).

[0129] The amino acid sequence of humanized GAB11 (HuGAB11)VH is MAVLGLLLCLVTFPSCVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (SEQ ID NO: 32). The amino acid sequences of CDR1, 2, and 3 of HuGAB11 VH are DYGVS (SEQ ID NO: 33), VIWGGGGTYYNSALKS (SEQ ID NO: 34), and HPYGHFGMDY (SEQ ID NO: 35), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuGAB11 VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0130] The amino acid sequence of humanized GAB11 (HuGAB11)VL is MRVLAELLGLLLFCFLGVRCDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIK (SEQ ID NO: 36). The amino acid sequences of CDR1, 2, and 3 of HuGAB11 VL are HASQNINVWLS (SEQ ID NO: 37), KASNLHT (SEQ ID NO: 38), and QQGQSYPLT (SEQ ID NO: 39), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuGAB11 VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0131] The expression vector pHuGAB11-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuGAB11 VH exon is positioned between the SpeI and HindIII sites, (ii) the HuGAB11 VL exon is positioned between the NheI and EcoRI sites, and (iii) two leucine residues at positions 234 and 235 (EU numbering) of CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., cited above). The novel vector pHuGAB11-IgG1.AA expresses a humanized anti-GITR IgG1 / kappa antibody (HuGAB11-IgG1.AA) in mammalian cells.

[0132] The mammalian expression vector pBS827, designed for the expression of a bispecific antibody that binds to both human EGFR (SEQ ID NO: 28) and human GITR (SEQ ID NO: 40), was constructed by modifying pCh225-IgG1 as follows: The VL and VH coding regions of the humanized anti-human GITR monoclonal antibody pHuGAB11-Ig1.AA were converted to scFv forms from the N-terminus to the C-terminus in the order of VL, polypeptide linker, and VH (HuGAB11.scFv; SEQ ID NO: 41). The N-terminus of HuGAB11.scFv was fused to the second-to-last glycine residue of CH3 of pCh225-IgG1 using a polypeptide linker (CH3-HuGAB11.scFv; SEQ ID NO: 42). Furthermore, two leucine residues at positions 234 and 235 (EU numbering) of CH2 were replaced with alanine residues (L234A and L235A, respectively). The resulting vector, pBS827, expresses a bispecific IgG antibody named BS827 that binds to both human EGFR and GITR.

[0133] The amino acid sequence of the mature heavy chain encoded in pBS827 is (SEQ ID NO: 43).

[0134] The amino acid sequence of the mature light chain encoded in pBS827 is DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 44).

[0135] The schematic structure of BS827 is shown in Figure 2.

[0136] Three expression vectors, pCh225-IgG1, pHuGAB11-IgG1.AA, and pBS827, were stably transfected into CHO-K1 as described above. Ch225-IgG1, HuGAB11-IgG1.AA, and BS827 were purified from the culture supernatant of their respective CHO-K1 stable transfectants using a protein A affinity column as described above. SDS-PAGE analysis under reducing conditions showed only two dominant bands for each of these three antibodies: a heavy chain of approximately 50 kD and a light chain of 25 kD (for Ch225-IgG1 and HuGAB11-IgG1.AA), or a heavy chain of approximately 75 kD and a light chain of 25 kD (for BS827).

[0137] Example 5: Biological activity of a bispecific antibody (BS827) that binds to EGFR and GITR

[0138] The biological activity of BS827, which binds to EGFR and GITR, was investigated using Jurkat Dual cells (InvivoGen) (JD / GITR) expressing human GITR on their surface and the mouse myeloma cell line NS0 (NS0 / EGFR) expressing human EGFR on its surface.

[0139] JD / GITR was prepared by stably transfecting Jurkat Dual cells with the human GITR expression vector pFCm343 via electroporation. The expression vector pFCm343 has the same structure as pFCm331 (Figure 1C), except that the SpeI-EagI fragment is replaced with a DNA fragment encoding a synthetic signal peptide (SEQ ID NO: 26) fused to mature human GITR (SEQ ID NO: 45). Promycin-resistant Jurkat Dual cells expressing GITR on their surface (JD / GITR) were maintained at 37°C in a 7.5% CO2 incubator using RPMI1640 medium containing 10% FBS. No EGFR expression was detected in JD / GITR cells by flow cytometry using Ch225-IgG1 and a secondary PE-labeled goat anti-human IgG antibody.

[0140] NS0 / EGFR cells were prepared by stably transfecting NS0 cells with the expression vector pFCm507 via electroporation. The expression vector pFCm507 has the same structure as pFCm331 (Figure 1C), except that the SpeI-EagI fragment is replaced from the N-terminus to the C-terminus with a DNA fragment encoding recombinant human EGFR, consisting of a synthetic signal peptide (SEQ ID NO: 26), the extracellular region of human EGFR (SEQ ID NO: 46), a FLAG polypeptide (SEQ ID NO: 9), and the GPI anchorage signal of human CD55 (SEQ ID NO: 10) (EGFR-FLAG-GPI). Promycin-resistant NS0 stable transfectants expressing EGFR-FLAG-GPI on their surface were maintained at 37°C in a 7.5% CO2 incubator using DME medium containing 10% FBS.

[0141] GITR, a member of the TNF receptor superfamily of immunocostimulatory molecules, requires trimerization on the cell surface to trigger the NF-κB pathway of intracellular signaling (Watts, Annu. Rev. Immunol. 23:23-68, 2005; Chattopadhyay et al., Proc. Natl. Acad. Sci. 104: 19452-19457, 2007; Croft et al., Nat. Rev. Drug Discov. 12:147-168, 2013). In JD / GITR cells, multimeric crosslinking of surface GITR upregulates the expression and secretion of lutialciferase. Approximately 200,000 JD / GITR cells were incubated in 200 μl of RPMI1640 medium containing 10% FBS, 0.5 μg / ml mouse anti-human CD3 IgG antibody OKT3 (BioLegend), and 5 μg / ml goat anti-mouse IgG antibody (human IgG absorption) (Jackson ImmunoResearch), along with approximately 200,000 NS0 or NS0 / EGFR cells, for 1 day in a 7.5% CO2 incubator at 37°C in 96-well plates, with the following to provide primary signals for T cell activation: (i) 1 μg / ml Ch225-IgG1 (anti-EGFR), (ii) 1 μg / ml HuGAB11-IgG1.AA (anti-GITR), (iii) 1 μg / ml BS827, or (iv) no antibody. Luciferase activity in the culture supernatant was measured in three separate steps using the QUANTI-Luc reagent (InvivoGen), following the vendor's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek). The results are shown in Figure 4.

[0142] The mean relative luciferase units (RLUs) in JD / GITR cells were 18,086 for NS0 cells alone, 17,183 for Ch225-IgG1 and NS0 cells, 18,504 for HuGAB11-IgG1.AA and NS0 cells, 17,490 for BS827 and NS0 cells, 18,662 for NS0 / EGFR cells alone, 20,748 for Ch225-IgG1 and NS0 / EGFR cells, 18,391 for HuGAB11-IgG1.AA and NS0 / EGFR cells, and 47,523 for BS827 and NS0 / EGFR cells. Luciferase activity in JD / GITR cells was significantly increased only in the presence of both a bispecific antibody (BS827) that binds to EGFR and GITR and EGFR-expressing cells (NS0 / EGFR). These results indicate that only BS827, which can bridge JD / GITR cells and NS0 / EGFR cells (and not Ch225-IgG1 or HuGAB11-IgG1.AA), clusters GITR on the surface of JD / GITR cells in proximity to NS0 / EGFR cells, activating JD / GITR cells and increasing luciferase expression.

[0143] Example 6: Bispecific antibody (BS828) that binds to CD33 and CD40

[0144] Mouse hybridomas producing ACS2, an IgG / kappa monoclonal antibody that binds to human and cynomolgus monkey CD40 (also known as TNFRSF5), were isolated as described in Example 1. As the immunogen, the extracellular region of human CD40 fused to the Fc region of the human gamma-1 heavy chain (hCD40-Fc; SEQ ID NO: 47) was used. Sequencing and humanization of ACS2 VH and VL were performed as described by Tsurushita et al. (Methods 36:69-83, 2005).

[0145] The amino acid sequence of humanized ACS2 (HuACS2)VH is MKLWLNWVFLLTLLHGIQCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS (SEQ ID NO: 48). The amino acid sequences of CDR1, 2, and 3 of HuACS2 VH are SYWLH (SEQ ID NO: 49), RIDPNSGDTKYNEKFKS (SEQ ID NO: 50), and YYYGRSYFDY (SEQ ID NO: 51), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuACS2 VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0146] The amino acid sequence of mature humanized ACS2 (HuACS2) VL is MDFQVQIFSFLLISAVIISRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO: 52). The amino acid sequences of CDR1, 2, and 3 of HuACS2 VL are SASSSVSYMH (SEQ ID NO: 53), DTSKLAS (SEQ ID NO: 54), and QQWSSNPLT (SEQ ID NO: 55), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuACS2 VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0147] The expression vector pHuACS2-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuACS2 VH exon is positioned between the SpeI and HindIII sites, (ii) the HuACS2 VL exon is positioned between the NheI and EcoRI sites, and (iii) two leucine residues at positions 234 and 235 (EU number) of CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., cited above). The novel vector pHuACS2-IgG1.AA expresses a humanized anti-CD40 IgG1 / kappa antibody (HuACS2-IgG1.AA) in mammalian cells.

[0148] A mammalian expression vector pBS828, designed for the expression of a bispecific antibody that binds to both human CD33 (SEQ ID NO: 1) and human CD40 (SEQ ID NO: 56), was constructed by modifying pHuM195-IgG1 as follows: The VL and VH coding regions of the humanized anti-human CD40 monoclonal antibody pHuACS2-Ig1.AA were converted to a single-stranded Fv (scFv) form from the N-terminus to the C-terminus in the order of VL, polypeptide linker, and VH (HuACS2.scFv; SEQ ID NO: 57). The N-terminus of HuACS2.scFv was fused to the second-to-last glycine residue of CH3 in pHuM195-IgG1 using a polypeptide linker (CH3-HuACS2.scFv; SEQ ID NO: 58). Furthermore, two leucine residues at positions 234 and 235 (Eu numbering) of CH2 were replaced with alanine residues (L234A and L235A, respectively). The resulting vector, pBS828, expresses a bispecific IgG antibody named BS828 that binds to both human CD33 and CD40.

[0149] The amino acid sequence of the mature heavy chain encoded in pBS828 is (SEQ ID NO: 59).

[0150] The amino acid sequence of the mature light chain encoded by pBS828 is the same as the mature light chain sequence (SEQ ID NO: 25) encoded by pBS824.

[0151] The schematic structure of BS828 is shown in Figure 2.

[0152] Each of the three expression vectors, pHuM195-IgG1, pHuACS2-IgG1.AA, and pBS828, was stably transfected into CHO-K1 cells as described above. HuM195-IgG1, HuACS2-IgG1.AA, and BS828 were purified from the culture supernatant of their respective CHO-K1 stable transfectants using a protein A affinity column as described above. SDS-PAGE analysis under reducing conditions showed only two dominant bands for each of the three antibodies: a heavy chain of approximately 50 kD and a light chain of 25 kD (for HuM195-IgG1 and HuACS2-IgG1.AA), or a heavy chain of approximately 75 kD and a light chain of 25 kD (for BS828).

[0153] The human Burkitt's B lymphoma cell line, Ramos, expresses CD40 on its surface. It is known that crosslinking CD40 on the surface of Ramos cells with the soluble trimer CD154 (also known as CD40L and TNFSF5), a natural CD40 ligand, induces increased CD95 expression (Henriquez et al., J. Immunol. 162:3298-3307, 1999). The biological activity of BS828, which increases CD95 expression in Ramos cells, was investigated in the presence or absence of the human promyelocytic leukemia cell line HL-60, which expresses CD33 on its surface.

[0154] Approximately 100,000 Ramos cells were incubated in 200 μl of RPMI1640 medium containing 10% FBS for 3 days in a 96-well plate in the presence or absence of approximately 50,000 HL-60 cells, with or without (i) 0.5 μg / ml HuM195-IgG1 (anti-CD33), (ii) 0.5 μg / ml HuACS2-IgG1.AA (anti-CD40), (iii) 0.5 μg / ml BS828, or (iv) no antibody, in 37°C, in a 7.5% CO2 incubator. CD95 expression in Ramos cells was analyzed by flow cytometry using a FITC-labeled mouse anti-human CD20 monoclonal antibody (clone 2H7, BioLegend) and a PE-labeled mouse anti-human CD95 antibody (clone DX2, BioLegend) to detect Ramos cells. Ramos cells are CD20-positive, while HL-60 cells are CD20-negative. The results of the FACS analysis are shown in Figure 5.

[0155] The proportion of CD95-positive Ramos cells within CD20-positive Ramos cells was 1.3% in the absence of antibody, 0.8% with HuM195-IgG1, 1.4% with HuACS2-IgG1.AA, and 1.6% with BS828. When Ramos cells were incubated in the presence of HL-60 cells, the proportion of CD95-positive Ramos cells was 1.0% in the absence of antibody, 1.1% with HuM195-IgG1, 0.7% with HuACS2-IgG1.AA, and 58.4% with BS828. CD95 expression in Ramos cells was significantly increased only in the presence of both a bispecific antibody (BS828) that binds to CD33 and CD40, and cells expressing CD33 (HL-60 cells). These results indicate that only BS828, which can bridge the gap between Ramos cells and HL-60 cells (and not HuM195-IgG1 or HuACS2-IgG1.AA), induces multimeric crosslinking of CD40 on the surface of Ramos cells in close proximity to HL-60 cells, leading to the upregulation of CD95 in Ramos cells.

[0156] Example 7: Bispecific antibody (BS809) that binds to PD-L1 and GITR

[0157] PD-L1 (also known as B7H1 and CD274) is a type I transmembrane protein that functions as a ligand for the checkpoint molecule PD-1. When PD-L1 binds to PD-1 on T cells, it suppresses the immune response. PD-L1 is highly expressed in cancer cells (Patel et l. 2015 Mol. Cancer Ther. 14:847-856; Brody et al. 2017 Lung Cancer 112:200-215; Sun et al. 2018 Immunity 48:434-452). Mouse hybridomas producing the IgG / kappa monoclonal antibody PRO1, which binds to human and cynomolgus monkey PD-L1 and blocks the interaction between PD-1 and PD-L1, were isolated as described in Example 1. As an immunogen, we used the extracellular region of human PD-L1 fused to the Fc region of human gamma-1 heavy chain (hPD-L1-Fc; SEQ ID NO: 60). Sequencing and humanization of PRO1 VH and VL were performed as described by Tsurushita et al. (cited above).

[0158] The amino acid sequence of humanized PRO1 (HuPRO1)VH is MEWNWVVLFLLSLTAGVYAQVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSS (SEQ ID NO: 61). The amino acid sequences of CDR1, 2, and 3 of HuPRO1 VH are SSYIS (SEQ ID NO: 62), WIYAGTGGTSYNQKFTG (SEQ ID NO: 63), and HEGVYWYFDV (SEQ ID NO: 64), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuPRO1 VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0159] The amino acid sequence of humanized PRO1 (HuPRO1) VL is MDFQVQIFSFLLISAVIMSRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK (SEQ ID NO: 65). The amino acid sequences of CDR1, 2, and 3 of HuPRO1 VL are SASSSVSYMH (SEQ ID NO: 66), DTSNLAS (SEQ ID NO: 67), and HQRSSYPWT (SEQ ID NO: 68), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuPRO1 VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0160] The expression vector pHuPRO1-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuPRO1 VH exon is positioned between the SpeI and HindIII sites, (ii) the HuPRO1 VL exon is positioned between the NheI and EcoRI sites, and (iii) two leucine residues at positions 234 and 235 (EU numbering) of CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) in order to eliminate effector function (Hezareh et al., cited above). The novel vector pHuPRO1-IgG1.AA expresses a humanized anti-PD-L1 IgG1 / kappa antibody (HuPRO1-IgG1.AA) in mammalian cells.

[0161] The mammalian expression vector pBS809, designed for the expression of bispecific antibodies that bind to both human PD-L1 (SEQ ID NO: 69) and human GITR (SEQ ID NO: 45), was constructed by replacing the CH3 coding region in pHuPRO1-IgG1.AA with the coding region of CH3-HuGAB11.scFv (SEQ ID NO: 42).

[0162] The amino acid sequence of the mature heavy chain encoded by pBS809 is (SEQ ID NO: 70).

[0163] The amino acid sequence of the mature light chain encoded by pBS809 is EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 71).

[0164] The schematic structure of BS809 is shown in Figure 2.

[0165] Each of the three expression vectors, pHuPRO1-IgG1.AA, pHuGAB11-IgG1.AA, and pBS809, was stably transfected into CHO-K1 as described above. HuPRO1-IgG1.AA, HuGAB11-IgG1.AA, and BS809 were purified from the culture supernatant of their respective CHO-K1 stable transfectants using a protein A affinity column as described above. SDS-PAGE analysis under reducing conditions showed only two dominant bands for each of the three antibodies: a heavy chain of approximately 50 kD and a light chain of 25 kD (for HuPRO1-IgG1.AA and HuGAB11-IgG1.AA), or a heavy chain of approximately 75 kD and a light chain of 25 kD (for BS809).

[0166] The biological activity of BS809, which enhances the immune response, was analyzed by measuring the expression level of IL-2 in human T cells (Malek, Annu. Rev. Immunol. 26:453-79, 2008; Liao et al., Immunity 38:13-25, 2013). Human peripheral blood mononuclear cells (PBMCs) from two donors (3124 and 4143) were obtained from iQ Biosciences (Berkeley, CA). PBMCs were cultured in RPMI-1640 containing 10% FBS and 5 μg / ml phytohemagglutinin-L (PHA-L) in a 7.5% CO2 incubator at 37°C for 3 days to enrich T cells (3-day-old PBMCs). The expression of PD-1, PD-L1, and GITR in 3-day-old PBMCs was confirmed by flow cytometry. Approximately 100,000 3-day-old PBMCs were pre-coated with 1 μg / ml of mouse anti-human CD3 monoclonal antibody OKT3 in the wells of a 96-well plate. The cells were incubated for 1 day in a 7.5% CO2 incubator at 37°C in the following conditions: (i) no additional antibody (control), (ii) 1 μg / ml of HuPRO1-IgG1.AA (anti-PD-L1), (iii) 1 μg / ml of HuGAB11-IgG1.AA (anti-GITR), (iv) 1 μg / ml of HuPRO1-IgG1.AA and 1 μg / ml of HuGAB11-IgG1.AA, and (v) 1 μg / ml of BS809. IL-2 concentrations in the culture supernatant were measured using the Human IL-2 ELISA MAX Standard Kit (BioLegend, San Diego, CA). The results are shown in Figure 6.

[0167] The mean IL-2 concentrations in T cells derived from PBMCs of donor 3124 were (i) 2,776 pg / ml without antibody (control), (ii) 3,075 pg / ml with HuPRO1-IgG1.AA, 2,577 pg / ml with HuGAB11-IgG1.AA, 2,747 pg / ml with a mixture of HuPRO1-IgG1.AA and HuGAB11-IgG1.AA, and 4,826 pg / ml with BS809. IL-2 expression was significantly increased compared to IL-2 levels in untreated control cells only in the presence of a bispecific antibody that binds to both PD-L1 and GITR. This is because BS809 bridges PD-L1-expressing cells and GITR-expressing T cells, causing multimeric crosslinking of surface GITR in close proximity to PD-L1-expressing cells, activating T cells and upregulating IL-2 expression.

[0168] The mean IL-2 concentrations in T cells derived from PBMCs of donor 4143 were (i) 658 pg / ml without antibody, (ii) 1,453 pg / ml with HuPRO1-IgG1.AA, (iii) 659 pg / ml with HuGAB11-IgG1.AA, (iv) 2,136 pg / ml with a mixture of HuPRO1-IgG1.AA and HuGAB11-IgG1.AA, and (v) 4,196 pg / ml with BS809. IL-2 expression was increased in the presence of HuPRO1-IgG1.AA compared to IL-2 levels in untreated control cells. This is thought to be because the interaction between PD-L1 and PD-1, which suppresses the immune response, was blocked by HuPRO1-IgG1.AA. The significant increase in IL-2 expression in the presence of BS809 is due to (a) its anti-PD-L1 antagonist activity and (b) its ability to bridge the gap between PD-L1-expressing cells and GITR-expressing T cells, thereby multimerically cross-linking GITR on the cell surface, resulting in potent activation of T cells.

[0169] Example 8: Bispecific antibody (BS813) that binds to PD-L1 and OX40

[0170] The mammalian expression vector pBS813, designed for the expression of bispecific antibodies that bind to both human PD-L1 (SEQ ID NO: 69) and human OX40 (SEQ ID NO: 21), was constructed by replacing the CH3 coding region of pHuPRO1-IgG1.AA with the coding region of CH3-HuOHX14DS.scFv (SEQ ID NO: 23).

[0171] The amino acid sequence of the mature heavy chain encoded in pBS813 is (SEQ ID NO: 72).

[0172] The amino acid sequence of the mature light chain encoded in pBS813 is the same as the mature light chain sequence (SEQ ID NO: 71) encoded in pBS809.

[0173] The schematic structure of BS813 is shown in Figure 2.

[0174] Each of the three expression vectors, pHuPRO1-IgG1.AA, pHuOHX14DS-IgG1.AA, and pBS813, was stably transfected into CHO-K1 cells as described above. HuPRO1-IgG1.AA, HuOHX14DS-IgG1.AA, and BS813 were purified from the culture supernatant of their respective CHO-K1 stable transfectants using a protein A affinity column as described above. SDS-PAGE analysis under reducing conditions showed only two dominant bands for each of the three antibodies: a heavy chain of approximately 50 kD and a light chain of 25 kD (for HuPRO1-IgG1.AA and HuOHX14DS-IgG1.AA), and a heavy chain of approximately 75 kD and a light chain of approximately 25 kD (for BS813). NS0 / PD-L1 cells expressing recombinant human PD-L1 on their surface were generated by stably transfecting NS0 cells with the expression vector pFCm370 via electroporation. The expression vector pFCm370 has the same structure as pFCm331, except that the SpeI-EagI fragment is replaced from the N-terminus to the C-terminus with a DNA fragment encoding recombinant human PD-L1 (PD-L1-FLAG-GPI), which consists of a synthetic signal peptide (SEQ ID NO: 26), the extracellular region of human PD-L1 (SEQ ID NO: 73), a FLAG polypeptide (SEQ ID NO: 9), and the GPI anchorage signal of human CD55 (SEQ ID NO: 10). (i) NS0 stable transfectants that survived in DME medium containing 10% FBS, 1 μg / ml mycophenolic acid, HT medium supplement, and 0.25 mg / ml xanthine, and (ii) expressed PD-L1-FLAG-GPI on their surface, were maintained in DME medium containing 10% FBS at 37°C in a 7.5% CO2 incubator.

[0175] Approximately 200,000 JD / OX40 cells were cultured in 200 μl of RPMI1640 medium containing 10% FBS, 0.5 μg / ml mouse anti-human CD3 IgG antibody OKT3 (BioLegend, San Diego, CA), and 5 μg / ml goat anti-mouse IgG antibody (human IgG-absorbed) (Jackson ImmunoResearch), with antibody-free (a and f) and 1 μg / ml HuPRO1-IgG1. AA (anti-PD-L1) (b and g), 1 μg / ml HuOHX14DS-IgG1.AA (anti-OX40) (c and h), 1 μg / ml HuPRO1-IgG1.AA and 1 μg / ml HuOHX14DS-IgG1.AA (d and i), and 1 μg / ml BS813 (e and j) were incubated in 96-well plates at 37°C in a 7.5% CO2 incubator for 1 day. 200,000 NS0 cells were added to reactions a, b, c, d, and e. 200,000 NS0 / PD-L1 cells were added to reactions f, g, h, i, and j. Luciferase activity in the culture supernatant was measured in three fractions using the QUANTI-Luc reagent (InvivoGen) according to the vendor's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek, Winooski, VT). The results are shown in Figure 7.

[0176] The mean relative luciferase units (RLUs) in JD / OX40 cells were 4,683 in NS0 cells, 6,193 in HuPRO1-IgG1.AA and NS0 cells (b), 7,230 in HuOHX14DS-IgG1.AA (c), and 7,671 in HuPRO1-IgG1.AA and HuOHX14DS-Ig1.AA (d). The levels were 7,671 (d) in AA, HuOHX14DS-Ig1.AA, and NS0 cells; 6,146 (e) in BS813 and NS0 cells; 3,842 (f) in NS0 / PD-L1 cells; 3,866 (g) in HuPRO1-IgG1.AA and NS0 / PD-L1 cells; 5,921 (h) in HuOHX14DS-IgG1.AA and NS0 / PD-L1 cells; 2,836 (i) in HuPRO1-IgG1.AA, HuOHX14DS-IgG1.AA, and NS0 / PD-L1 cells; and 23,715 (j) in BS813 and NS0 / PD-L1 cells. Luciferase activity in JD / OX40 cells was significantly increased only when both a bispecific antibody (BS813) that binds to PD-L1 and OX40 and PD-L1-expressing cells (NS0 / PD-L1) were present. This result indicates that only BS813, which can bridge the gap between JD / OX40 cells and NS0 / PD-L1 cells (and not HuPRO1-IgG1.AA, HuOHX14DS-IgG1.AA, or combinations of these two monospecific antibodies), induces multimeric crosslinking of OX40 on the surface of JD / OX40 cells in close proximity to NS0 / PD-L1 cells, resulting in activation of JD / OX40 cells and increased luciferase expression.

[0177] Example 9: Preparation of a bispecific antibody (BS841) that binds to PD-L1 and OX40.

[0178] (i) Mouse hybridomas producing an IgG / kappa monoclonal antibody PRO2 that binds to human and cynomolgus monkey PD-L1 and (ii) blocks the interaction between PD-1 and PD-L1 were isolated as described in Example 1. The VH and VL sequences of PRO2 were sequenced and humanized as described in Tsurushita et al. (cited above).

[0179] The amino acid sequence of humanized PRO2 (HuPRO2)VH is MGWNWIFLFLSGTAGVHCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSS (SEQ ID NO: 74). The amino acid sequences of CDR1, 2, and 3 of HuPRO2 VH are SYGIN (SEQ ID NO: 75), YIYPGSGGPVYNQKFKG (SEQ ID NO: 76), and ENYRYWYFDV (SEQ ID NO: 77), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuPRO2 VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0180] The amino acid sequence of humanized PRO2 (HuPRO2) VL is MHFQVQIFSFLLISASVIMSRGDIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK (SEQ ID NO: 78). The amino acid sequences of CDR1, 2, and 3 of HuPRO2 VL are SASSSVNYMH (SEQ ID NO: 79), STSNLAS (SEQ ID NO: 80), and QQRSSYPLT (SEQ ID NO: 81), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuPRO2 VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0181] The expression vector pHuPRO2-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuPRO2 VH exon is positioned between the SpeI site and the HindIII site, (ii) the HuPRO2 VL exon is positioned between the NheI site and the EcoRI site, and (iii) two leucine residues at positions 234 and 235 (Eu numbering) of CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) in order to remove the effector function (Hezareh et al., cited above). The novel vector pHuPRO2-IgG1.AA expresses a humanized anti-PD-L1 IgG1 / kappa antibody (HuPRO2-IgG1.AA) in mammalian cells.

[0182] The mammalian expression vector pBS841, designed for the expression of bispecific antibodies that bind to both human PD-L1 (SEQ ID NO: 69) and human OX40 (SEQ ID NO: 21), was constructed by replacing the CH3 coding region of pHuPRO2-IgG1.AA with the coding region of CH3-HuOHX14DS.scFv (SEQ ID NO: 23).

[0183] The amino acid sequence of the mature heavy chain encoded by pBS841 is (SEQ ID NO: 82).

[0184] The amino acid sequence of the mature light chain encoded by pBS841 is DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 83).

[0185] Example 10: Preparation of a bispecific antibody (BS839) that binds to PD-L1 and OX40.

[0186] (i) Mouse hybridomas producing an IgG / kappa monoclonal antibody PRO5 that binds to human and cynomolgus monkey PD-L1 and (ii) blocks the interaction between PD-1 and PD-L1 were isolated as described in Example 1. The VH and VL sequences of PRO5 were sequenced and humanized as described in Tsurushita et al. (cited above).

[0187] The amino acid sequence of humanized PRO5 (HuPRO5)VH is MMVLSLLYLLTALPGILSQVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSS (SEQ ID NO: 84). The amino acid sequences of CDR1, 2, and 3 of HuPRO5 VH are SGYWN (SEQ ID NO: 85), YISYTGSTYSNPSLKS (SEQ ID NO: 86), and SQNWERAWFAY (SEQ ID NO: 87), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuPRO5 VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0188] The amino acid sequence of humanized PRO5 (HuPRO5) VL is MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK (SEQ ID NO: 88). The amino acid sequences of CDR1, 2, and 3 of HuPRO5 VL are SASSSVSYMH (SEQ ID NO: 89), DTSKLAS (SEQ ID NO: 90), and FQGSGYPFT (SEQ ID NO: 91), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuPRO5 VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0189] The expression vector pHuPRO5-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuPRO5 VH exon is positioned between the SpeI site and the HindIII site, (ii) the HuPRO5 VL exon is positioned between the NheI site and the EcoRI site, and (iii) two leucine residues at positions 234 and 235 (Eu numbering) of CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) in order to eliminate effector function (Hezareh et al., cited above). The novel vector pHuPRO5-IgG1.AA expresses a humanized anti-PD-L1 IgG1 / kappa antibody (HuPRO5-IgG1.AA) in mammalian cells.

[0190] The mammalian expression vector pBS839, designed for the expression of bispecific antibodies that bind to both human PD-L1 (SEQ ID NO: 69) and human OX40 (SEQ ID NO: 21), was constructed by replacing the CH3 coding region of pHuPRO5-IgG1.AA with the coding region of CH3-HuOHX14DS.scFv (SEQ ID NO: 23).

[0191] The amino acid sequence of the mature heavy chain encoded by pBS839 is (SEQ ID NO: 92).

[0192] The amino acid sequence of the mature light chain encoded by pBS839 is DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93).

[0193] Example 11: Expression and characterization of bispecific antibodies BS839 and BS841 that bind to PD-L1 and OX40.

[0194] The four expression vectors, pHuPRO2-IgG1.AA, pHuPRO5-IgG1.AA, pBS839, and pBS841, stably transfect CHO-K1 as described above. HuPRO2-IgG1AA, HuPRO5-IgG1.AA, BS839, and BS841 are purified from the culture supernatant of each CHO-K1 transfectant using a protein A affinity column as described above. SDS-PAGE analysis is performed under reducing conditions to determine the heavy and light chain sizes of these four antibodies.

[0195] The biological activity of BS839 and BS841, which enhance the immune response, will be analyzed by measuring the expression level of IL-2 in human T cells. Human PBMCs will be cultured for 3 days in RPMI-1640 containing 10% FBS and 10 μg / ml PHA-L as described above (3-day-old PBMCs). Approximately 100,000 3-day-old PBMCs were incubated for 1 day in a 7.5% CO2 incubator at 37°C in wells of a 96-well plate pre-coated with 1 μg / ml mouse anti-human CD3 monoclonal antibody OKT3, in the following conditions: (i) no additional antibody, (ii) 1 μg / ml HuPRO2-IgG1.AA, (iii) 1 μg / ml HuPRO5-IgG1.AA, (iv) 1 μg / ml HuOHX14DS-IgG1.AA, (v) 1 μg / ml HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA, (vi) 1 μg / ml HuPRO5-IgG1.AA and HuOHX14DS-IgG1.AA, (vii) 1 μg / ml S841, and iii) 1 μg / ml S839. The IL-2 concentration in the culture supernatant is measured using the Human IL-2 ELISA MAX Standard Kit (BioLegend).

[0196] Example 12: Bispecific antibody that binds to cancer cells and costimulatory molecules

[0197] Monoclonal antibodies that bind to cancer cells are isolated using hybridoma technology or display technology (Akamatsu et al., J. Immunol. Methods, 327:40-52, 2007; Bradbury et al., Nat. Biotechnol. 29:245-254, 2011; Hammers et al., J. Invest. Dermatol. 134:e17, 2014; Cherf et al., Methods Mol. Biol. 1319:155-175, 2015; Saeed et al., MOJ Immunol. 3:00099, 2016; Mahmuda et al., Trop. J. Pharm. Res. 16:713-722, 2017). The VH and VL regions of such isolated anti-cancer antibodies, or their humanized forms, are cloned into expression vectors such as pHuM195-IgG1 (Figure 1A), as described above. Additionally, another monoclonal antibody that binds to an immunocostimulatory molecule (Bakdash et al., Front. Immunol. 4: Article 53, 2013; Mahoney et al., Nat. Rev. Drug Discov. 14:561-584, 2015) is isolated using hybridoma or display technology. The VH and VL regions of such isolated anticostimulatory molecule antibodies, or their humanized forms, are converted to single-stranded Fv (scFv) forms (Nelson, mAbs 2:77-83, 2010; Ahmad et al., Clin. Dev. Immunol. Article ID 980250, 2012). A novel vector is created that expresses a bispecific antibody that binds to cancer cells and costimulatory molecules by fusing the scFv form of an anti-costimulatory molecule antibody to the C-terminus of the heavy chain of an anti-cancer antibody.The biological activity of the bispecific antibodies thus produced, which (i) bridge the gap between cancer cells and immune cells and (ii) activate such immune cells through multimeric crosslinking of costimulatory molecules, will be tested in an appropriate disease model, such as a tumor xenograft model using humanized mice with human-derived immune cells (Day et al., Cell 163:39-53, 2015; Morton et al., Cancer Res. 76:6153-6158, 2016).

[0198] Example 13: Bispecific antibody that binds to pathogen-derived molecules and costimulatory molecules

[0199] Monoclonal antibodies that bind to the pathogen expressed on the surface of host cells by the infected organism are isolated using hybridoma technology or display technology as described above. The VH and VL regions of these isolated anti-pathogen antibodies, or their humanized forms, are cloned into an expression vector such as pHuM195-IgG1 (Figure 1A) as described above. Another monoclonal antibody that binds to an immunocostimulatory molecule is isolated using hybridoma technology or display technology. The VH and VL regions of such isolated anticostimulatory molecule antibodies, or their humanized forms, are converted to scFv forms as described above. The scFv form of the anticostimulatory antibody is fused to the C-terminus of the heavy chain of the anti-pathogen antibody in the expression vector of the anti-pathogen IgG antibody as described above. The novel vector thus obtained expresses a bispecific IgG antibody that can bind to pathogen-infected cells and the costimulatory molecule. The biological activity of the bispecific antibodies thus produced, which (i) can bridge the gap between pathogen-infected cells and immune cells, and (ii) can activate such immune cells by multimeric crosslinking of costimulatory molecules, is tested in an appropriate disease model, for example, a humanized mouse having immune cells derived from a human infected with a pathogen (Shultz et al., Ann. NY Acad. Sci. 1245:50-54, 2011; Leung et al., Eur. J. Immunol. 43: 2246-2254, 2013).

[0200] Example 14: Replacement of scFv with receptor-binding modules in different formats

[0201] The scFv region of the bispecific antibody of the present invention, which provides a second antigen-binding site to the IgG molecule, is replaced with a polypeptide capable of binding to cell surface receptors (e.g., growth factors, cytokines, chemokines, soluble forms of receptors), a single-domain antibody such as VH, VL, or VHH (Holt et al., Trends Biotechnol. 21:484-490, 2003; Bannas et al, Front. Immunol. 8: Article 1603, 2017), or an antibody mimetic (Yu et al., Annu. Rev. Anal. Chem. 10: 293-320, 2017). The bispecific antibody thus produced is then tested for its biological activity in activating immune cells via multivalent crosslinking of costimulatory molecules in appropriate disease model animals for cancer and infectious diseases as described above.

[0202] Example 15: Fusion of scFv at the C-terminus of the light chain

[0203] The scFv region of an anti-co-stimulatory molecule is fused to the C-terminus of the light chain of an IgG antibody against a checkpoint molecule, cancer cells, or pathogen. The resulting bispecific antibody is then tested for its biological activity in activating immune cells via multivalent crosslinking of the co-stimulatory molecule in appropriate animal models for cancer and infectious diseases, as described above.

[0204] Example 16: Activation of T cells by BS809

[0205] Approximately 100,000 JD / GITR cells were cultured in 200 μl / well RPMI1640 medium containing 10% FBS, 0.5 μg / ml mouse anti-human CD3 IgG antibody OKT3 (BioLegend, San Diego, CA), and 5 μg / ml goat anti-mouse IgG antibody (human IgG-adsorbed) (Jackson ImmunoResearch) under the following conditions: no antibody (a and f), 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1 in Figure 8) (b and g), and 1 μg / ml HuGAB11-IgG1. AA (anti-GITR in Figure 8) (c and h), 1 μg / ml HuPRO1-IgG1.AA and 1 μg / ml HuGAB11-IgG1.AA (d and i), and 1 μg / ml BS809 (e and j) were incubated in 96-well plates at 37°C in a 7.5% CO2 incubator. Reactions a, b, c, d, and e contained 100,000 NS0 cells. Reactions f, g, h, i, and j contained 100,000 NS0 / PD-L1 cells. After 1 day of incubation, luciferase activity in the culture supernatant was measured in three fractions using QUANTI-Luc reagent (InvivoGen) according to the vendor's protocol. Luminescence was measured using a Synergy HT microplate reader equipped with Gen5 software (BioTek, Winooski, VT). The results are shown in Figure 8.

[0206] The mean relative luciferase units (RLU) in the culture supernatant of JD / GITR cells were: (a) 479,187 in NS0 cells, (b) 400,158 in HuPRO1-IgG1.AA and NS0 cells, (c) 472,050 in HuGAB11-IgG1.AA and NS0 cells, (d) 531,275 in HuPRO1-IgG1.AA, HuGAB11-IgG1.AA and NS0 cells, and (e) 422,9 in BS809 and NS0 cells. The values ​​were 55, (f) 405,780 in NS0 / PD-L1 cells, (g) 443,588 in HuPRO1-IgG1.AA and NS0 / PD-L1 cells, (h) 568,268 in HuGAB11-IgG1.AA and NS0 / PD-L1 cells, (i) 569,172 in HuPRO1-IgG1.AA, HuGAB11-Ig1.AA and NS0 / PD-L1 cells, and (j) 1,035,453 in BS809 and NS0 / PD-L1 cells. Luciferase activity in JD / GITR cells was significantly increased only when both a bispecific antibody (BS809) that binds to PD-L1 and GITR and PD-L1-expressing cells (NS0 / PD-L1) were present. These results indicate that only BS809, which can bridge the gap between JD / GITR cells and NS0 / PD-L1 cells (and not HuPRO1-IgG1.AA, HuGAB11-IgG1.AA, or any combination of these two monospecific antibodies), induces multimeric crosslinking of GITR on the surface of JD / GITR cells in close proximity to NS0 / PD-L1 cells, resulting in activation of JD / GITR cells and increased luciferase expression.

[0207] Example 17: Activation of PHA-L treated PBMCs with BS813, BS841, and BS839

[0208] The biological activity of BS813, BS841, and BS839, which enhance the immune response, was analyzed by measuring IL-2 expression in human T cells. The bispecific antibodies BS813, BS841, and BS839, which bind to PD-L1 and OX40 respectively, were purified from the culture supernatant of CHO-K1 cells stably transfected with pBS813, pBS841, and pBS839, respectively, using a protein A affinity column. Human PBMCs were cultured for 3 days in RPMI-1640 medium containing 10% FBS and 10 μg / ml PHA-L as described above (3-day-old PBMCs). Approximately 100,000 3-day-old PBMC cells were pre-coated with 1 μg / ml mouse anti-human CD3 monoclonal antibody OKT3 in 96-well plates, with 200 μl / well of each antibody used to perform the following tests: (a) No additional antibody; (b) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1 #1 in Figure 9) and 1 μg / ml HuOHX14DS-IgG1.AA (anti-OX40 in Figure 9); (c) 1 μg / ml HuPRO2-IgG1.AA (anti-PD-L1 #2 in Figure 9) and 1 μg / ml HuOHX14DS-IgG1.AA; (d) HuPRO5-IgG1.AA (anti-PD-L1 in Figure 9) (5) and 1 μg / ml HuOHX14DS-IgG1.AA, (e) 1 μg / ml BS813, (f) 1 μg / ml BS841, and (g) 1 μg / ml BS839 were incubated in a 7.5% CO2 incubator at 37°C for 1 day. The IL-2 concentration in the culture supernatant was measured in three separate steps using the Human IL-2 ELISA MAX Standard Kit (BioLegend). The results are shown in Figure 9.

[0209] The mean IL-2 concentrations in human PBMC-derived T cells were as follows: (a) 439 pg / ml with no test antibody (control), (b) 324 pg / ml with the combination of HuPRO1-IgG1.AA and HuOHX14DS-IgG1.A, (c) 281 pg / ml with the combination of HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA, (d) 53 pg / ml with the combination of HuPRO5-IgG1.AA and HuOHX14DS-IgG1.AA, (e) 2,202 pg / ml with BS813, (f) 1,698 pg / ml with BS841, and (g) 1,259 pg / ml with BS839. In the presence of only one of the bispecific antibodies (BS813, BS841, and BS839) that bind to both PD-L1 and OX40, IL-2 expression was significantly increased compared to the control or to each of the two parental antibody combinations of BS813, BS841, and BS839. This is because bridging between PD-L1-expressing cells and OX40-expressing T cells by each of BS813, BS841, and BS839 leads to multimeric crosslinking of OX40 on the cell surface in close proximity to PD-L1-expressing cells, resulting in activation of OX40-expressing T cells and upregulating IL-2 expression.

[0210] Example 18: Activation of SEB-treated PBMC with BS813, BS841, and BS839

[0211] Superantigens such as SEB (Staphylococcus enterotoxin B) activate T cells by linking MHC class II molecules on antigen-presenting cells to the νβ element of the T cell receptor, resulting in the production of cytokines including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interferon gamma (IFNγ) (see, e.g., Krakauer et al., Toxins (Basel). 2010 Aug; 2(8): 1963-1983). SEB can activate up to 10–20% of T cells in human blood, depending on the proportion of T cells with νβ3, νβ12, νβ14, and νβ17 found in each particular blood donor. Therefore, SEB can be used in T cell-based cytokine secretion assays to monitor the activation of immunocostimulatory molecules.

[0212] Approximately 400,000 human PBMCs were plated in 96-well plates with 200 μl / well of RPMI-1640 medium containing 10% FBS and 1 μg / ml SEB, and the following were performed: (a) no antibody (control), (b) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1 #1 in Figure 10) and HuOHX14DS-IgG1.AA (anti-OX40 in Figure 10), (c) 1 μg / ml HuPRO2-IgG1.AA (anti-PD-L1 #2 in Figure 10) and 1 μg / ml HuOHX14DS-IgG1.AA, (d) HuPRO5-IgG1.AA (anti-PD-L1 (5) and 1 μg / ml HuOHX14DS-IgG1.AA, (e) 1 μg / ml BS813, (f) 1 μg / ml BS841, and (g) 1 μg / ml BS839 were incubated in a 96-well plate. After incubation at 37°C for 1 day in a 7.5% CO2 incubator, the IL-2 concentration in the culture supernatant was measured in three separate steps using the Human IL-2 ELISA MAX Standard Kit (BioLegend). The results are shown in Figure 10.

[0213] The mean IL-2 concentrations were (a) 11.3 ng / ml without test antibody, (b) 10.5 ng / ml with the combination of HuPRO1-IgG1.AA and HuOHX14DS-IgG1.A, (c) 10.0 ng / ml with the combination of HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA, (d) 10.0 ng / ml with the combination of HuPRO5-IgG1.AA and HuOHX14DS-IgG1.AA, (e) 17.7 ng / ml with BS813, (f) 16.5 ng / ml with BS841, and (g) 15.8 ng / ml with BS839. Compared to the IL-2 levels of the control (no antibody added to SEB-treated PBMCs), IL-2 expression was significantly increased only in the presence of one of the bispecific antibodies (BS813, BS841, BS839) that bind to both PD-L1 and OX40. Furthermore, no combination of two parental antibodies for BS813, BS841, and BS839 increased IL-2 expression levels above the control level. This result indicates that each of BS813, BS841, and BS839 can bridge the gap between PD-L1-expressing cells and OX40-expressing T cells, leading to multimeric crosslinking of OX40 on the cell surface in close proximity to PD-L1-expressing cells, resulting in activation of OX40-expressing T cells and upregulating IL-2 expression.

[0214] Example 19: Bispecific antibody having disulfide-bonded single-chain Fv

[0215] Brinkman et al. previously reported that the binding of VH and VL in the Fv format (dsFv) is stabilized by introducing a disulfide bond by converting the amino acid residue at position 100 of VL to a cysteine ​​residue and another amino acid residue at position 44 of VH to a cysteine ​​residue (Eu numbering) (Proc. Natl. Acad. Sci. 90:7538-7542, 1993). It has also been reported that different positions on VH and VL can be used to introduce a pair of cysteine ​​residues to stabilize the Fv structure (Brinkman et al. supra; Young et al. FEBS Lett. 377:135-139, 1995; Schmiedl et al. Protein Eng. 13:724-730, 2000).

[0216] The bispecific anti-PD-L1 / GITR antibody (BS809) of the present invention was modified to stabilize the single-stranded Fv structure by introducing two cysteine ​​residues (underlined) into the anti-GITR variable region (one at position 44 of VH and the other at position 100 of VL) through site-directed mutagenesis of pBS809. The resulting expression vector pBS853 has the same structure as pBS809, except that the amino acid sequence of the anti-GITR single-stranded Fv region differs between pBS853 and pBS809. Vectors pBS809 and pBS853 have the same light chain sequence (SEQ ID NO: 71). The amino acid sequence of the mature heavy chain encoded in pBS853 is QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK CThis is LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (Sequence ID 106). Cysteine ​​substitutions are underlined.

[0217] A modified bispecific antibody (BS853) encoded by pBS853 was transiently expressed in HEK293 cells according to the procedure of Durocher et al. (Nucl. Acids Res. 30:e9, 2002). BS853 showed comparable binding to PD-L1 and GITR by ELISA to BS809.

[0218] Example 20: Bispecific antibody (BS859) that binds to PD-L1 and ICOS

[0219] ICOS (inducible T-cell costimulatory), also known as CD278, is an immunocostimulatory molecule belonging to the CD28 family. Multimeric crosslinking of ICOS on the cell surface of immune cells is required to initiate intracellular signaling for enhancing the immune response (Wikenheiser et al., Front. Immunol. 7: Article 304, 2016).

[0220] Rat hybridomas producing the IgG / kappa monoclonal antibody TAM14, which specifically binds to human and cynomolgus monkey ICOS, were isolated as described in Example 1, except that Sprague Dawley rats were used for immunization. As the immunogen, the extracellular region of human ICOS fused to the Fc region of the human gamma-1 heavy chain (hICOS-Fc; SEQ ID NO: 107) was used. Sequencing and humanization of TAM14 VH and VL were performed as described by Tsurushita et al. (cited above).

[0221] The amino acid sequence of humanized TAM14 (HuTAM14)VH is MAVLVLLLCLVTFPSCALSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS (SEQ ID NO: 108). The amino acid sequences of CDR1, 2, and 3 of HuTAM14 VH are SNSVS (SEQ ID NO: 109), AIWSGGSTDYNSALKS (SEQ ID NO: 110), and WEQPYYFDY (SEQ ID NO: 111), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuTAM14 VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0222] The amino acid sequence of humanized TAM14 (HuTAM14)VL is MRTSIQLLGLLLFWLHDAQCDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIK (SEQ ID NO: 112). The amino acid sequences of CDR1, 2, and 3 of HuTAM14 VL are QASQNIYKYIA (SEQ ID NO: 113), YTSTLES (SEQ ID NO: 114), and LQYVNLYT (SEQ ID NO: 115), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuTAM14 VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0223] The expression vector pHuTAM14-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuTAM14 VH exon is positioned between the SpeI and HindIII sites, (ii) the HuTAM14 VL exon is positioned between the NheI and EcoRI sites, and (iii) two leucine residues at positions 234 and 235 of CH2 (Kabat et al.'s EU numbering, cited above) are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., J. Virol. 75:12161-12168, 2001). The novel vector pHuTAM14-IgG1.AA expresses a humanized anti-ICOS IgG1 / kappa antibody (HuTAM14-IgG1.AA) in mammalian cells.

[0224] A mammalian expression vector designed for the expression of a bispecific antibody that binds to both human PD-L1 (SEQ ID NO: 69) and human ICOS (SEQ ID NO: 116) was constructed by modifying pHuPRO1-IgG1.AA as follows: The VL and VH coding regions of the humanized anti-human ICOS monoclonal antibody of pHuTAM14-IgG1.AA were converted to a single-stranded Fv (scFv) form from the N-terminus to the C-terminus in the order of VL, polypeptide linker, and VH (HuTAM14.scFv; SEQ ID NO: 117). The N-terminus of HuTAM14.scFv was fused to the second-to-last glycine residue of CH3 in pHuPRO1-IgG1.AA, separated by a polypeptide linker (CH3-HuTAM14.scFv; SEQ ID NO: 118). The resulting vector pBS859 expresses a bispecific IgG antibody named BS859 that binds to both human PD-L1 and ICOS.

[0225] The amino acid sequence of the mature heavy chain encoded by pBS859 is (SEQ ID NO: 119).

[0226] The amino acid sequence of the mature light chain encoded by pBS859 is the same as the mature light chain sequence (SEQ ID NO: 71) encoded by pBS809.

[0227] Figure 2 shows the schematic structure of a bispecific IgG antibody of the present invention, such as BS859.

[0228] Each of the three expression vectors, pHuPRO1-IgG1.AA, pHuTAM14-IgG1.AA, and pBS859, was stably transfected into CHO-K1 cells as described above. HuPRO1-IgG1.AA, HuTAM14-IgG1.AA, and BS859 were purified from the culture supernatant of the respective CHO-K1 stable transfectants using a protein A affinity column as described above. SDS-PAGE analysis under reducing conditions showed only two dominant bands for each of the three antibodies: a heavy chain of approximately 50 kD and a light chain of 25 kD (for HuPRO1-IgG1.AA and HuTAM14-IgG1.AA), or a heavy chain of approximately 75 kD and a light chain of 25 kD (for BS859).

[0229] The biological activity of BS859 in enhancing the immune response was analyzed by measuring the expression levels of IL-2 and IL-10 in human T cells. Human peripheral blood mononuclear cells (PBMCs) from two donors (3486 and 4239) were obtained from iQ Biosciences (Berkeley, CA). Human PBMCs were cultured in RPMI-1640 containing 10% FBS and 10 μg / ml phytohemagglutinin-L (PHA-L) in a 7.5% CO2 incubator at 37°C for 3 days to enrich T cells (3-day-old PBMCs).

[0230] Approximately 200,000 3-day-old PBMC cells were pre-coated with 1 μg / ml mouse anti-human CD3 monoclonal antibody OKT3 and 1 μg / ml human PD-L1-Fc fusion protein (Recombinant Human B7-H1-Fc Chimera, BioLegend, San Diego, CA) in wells of a 96-well plate. These were incubated for 1 day in a 7.5% CO2 incubator at 37°C in the presence of (i) no additional antibody (control), (ii) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1), (iii) 1 μg / ml HuTAM14-IgG1.AA (anti-ICOS), (iv) 1 μg / ml HuPRO1-IgG1.AA and 1 μg / ml HuTAM14-IgG1.AA, and (v) 1 μg / ml BS859. The concentrations of IL-2 and IL-10 in the culture supernatant were measured using ELISA MAX Standard Set Human IL-2 and ELISA MAX Standard Set Human IL-10 (BioLegend, San Diego, CA), respectively. The results are shown in Figure 11.

[0231] The mean IL-2 concentrations in PBMC-derived T cells from donor 3486 were: (i) 3.3 ng / ml in the control group (no antibody), (ii) 13.3 ng / ml in the HuPRO1-IgG1.AA group, (iii) 3.6 ng in the HuTAM14-IgG1.AA group, (iv) 9.9 ng / ml in the mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA, and (v) 46.2 ng / ml in the BS859 group.

[0232] The mean IL-10 concentrations in T cells derived from PBMCs of donor 3486 were: (i) 604 pg / ml in the control group (no antibody), (ii) 676 pg / ml with HuPRO1-IgG1.AA, (iii) 527 pg / ml with HuTAM14-IgG1.AA, (iv) 699 pg / ml with a mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA, and (v) 1,201 pg / ml with BS859.

[0233] The mean IL-2 concentrations in T cells derived from PBMCs of donor 4239 were: (i) 0.01 ng / ml in the control group without antibody, (ii) 0.15 ng / ml with HuPRO1-IgG1.AA, (iii) 0.01 ng / ml with HuTAM14-IgG1.AA, (iv) 0.06 ng / ml with a mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA, and (v) 13.6 ng / ml with BS859.

[0234] The mean IL-10 concentrations in T cells derived from PBMCs of donor 4239 were: (i) 238 pg / ml in the control group without antibody, (ii) 252 pg / ml with HuPRO1-IgG1.AA, (iii) 217 ​​pg / ml with HuTAM14-IgG1.AA, (iv) 220 pg / ml with a mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA, and (v) 537 pg / ml with BS859.

[0235] HuPRO1-IgG1.AA alone was able to increase IL-2 expression in PHA-L treated PBMCs from two different donors compared to their respective control groups that did not receive antibody treatment. BS859 further significantly increased IL-2 expression in these two donors.

[0236] IL-10 expression in PHA-L-treated PBMCs from two different donors was significantly increased only in the presence of the bispecific antibody (BS859) of the present invention, which binds to both PD-L1 and ICOS.

[0237] Example 21: Bispecific antibody (BS840) that binds to PD-L1 and GITR

[0238] The mammalian expression vector pBS840, designed for the expression of a bispecific antibody that binds to both human PD-L1 (SEQ ID NO: 69) and human GITR (SEQ ID NO: 40), has the same structure as pBS841, except that CH3-HuOHX14DS.scFv (SEQ ID NO: 23) is substituted with CH3-HuGAB11.scFv (SEQ ID NO: 42). Vector pBS840 expresses a bispecific IgG antibody named BS840 that binds to both human PD-L1 and GITR.

[0239] The amino acid sequence of the mature heavy chain encoded in pBS840 is (SEQ ID NO: 120).

[0240] The amino acid sequence of the mature light chain encoded by pBS840 is the same as the amino acid sequence of the light chain encoded by pBS841 (SEQ ID NO: 83).

[0241] The expression vector pBS840 was stably transfected into CHO-K1 cells as described above. BS840 was purified from the culture supernatant of the obtained CHO-K1 stable transfectant using a protein A affinity column as described above. BS840 purified with protein A showed a single dominant peak of the expected size (approximately 200 kDa) by gel filtration using a Superose 6 size exclusion 10 / 300 column (GE Healthcare Life Sciences, Pittsburgh, PA). SDS-PAGE analysis under reducing conditions showed only two dominant bands: a heavy chain of approximately 75 kDa and a light chain of 25 kDa.

[0242] As described above, we investigated the biological activity of BS840 in enhancing the immune response using Jurkat Dual cells (JD / GITR) that stably express human GITR. The data is shown in Figure 12. The mean relative luciferase units (RLUs) in the culture supernatant of JD / GITR cells were as follows: (a) 354,057 in NS0 cells; (b) 341,277 in HuPRO2-IgG1.AA (anti-PD-L1 #2 in Figure 12; parental monospecific IgG antibody of BS840), HuGAB11-IgG1.AA (anti-GITR in Figure 12), and NS0 cells; (c) 326,577 in BS840 and NS0 cells; (d) 282,554 in NS0 / PD-L1 cells; (e) 386,137 in HuPRO2-IgG1.AA, HuGAB11-Ig1.AA, and NS0 / PD-L1 cells; and (f) 989,407 in BS840 and NS0 / PD-L1 cells. Luciferase activity in JD / GITR cells was significantly increased only when both a bispecific antibody (BS840) that binds to PD-L1 and GITR, and PD-L1-expressing cells (NS0 / PD-L1), were present.

[0243] Example 22: Bispecific antibody (BS846) that binds to PD-L1 and CD40

[0244] The mammalian expression vector pBS846, designed for the expression of a bispecific antibody that binds to both human PD-L1 (SEQ ID NO: 69) and human CD40 (SEQ ID NO: 56), has the same structure as pBS809, except that CH3-HuGAB11.scFv (SEQ ID NO: 42) is substituted with CH3-HuACS2.scFv (SEQ ID NO: 58). Vector pBS846 expresses a bispecific IgG antibody named BS846 that binds to both human PD-L1 and CD40.

[0245] The amino acid sequence of the mature heavy chain encoded in pBS846 is (SEQ ID NO: 121).

[0246] The amino acid sequence of the mature light chain encoded by pBS846 is the same as the amino acid sequence of the light chain encoded by pBS809 (SEQ ID NO: 71).

[0247] The expression vector pBS846 was stably transfected into CHO-K1 cells as described above. From the culture supernatant of the obtained CHO-K1 stable transfectant, BS846 was purified using a protein A affinity column as described above. BS846 purified with protein A showed a single dominant peak of the expected size (approximately 200 kDa) by gel filtration using a Superose 6 size exclusion 10 / 300 column (GE Healthcare Life Sciences, Pittsburgh, PA). SDS-PAGE analysis under reducing conditions showed only two dominant bands: a heavy chain of approximately 75 kD and a light chain of 25 kD.

[0248] The biological activity of BS846 in enhancing the CD40-mediated immune response was investigated using Ramos cells as described above. Ramos cells were prepared in DME medium containing 10% FBS in the following conditions: (a) no antibody and NS0 cells, (b) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1) and NS0 cells, (c) 1 μg / ml HuACS2-IgG1.AA (anti-CD40) and NS0 cells, and (d) 1 μg / ml HuPRO1-IgG1.AA and 1 μg / ml HuACS2-IgG1. AA and NS0 cells were incubated for 3 days in the presence of (e) BS846 and NS0 cells at 1 μg / ml, (f) NS0 / PD-L1 cells without antibody, (g) HuPRO1-IgG1.AA and NS0 / PD-L1 cells at 1 μg / ml, (h) HuACS2-IgG1.AA and NS0 / PD-L1 cells at 1 μg / ml, (i) HuPRO1-IgG1.AA, 1 μg / ml of HuACS2-IgG1.AA and NS0 / PD-L1 cells, and (j) BS846 and NS0 / PD-L1 cells at 1 μg / ml. CD95 expression in Ramos cells was analyzed by flow cytometry using a FITC-labeled mouse anti-human CD20 monoclonal antibody (Clone 2H7, BioLegend) to detect Ramos cells, and a PE-labeled mouse anti-human CD95 antibody (Clone DX2, BioLegend) to monitor CD95 expression.

[0249] The proportion of CD95-positive cells among CD20-positive Ramos cells was 0.3% in the antibody-free and NS0 cells (a), 0.3% in NS0 cells with 1 μg / ml HuPRO1-IgG1.AA (b), 0.3% in NS0 cells with 1 μg / ml HuACS2-IgG1.AA (c), 0.3% in NS0 cells with 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA, and NS0 cells (d), and 2.5% in NS0 cells with 1 μg / ml BS846 (e). The concentrations were 0.2% (f) in S0 / PD-L1 cells, 0.3% (g) in NS0 / PD-L1 cells with 1 μg / ml HuPRO1-IgG1.AA, 0.3% (h) in NS0 / PD-L1 cells with 1 μg / ml HuACS2-IgG1.AA, 0.2% (i) in NS0 / PD-L1 cells with 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA, and NS0 / PD-L1 cells, and 59.5% (j) in NS0 / PD-L1 cells with 1 μg / ml BS846 (Figure 13). Only BS846, which can bridge the gap between Ramos cells and NS0 / PD-L1 cells, was able to significantly increase CD95 expression in Ramos cells. This is because BS846 can multimerically crosslink CD40 on the surface of Ramos cells at the intercellular junction with NS0 / PD-L1 cells, triggering intracellular signaling via CD40 and upregulating CD95 expression in Ramos cells.

[0250] Example 24: Disulfide bond single-strand Fv

[0251] The single-chain Fv (scFv) antibodies against OX40 (HuOHX14DS.scFv; SEQ ID NO: 22), CD40 (HuACS2.scFv; SEQ ID NO: 57), and ICOS (HuTAM14.scFv) were stabilized by site-directed mutagenesis, with the amino acid residue at position 44 of VH being replaced with a cysteine ​​residue and the amino acid residue at position 100 of VL being replaced with a cysteine ​​residue (Eu numbering) (Brinkman et al., Proc. Natl. Acad. Sci. 90:7538-7542, 1993).

[0252] The amino acid sequence of mature HuOHX14DS.scFv (HuOHX14DS.scFv.ds) with two cysteine ​​substitutions is DIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQ C This is LEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS (sequence number 122).

[0253] The amino acid sequence of mature HuACS2.scFv with two cysteine ​​substitutions (HuACS2.scFv.ds) is EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQ C LEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS (Sequence ID 123).

[0254] The amino acid sequence of mature HuTAM14.scFv with two cysteine ​​substitutions (HuTAM14.scFv.ds) is DIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGK CThis is LEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS (sequence number 124).

[0255] The amino acid sequence of mature HuGAB11.scFv with two cysteine ​​substitutions (HuGAB11.scFv.ds) (preparation is described in Example 19) is DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C This is LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (Sequence ID 125).

[0256] In the amino acid sequence shown in Example 24, cysteine ​​substitutions are underlined.

[0257] Example 25: Construction of bispecific antibodies (BS883 and BS884) that bind to PD-L1 and 4-1BB.

[0258] 4-1BB, also known as CD137 or TNFRSF9, is an immunocostimulatory molecule belonging to the TNF receptor superfamily. Cross-linking of 4-1BB on the cell surface of immune cells is necessary to initiate intracellular signaling for enhancing the immune response (Chester et al. 2016 Cancer Immunol. Immunother. 65:1243-1248).

[0259] Mouse hybridomas producing FOB5, an IgG / kappa monoclonal antibody that specifically binds to human and cynomolgus monkey 4-1BB, were isolated as described in Example 1. The extracellular region of human 4-1BB fused to the Fc region of the human gamma-1 heavy chain (h4-1BB-Fc; SEQ ID NO: 126) was used as the immunogen. Sequencing and humanization of FOB5 VH and VL were performed as described by Tsurushita et al. (cited above).

[0260] The amino acid sequence of humanized FOB5 (HuFOB5)VH is MERHWIFLFLFSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQGLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS (SEQ ID NO: 127). The amino acid sequences of CDR1, 2, and 3 of HuFOB5 VH are NYWMH (SEQ ID NO: 128), YINPSTGYTESNQKFKD (SEQ ID NO: 129), and SYVGYYYAVDY (SEQ ID NO: 130), respectively, according to the definition by Kabat et al. (cited above). The gene encoding HuFOB5 VH was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment.

[0261] The amino acid sequence of humanized FOB5 (HuFOB5) VL is MDSQAQVLMLLLLWVSGTCGDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK (SEQ ID NO: 131). The amino acid sequences of CDR1, 2, and 3 of HuFOB5 VL, according to the definition by Kabat et al. (cited above), are KSSQSLLYSNNEKNYLA (SEQ ID NO: 132), WASTRES (SEQ ID NO: 133), and QQYYSYPYT (SEQ ID NO: 134), respectively. The gene encoding HuFOB5 VL was synthesized as an exon containing a splice donor signal at the 3' end of the coding region, an NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment.

[0262] The expression vector pHuFOB5-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuFOB5 VH exon is positioned between the SpeI and HindIII sites, (ii) the HuFOB5 VL exon is positioned between the NheI and EcoRI sites, and (iii) two leucine residues at positions 234 and 235 of CH2 (Kabat et al.'s EU numbering, cited above) are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., J. Virol. 75:12161-12168, 2001). The novel vector pHuFOB5-IgG1.AA expresses a humanized anti-4-1BB IgG1 / kappa antibody (HuFOB5-IgG1.AA) in mammalian cells.

[0263] A mammalian expression vector designed for the expression of bispecific antibodies that bind to both human PD-L1 (SEQ ID NO: 69) and human 4-1BB (SEQ ID NO: 135) was constructed by modifying pHuPRO1-IgG1.AA as follows: The VL and VH coding regions of the humanized anti-human 4-1BB monoclonal antibody pHuFOB5-IgG1.AA were converted to a single-stranded Fv (scFv) form from the N-terminus to the C-terminus, in the order of VL, polypeptide linker, and VH. Furthermore, the glycine residue at position 44 of HuFOB5 VH was changed to a cysteine ​​residue, and the glycine residue at position 100 of HuFOB5 VL was changed to a cysteine ​​residue to construct disulfide-linked HuFOB5 scFv (HuFOB5.scFv.LH.ds; SEQ ID NO: 136). The N-terminus of HuFOB5.scFv was fused to the second-to-last glycine residue of CH3 in pHuPRO1-IgG1.AA across a polypeptide linker (CH3-HuFOB5.scFv.LH.ds; SEQ ID NO: 137). The CH3 coding region of pHuPRO1-IgG1.AA was replaced with the coding region of CH3-FOB5.scFv.LH.ds. The resulting vector pBS883 expresses a bispecific IgG antibody named BS883 that binds to both human PD-L1 and 4-1BB.

[0264] The amino acid sequence of the mature heavy chain encoded in pBS883 is QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQ C This is LEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS (Sequence ID 138). Cysteine ​​substitutions are underlined.

[0265] The amino acid sequence of the mature light chain encoded in pBS883 is the same as the sequence of the mature light chain encoded in pBS809 (SEQ ID NO: 71).

[0266] Another mammalian expression vector designed for the expression of a bispecific antibody that binds to both human PD-L1 (SEQ ID NO: 69) and human 4-1BB (SEQ ID NO: 135) was constructed by modifying pHuPRO1-IgG1.AA as follows: The VH and VL coding regions of the humanized anti-human 4-1BB monoclonal antibody of pHuFOB5-IgG1.AA were converted to scFv forms from the N-terminus to the C-terminus in the order of VH, polypeptide linker, and VL. Furthermore, the glycine residue at position 44 of HuFOB5 VH was changed to a cysteine ​​residue, and the glycine residue at position 100 of HuFOB5 VL was changed to a cysteine ​​residue to construct disulfide-linked HuFOB5 scFv (HuFOB5.scFv.HL.ds; SEQ ID NO: 139). The N-terminus of HuFOB5.scFv was fused to the second-to-last glycine residue of CH3 in pHuPRO1-IgG1.AA across a polypeptide linker (CH3-FOB5.scFv.HL.ds; SEQ ID NO: 140). The CH3 coding region of pHuPRO1-IgG1.AA was replaced with the coding region of CH3-HuFOB5.scFv.HL.ds. The resulting vector pBS884 expresses a bispecific IgG antibody named BS884 that binds to both human PD-L1 and 4-1BB.

[0267] The amino acid sequence of the mature heavy chain encoded in pBS884 is QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQ C LEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFG C This is GTKVEIK (sequence number 141). Cysteine ​​substitutions are underlined.

[0268] The amino acid sequence of the mature light chain encoded in pBS884 is the same as the mature light chain sequence (SEQ ID NO: 71) encoded in pBS809.

[0269] The schematic structures of the bispecific IgG antibodies of the present invention, such as BS883 and BS884, are shown in Figure 2.

[0270] Example 26: Blocking the interaction between PD-L1 and PD-1

[0271] The activities of HuPRO1-IgG1.AA, HuPRO2-IgG1.AA and HuPRO5-IgG1.AA that block the interaction between human PD-1 (SEQ ID NO: 142) and human PD-L1 were analyzed by flow cytometry using NS0 / PD-L1 cells and a PD-1-Fc fusion protein. For use as a ligand that binds to PD-L1, the extracellular region of human PD-1 was fused to the Fc region of the human gamma-1 heavy chain (hPD-1-Fc; SEQ ID NO: 143), expressed in NS0 cells, and purified by protein A affinity chromatography. To monitor binding to NS0 / PD-L1 cells, hPD-1-Fc was labeled with fluorescein isothiocyanate (FITC) using standard conjugation methods. NS0 / PD-L1 cells were incubated with sub-saturating concentrations of FITC-labeled hPD-1-Fc and various concentrations of test antibodies (HuPRO1-IgG1.AA, HuPRO2-IgG1.AA or HuPRO5-IgG1.AA) in FACS Buffer (PBS containing 0.5% BSA and 0.05% sodium azide) for 60 minutes at 4°C. After washing and resuspending in FACS Buffer, the cells were subjected to flow cytometry analysis. The half maximal inhibitory concentration (IC50) for blocking the binding of hPD-1-Fc to NS0 / PD-L1 cells was 106 ng / ml for HuPRO1-IgG1.AA, 55 ng / ml for HuPRO2-IgG1.AA, and 87 ng / ml for HuPRO5-IgG1.AA. Sequence Listing

[0272] SEQ ID NO: 1

[0273] Amino acid sequence of human CD33

[0274] MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSA APTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0275] Sequence ID 2

[0276] Amino acid sequence of HuM195 VH

[0277] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS

[0278] Sequence ID 3

[0279] Amino acid sequence of the CH1 region of the human gamma-1 heavy chain encoded by pHuM195-IgG1

[0280] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0281] Sequence ID 4

[0282] The amino acid sequence of the hinge region of the human gamma-1 heavy chain encoded by pHuM195-IgG1.

[0283] EPKSCDKTHTCPPCP

[0284] Sequence ID 5

[0285] Amino acid sequence of the CH2 region of the human gamma-1 heavy chain encoded by pHuM195-IgG1

[0286] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0287] Sequence ID 6

[0288] Amino acid sequence of the CH3 region of the human gamma-1 heavy chain encoded by pHuM195-IgG1

[0289] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0290] Sequence ID 7

[0291] Amino acid sequence of HuM195 VL

[0292] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK

[0293] Sequence ID 8

[0294] Amino acid sequence of the human kappa constant region encoded by pHuM195-IgG1

[0295] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0296] SEQ ID NO: 9

[0297] Amino acid sequence of the FLAG peptide

[0298] DYKDDDDK

[0299] SEQ ID NO: 10

[0300] Amino acid sequence of the GPI-anchorage signal

[0301] PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0302] SEQ ID NO: 11

[0303] Amino acid sequence of mature OX40-FLAG-GPI

[0304] LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRATGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0305] SEQ ID NO: 12

[0306] Amino acid sequence of HuOHX14DS VH

[0307] MGRLTSSFLLLIVPAYVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0308] Sequence ID 13

[0309] Amino acid sequence of CDR1 in HuOHX14DS VH

[0310] SYIMH

[0311] Sequence ID 14

[0312] Amino acid sequence of CDR2 in HuOHX14DS VH

[0313] YINPYNSGTKYNEKFKG

[0314] Sequence ID 15

[0315] Amino acid sequence of CDR3 in HuOHX14DS VH

[0316] YYGSTFTMDY

[0317] Sequence ID 16

[0318] Amino acid sequence of HuOHX14DS VL

[0319] MMSSAQFLGLLLLCFQGTRCDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIK

[0320] Sequence ID 17

[0321] Amino acid sequence of CDR1 in HuOHX14DS VL

[0322] RASQDIRTYLN

[0323] Sequence ID 18

[0324] Amino acid sequence of CDR2 in HuOHX14DS VL

[0325] YTSRLHS

[0326] Sequence ID 19

[0327] Amino acid sequence of CDR3 in HuOHX14DS VL

[0328] QQGNTLPWT

[0329] Sequence ID 20

[0330] Amino acid sequences of the CH2 region with L234A and L235 mutations

[0331] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0332] Sequence ID 21

[0333] Amino acid sequence of human OX40

[0334] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFPSPGDN QACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0335] Sequence ID 22

[0336] Amino acid sequence of HuOHX14DS.scFv

[0337] DIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGGSGGGGSGGG GSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0338] Sequence ID 23

[0339] The amino acid sequence of the CH3-HuOHX14DS.scFv (CH3-HuOHX14DS.scFv) fusion encoded by pBS824

[0340] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRF SGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0341] Sequence ID 24

[0342] Amino acid sequence of the mature heavy chain encoded by pBS824

[0343] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSG SGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0344] Sequence ID 25

[0345] Amino acid sequence of the mature light chain encoded by pBS824

[0346] DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0347] Sequence ID 26

[0348] Amino acid sequence of synthetic signal peptide

[0349] MGWSWIFFFLLSGTASVLS

[0350] Sequence ID 27

[0351] Amino acid sequence of the extracellular region of human CD33 encoded by pFCm267

[0352] MDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVH VTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPDGSGKQETRAGVVH

[0353] Sequence ID 28

[0354] Amino acid sequence of human epidermal growth factor receptor (EGFR)

[0355]

[0356] Sequence ID 29

[0357] Amino acid sequence of mouse 225 (Ch225) VH

[0358] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

[0359] Sequence ID 30

[0360] Amino acid sequence of mouse 225 (Ch225) VL

[0361] MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

[0362] Sequence ID 31

[0363] Amino acid sequence of hGITR-Fc

[0364] QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPAEPLGTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0365] Sequence ID 32

[0366] Amino acid sequence of HuGAB11 VH

[0367] MAVLGLLLCLVTFPSCVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0368] Sequence ID 33

[0369] Amino acid sequence of CDR1 in HuGAB11 VH

[0370] DYGVS

[0371] Sequence ID 34

[0372] Amino acid sequence of CDR2 in HuGAB11 VH

[0373] VIWGGGGTYYNSALKS

[0374] Sequence ID 35

[0375] Amino acid sequence of HuGAB11 VH CDR3

[0376] HPYGHFGMDY

[0377] Sequence ID 36

[0378] Amino acid sequence of HuGAB11 VL

[0379] MRVLAELLGLLLFCFLGVRCDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIK

[0380] Sequence ID 37

[0381] Amino acid sequence of CDR1 in HuGAB11 VL

[0382] HASQNINVWLS

[0383] Sequence ID 38

[0384] Amino acid sequence of CDR2 in HuGAB11 VL

[0385] KASNLHT

[0386] Sequence ID 39

[0387] Amino acid sequence of CDR3 in HuGAB11 VL

[0388] QQGQSYPLT

[0389] Sequence ID 40

[0390] Amino acid sequence of human GITR

[0391] MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHHPCPPGQGVQSQGKFSFGFQCIDCASGTF SGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0392] Sequence ID 41

[0393] Amino acid sequence of HuGAB11.scFv

[0394] DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGGSGGGGSGGG GSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0395] Sequence ID 42

[0396] The amino acid sequence of the CH3-HuGAB11.scFv (CH3-HuGAB11.scFv) fusion encoded in pBS827

[0397] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSR FSSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0398] Sequence ID 43

[0399] Amino acid sequence of the mature heavy chain encoded by pBS827

[0400] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0401] Sequence ID 44

[0402] Amino acid sequence of the mature light chain encoded by pBS827

[0403] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0404] Sequence ID 45

[0405] Amino acid sequence of mature human GITR

[0406] QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTD CTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0407] Sequence ID 46

[0408] Amino acid sequence of the extracellular region of human EGFR encoded by pFCm507

[0409] LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMD FQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPLMLYNPTTYQMDVNPEGKYSFGATCVKCCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCR KVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTK IISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0410] Sequence ID 47

[0411] Amino acid sequence of hCD40-Fc

[0412] EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRTGGGEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0413] Sequence ID 48

[0414] Amino acid sequence of HuACS2 VH

[0415] MKLWLNWVFLLTLLHGIQCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0416] Sequence ID 49

[0417] Amino acid sequence of CDR1 in HuACS2 VH

[0418] SYWLH

[0419] Sequence ID 50

[0420] Amino acid sequence of CDR2 in HuACS2 VH

[0421] RIDPNSGDTKYNEKFKS

[0422] Sequence ID 51

[0423] Amino acid sequence of CDR3 in HuACS2 VH

[0424] YYYGRSYFDY

[0425] Sequence ID 52

[0426] Amino acid sequence of HuACS2 VL

[0427] MDFQVQIFSFLLISAVIISRGEIVLTQSPATLSLSPGERATLSCSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIK

[0428] Sequence ID 53

[0429] Amino acid sequence of CDR1 in HuACS2 VL

[0430] SASSSVSYMH

[0431] Sequence ID 54

[0432] Amino acid sequence of CDR2 in HuACS2 VL

[0433] DTSKLAS

[0434] Sequence ID 55

[0435] Amino acid sequence of CDR3 in HuACS2 VL

[0436] QQWSSNPLT

[0437] Sequence ID 56

[0438] Amino acid sequence of human CD40

[0439] MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0440] Sequence ID 57

[0441] Amino acid sequence of HuACS2.scFv

[0442] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGG SQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0443] Sequence ID 58

[0444] Amino acid sequence of the CH3-HuACS2.scFv (CH3-HuACS2.scFv) fusion encoded in pBS828

[0445] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARF SGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0446] Sequence ID 59

[0447] Amino acid sequence of the mature heavy chain encoded by pBS828

[0448] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGS GTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0449] Sequence ID 60

[0450] Amino acid sequence of hPD-L1-Fc

[0451] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA PYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHTGGGEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0452] Sequence ID 61

[0453] Amino acid sequence of HuPRO1 VH

[0454] MEWNWVVLFLLSLTAGVYAQVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSS

[0455] Sequence ID 62

[0456] Amino acid sequence of CDR1 in HuPRO1 VH

[0457] SSYIS

[0458] Sequence ID 63

[0459] Amino acid sequence of CDR2 in HuPRO1 VH

[0460] WIYAGTGGTSYNQKFTG

[0461] Sequence ID 64

[0462] Amino acid sequence of CDR3 in HuPRO1 VH

[0463] HEGVYWYFDV

[0464] Sequence ID 65

[0465] Amino acid sequence of HuPRO1 VL

[0466] MDFQVQIFSFLLISAVIMSRGEIVLTQSPATLSLSPGERATLSCSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK

[0467] Sequence ID 66

[0468] Amino acid sequence of CDR1 in HuPRO1 VL

[0469] SASSSVSYMH

[0470] Sequence ID 67

[0471] Amino acid sequence of CDR2 in HuPRO1 VL

[0472] DTSNLAS

[0473] Sequence ID 68

[0474] Amino acid sequence of CDR3 in HuPRO1 VL

[0475] HQRSSYPWT

[0476] Sequence ID 69

[0477] Amino acid sequence of human PD-L1

[0478] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD PVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

[0479] Sequence ID 70

[0480] Amino acid sequence of the mature heavy chain encoded by pBS809

[0481] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0482] Sequence ID 71

[0483] Amino acid sequences of mature light chains encoded by pBS809 and pBS813

[0484] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0485] Sequence ID 72

[0486] Amino acid sequence of the mature heavy chain encoded by pBS813

[0487] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSG SGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0488] Sequence ID 73

[0489] Amino acid sequence of the extracellular region of human PD-L1

[0490] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTH

[0491] Sequence ID 74

[0492] Amino acid sequence of HuPRO2 VH

[0493] MGWNWIFLFLSGTAGVHCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSS

[0494] Sequence ID 75

[0495] Amino acid sequence of CDR1 in HuPRO2 VH

[0496] SYGIN

[0497] Sequence ID 76

[0498] Amino acid sequence of CDR2 in HuPRO2 VH

[0499] YIYPGSGGPVYNQKFKG

[0500] Sequence ID 77

[0501] Amino acid sequence of CDR3 in HuPRO2 VH

[0502] ENYRYWYFDV

[0503] Sequence ID 78

[0504] Amino acid sequence of HuPRO2 VL

[0505] MHFQVQIFSFLLISASVIMSRGDIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK

[0506] Sequence ID 79

[0507] Amino acid sequence of CDR1 in HuPRO2 VL

[0508] SASSSVNYMH

[0509] Sequence ID 80

[0510] Amino acid sequence of CDR2 in HuPRO2 VL

[0511] STSN LAS

[0512] Sequence ID 81

[0513] Amino acid sequence of CDR3 in HuPRO2 VL

[0514] QQRSSYPLT

[0515] Sequence ID 82

[0516] Amino acid sequence of the mature heavy chain encoded by pBS841

[0517] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSG SGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0518] Sequence ID 83

[0519] Amino acid sequence of the mature light chain encoded by pBS841

[0520] DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0521] Sequence ID 84

[0522] Amino acid sequence of HuPRO5 VH MMVLSLLYLLTALPGILSQVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSS

[0523] Sequence ID 85

[0524] Amino acid sequence of CDR1 in HuPRO5 VH

[0525] SGYWN

[0526] Sequence ID 86

[0527] Amino acid sequence of CDR2 in HuPRO5 VH

[0528] YISYTGSTYSNPSLKS

[0529] Sequence ID 87

[0530] Amino acid sequence of CDR3 in HuPRO5 VH

[0531] SQNWERAWFAY

[0532] Sequence ID 88

[0533] Amino acid sequence of HuPRO5 VL

[0534] MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK

[0535] Sequence ID 89

[0536] Amino acid sequence of CDR1 in HuPRO5 VL

[0537] SASSSVSYMH

[0538] Sequence ID 90

[0539] Amino acid sequence of CDR2 in HuPRO5 VL

[0540] DTSKLAS

[0541] Sequence ID 91

[0542] Amino acid sequence of CDR3 in HuPRO5 VL

[0543] FQGSGYPFT

[0544] Sequence ID 92

[0545] Amino acid sequence of the mature heavy chain encoded by pBS839

[0546] QVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSG SGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0547] Sequence ID 93

[0548] Amino acid sequence of the mature light chain encoded by pBS839

[0549] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0550] Sequence ID 94

[0551] Amino acid sequence of CDR1 in HuM195 VH

[0552] DYNMH

[0553] Sequence ID 95

[0554] Amino acid sequence of CDR2 in HuM195 VH

[0555] YIYPYNGGTGYNQKFKS

[0556] Sequence ID 96

[0557] Amino acid sequence of CDR3 in HuM195 VH

[0558] GRPAMDY

[0559] Sequence ID 97

[0560] Amino acid sequence of CDR1 in HuM195 VL

[0561] RASESVDNYGISFMN

[0562] Sequence ID 98

[0563] Amino acid sequence of CDR2 in HuM195 VL

[0564] AASNQGS

[0565] Sequence ID 99

[0566] Amino acid sequence of CDR3 in HuM195 VL

[0567] QQSKEVPWT

[0568] Sequence ID 100

[0569] Amino acid sequence of Ch225 VH CDR1

[0570] NYGVH

[0571] Sequence ID 101

[0572] Amino acid sequence of Ch225 VH CDR2

[0573] VIWSGGNTDYNTPFTS

[0574] Sequence ID 102

[0575] Amino acid sequence of Ch225 VH CDR3

[0576] ALTYYDYEFAY

[0577] Sequence ID 103

[0578] Amino acid sequence of CDR1 in Ch225 VL

[0579] RASQSIGTNIH

[0580] Sequence ID 104

[0581] Amino acid sequence of CDR2 in Ch225 VL

[0582] YASESIS

[0583] Sequence ID 105

[0584] Amino acid sequence of Ch225 VL CDR3

[0585] QQNNNWPTT

[0586] Sequence ID 106

[0587] Amino acid sequence of the mature heavy chain encoded by pBS853

[0588] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0589] Sequence ID 107

[0590] Amino acid sequence of hICOS-Fc

[0591] GEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0592] Sequence ID 108

[0593] Amino acid sequence of HuTAM14 VH

[0594] MAVLVLLLCLVTFPSCALSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0595] Sequence ID 109

[0596] Amino acid sequence of CDR1 in HuTAM14 VH

[0597] SNSVS

[0598] Sequence ID 110

[0599] Amino acid sequence of CDR2 in HuTAM14 VH

[0600] AIWSGGSTDYNSALKS

[0601] Sequence ID 111

[0602] Amino acid sequence of CDR3 in HuTAM14 VH

[0603] WEQPYYFDY

[0604] Sequence ID 112

[0605] Amino acid sequence of HuTAM14 VL

[0606] MRTSIQLLGLLLFWLHDAQCDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIK

[0607] Sequence ID 113

[0608] Amino acid sequence of CDR1 in HuTAM14 VL

[0609] QASQNIYKYIA

[0610] Sequence ID 114

[0611] Amino acid sequence of CDR2 in HuTAM14 VL

[0612] YTSTLES

[0613] Sequence ID 115

[0614] Amino acid sequence of CDR3 in HuTAM14 VL

[0615] LQYVNLYT

[0616] Sequence ID 116

[0617] Amino acid sequence of human ICOS

[0618] MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

[0619] Sequence ID 117

[0620] Amino acid sequence of HuTAM14.scFv

[0621] DIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGGSGGGGSGGG GSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0622] Sequence ID 118

[0623] The amino acid sequence of the CH3-HuTAM14.scFv (CH3-HuTAM14.scFv) fusion encoded by pBS859

[0624] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPS RFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0625] Sequence ID 119

[0626] Amino acid sequence of the mature heavy chain encoded by pBS859

[0627] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFS GSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0628] Sequence ID 120

[0629] Amino acid sequence of the mature heavy chain encoded in pBS840

[0630] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0631] Sequence ID 121

[0632] Amino acid sequence of the mature heavy chain encoded by pBS846

[0633] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSG SGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0634] Sequence ID 122

[0635] Amino acid sequence of HuOHX14DS.scFv.ds

[0636] DIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGCGTKVEIKGGGGGSGGGGSGGG GSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQCLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0637] Sequence ID 123

[0638] Amino acid sequence of HuACS2.scFv.ds

[0639] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGCGTKVEIKGGGGSGGGGSGGGG SQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQCLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0640] Sequence ID 124

[0641] Amino acid sequence of HuTAM14.scFv.ds

[0642] DIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGCGTKVEIKGGGGGSGGGGSGGG GSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKCLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0643] Sequence ID 125

[0644] Amino acid sequence of HuGAB11.scFv.ds

[0645] DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTFTLTISSLQPEDVATYYCQQGQSYPLTFGCGTKVEIKGGGGGSGGGGSGGG GSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKCLEWLGVIWGGGGTYYNSALKSRLTISKDTTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0646] Sequence ID 126

[0647] Amino acid sequence of h4-1BB-Fc

[0648] FERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0649] Sequence ID 127

[0650] Amino acid sequence of HuFOB5 VH

[0651] MERHWIFLFLFSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQGLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0652] Sequence ID 128

[0653] Amino acid sequence of CDR1 in HuFOB5 VH

[0654] NYWMH

[0655] Sequence ID 129

[0656] Amino acid sequence of CDR2 in HuFOB5 VH

[0657] YINPSTGYTESNQKFKD

[0658] Sequence ID 130

[0659] Amino acid sequence of HuFOB5 VH CDR3

[0660] SYVGYYYAVDY

[0661] Sequence ID 131

[0662] Amino acid sequence of HuFOB5 VL

[0663] MDSQAQVLMLLLLWVSGTCGDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK

[0664] Sequence ID 132

[0665] Amino acid sequence of CDR1 in HuFOB5 VL

[0666] KSSQSLLYSNNEKNYLA

[0667] Sequence ID 133

[0668] Amino acid sequence of CDR2 in HuFOB5 VL

[0669] WASTRES

[0670] Sequence ID 134

[0671] Amino acid sequence of CDR3 in HuFOB5 VL

[0672] QQYYSYPYT

[0673] Sequence ID 135

[0674] Amino acid sequence of human 4-1BB

[0675] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFND QKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0676] Sequence ID 136

[0677] Amino acid sequence of HuFOB5.scFv.LH.ds

[0678] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGS GGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0679] Sequence ID 137

[0680] The amino acid sequence of the CH3-HuFOB5.scFv.LH.ds (CH3-HuFOB5.scFv.LH.ds) fusion encoded by pBS883

[0681] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVP DRFSSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0682] Sequence ID 138

[0683] Amino acid sequence of the mature heavy chain encoded by pBS883

[0684] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSG SGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0685] Sequence ID 139

[0686] Amino acid sequence of HuFOB5.scFv.HL.ds

[0687] QVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGG SGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0688] Sequence ID 140

[0689] The amino acid sequence of the fusion of CH3 and HuFOB5.scFv.HL.ds (CH3-HuFOB5.scFv.HL.ds) encoded in pBS884

[0690] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0691] Sequence ID 141

[0692] Amino acid sequence of the mature heavy chain encoded by pBS884

[0693] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTIT ADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0694] Sequence ID 142

[0695] Amino acid sequence of human PD-1

[0696] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSPARRGSADGPRSAQPLRPEDGHCSWPL

[0697] Sequence ID 143

[0698] Amino acid sequence of hPD-1-Fc

[0699] LDSPPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. It includes a first binding site that specifically binds to PD-L1 and a second binding site that specifically binds to OX40, The first binding site antagonistizes the binding of PD-L1 to PD-1, and the second binding site agonizes OX40. The heavy chain variable region and light chain variable region of the first pair form a first binding site, the heavy chain variable region and light chain variable region of the second pair form a second binding site, the C-terminuses of the heavy chain variable region and light chain variable region of the first pair fuse to the N-terminuses of the heavy chain steady region and light chain steady region, respectively, the heavy chain variable region and light chain variable region of the second pair form an scFv, and the light chain variable region of the scFv fuses to the C-terminus of the heavy chain steady region. The heavy chain constant region has at least one mutation that reduces FcRγ binding, A bispecific antibody containing two primary binding sites and two secondary binding sites.

2. The first binding site is, It includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 62-64, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 66-68; or It includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 75-77, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 79-81; or It includes a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 85-87, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 89-91. The bispecific antibody according to claim 1, wherein the second binding site comprises a mature heavy chain variable region containing CDR H1, H2, and H3 of SEQ ID NOs. 13 to 15, and a mature light chain variable region containing CDR L1, L2, and L3 of SEQ ID NOs. 17 to 19, respectively.

3. The first binding site is, It comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 61, and a mature light chain variable region including residues 23-127 of SEQ ID NO: 65; or It comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 74, and a mature light chain variable region including residues 23-128 of SEQ ID NO: 78; or It includes a mature heavy chain variable region containing residues 19-137 of SEQ ID NO: 84, and a mature light chain variable region containing residues 23-128 of SEQ ID NO:

88. The bispecific antibody according to claim 2, wherein the second binding site comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region including residues 21-127 of SEQ ID NO:

16.

4. The first binding site is, It comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 61, and a mature light chain variable region including residues 23-127 of SEQ ID NO: 65; or It comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 74, and a mature light chain variable region including residues 23-128 of SEQ ID NO: 78; or It includes a mature heavy chain variable region containing residues 19-137 of SEQ ID NO: 84, and a mature light chain variable region containing residues 23-128 of SEQ ID NO:

88. The bispecific antibody according to claim 2, wherein the second binding site comprises a single-stranded Fv fragment including SEQ ID NO: 22 or 122.

5. The bispecific antibody according to any one of claims 1 to 4, wherein the first and second binding sites are a humanization binding site, a veneer binding site, or a human binding site, and the heavy chain constant region and the light chain constant region are a human heavy chain constant region and a human light chain constant region.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the isotype of the heavy chain constant region is human IgG1 and the isotype of the light chain constant region is kappa.

7. The bispecific antibody according to any one of claims 1 to 6, wherein the heavy chain constant region has at least one mutation that increases binding to FcRn.

8. comprising the heavy chain of SEQ ID NO: 72 and the light chain of SEQ ID NO: 71; or Including the heavy chain of SEQ ID NO: 82 and the light chain of SEQ ID NO: 83; or The bispecific antibody according to claim 1, comprising the heavy chain of SEQ ID NO: 92 and the light chain of SEQ ID NO:

93.

9. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. A pharmaceutical composition for treating cancer, comprising a bispecific antibody as described in any one of claims 1 to 8.

11. A pharmaceutical composition for treating infectious diseases, comprising a bispecific antibody as described in any one of claims 1 to 8.

Images