Bispecific antibodies that activate immune cells

Abstract

The present invention provides bispecific antibodies, one arm of which binds to a cancer-associated antigen on a cancer cell, such as CD33, EGFR or PD-L1, and the second arm binds to a co-stimulatory molecule, such as OX40, CD40, GITR, ICOS or 4-1BB. The bridging of bispecific antibodies between cancer cells expressing cancer-associated antigens and immune cells expressing co-stimulatory molecules leads to the aggregation of co-stimulatory molecules and the selective activation of immune cells in close proximity to cancer cells. Therefore, immune cells can exert an immunotherapeutic effect on cancer cells while reducing toxicity to healthy tissues.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a non-provisional application of US 62 / 760,328, filed November 13, 2018 and US 62 / 838,579, filed April 25, 2019, the entire contents of which are incorporated herein by reference for all purposes.

[0003] By referencing and incorporating into the sequence list

[0004] This application includes a 438KB sequence list written in txt file 530874-ST25, which is incorporated by reference. Background Technology

[0005] Antigen-specific immune responses are complex biological processes controlled by multiple layers of positive and negative regulators. T cells are initially stimulated via T cell receptors (TCRs) by recognizing cognate peptide 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 of the CD28 superfamily, such as CD28 and ICOS. The immune response is further positively regulated by other co-stimulatory molecules (e.g., CD40, OX40, GITR, CD27, HVEM, and 4-1BB, belonging to the TNF receptor superfamily), which are expressed in cell-type and developmental-stage-related ways, and negatively regulated by checkpoint molecules (e.g., PD-1, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD96, and CD112R). Checkpoint molecules function to prevent unintended overreactions by the human immune system. However, they also limit the immune system's ability to effectively combat cancer and infectious diseases. 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 etal.Ann.Transl.Med.3:267,2015; Mercier et al., Front.Immunol.6:418,2015; Topalianet al., Cancer Cell 27:450-461,2015; Baumeister et al., Annu.Rev.Immunol.34:539-573,2016; Ward-Kavanagh et al. al., Immunity 44:1005-1019, 2016; Torphy et al., Int. J. Mol. Sci. 18: 2642, 2017.

[0006] It has been reported that antagonistic monoclonal antibodies blocking the function of checkpoint molecules can effectively enhance the immune system (Mercier et al., ibid.; Baumeister et al., ibid.). For example, the interaction between PD-1 (also known as CD279) expressed on the surface of T cells and its ligand PD-L1 (also known as CD274) expressed on antigen-presenting cells suppresses the body's immune response. The ligands of checkpoint molecules expressed on cancer cells interact with their respective receptors on immune cells, weakening the immune response in the body, thus allowing cancer cells to survive and proliferate. Inhibiting the interaction between PD-1 and PD-L1 with monoclonal antibodies has been shown to be effective in treating cancer. To date, the US FDA has approved three anti-PD-1 monoclonal IgG antibodies (nivolumab, pembrolizumab, and cemiplimab) and three anti-PD-L1 monoclonal IgG antibodies (durvalumab, atezolizumab, and avelumab) as cancer therapeutics. In addition, the therapeutic efficacy of monoclonal IgG antibodies targeting other checkpoint molecules (including TIGIT, TIM-3, LAG-3, and VISTA) that block their interactions with their respective ligands has been investigated in clinical studies (Marin-Acevedo et al., J. Hematol. Oncol. 11:39, 2018).

[0007] The anti-CTLA-4 monoclonal IgG antibody (ipilimumab) has been used as a cancer treatment drug. This antibody can prevent the interaction between CTLA-4 on T cells and B7-1 and B7-2 (also known as CD80 and CD86, respectively, which are ligands of the co-stimulatory molecule CD28) on antigen-presenting cells, thereby stimulating CD28-mediated T cell activation. Anti-co-stimulatory molecules (such as ICOS, CD40, OX40, GITR, CD27, and 4-1BB) agonist IgG antibodies can also 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). Co-stimulatory molecules belonging to the TNF receptor superfamily and ICOS require polymeric cross-links on the surface of immune cells to initiate intracellular signal transduction and 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 bivalent for antigen binding, it cannot effectively trigger signal transduction through these co-stimulatory molecules unless the IgG molecule bound to the cell surface is conjugated, for example, by binding to an Fcγ receptor on another cell. In mouse xenograft tumor models, anti-CD40 IgG2 antibodies with Fc mutations to enhance binding to FcγRIIB (also known as CD32B) exhibited superior antitumor activity compared to parental IgG2 antibodies (Dahan et al., Cancer Cell 29:820-831, 2016). As another method to promote multivalent binding of surface molecules, Tso et al. engineered IgG antibodies by fusing the CH3 and CH4 regions of the human μ heavy chain to the C-terminus of the human γ heavy chain (US Patent 10,053,517). Such engineered hexameric IgG antibodies effectively crosslink co-stimulatory molecules on the cell surface to trigger intracellular signal transduction.

[0008] Bispecific antibodies are artificially engineered antibodies capable of binding to two different antigens or two different epitopes on the same antigen. Various forms of bispecific antibodies have been developed (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., MAbs9:182-212, 2017; Sedykh et al., Drug Des. Dev. Ther. 12:195-208, 2018). Some bispecific antibodies, such as blinatumomab which binds to both CD3 and CD19, are designed to bring T cells close to cancer cells carrying CD19 and trigger T cell-mediated cytotoxicity against cancer cells (Viardot et al., Cancer Treat. Rev. 65:87-95, 2018). Summary of the Invention

[0009] This 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 activates OX40 or CD40. Optionally, the first binding site comprises mature heavy chain variable regions including CDR H1, H2, and H3 of SEQ ID NO. 94-96 and mature light chain variable regions including CDR L1, L2, and L3 of SEQ ID NO. 97-99, and the second binding region comprises mature heavy chain variable regions including CDR H1, H2, and H3 of SEQ ID NO. 33-35 and mature light chain variable regions including CDR L1, L2, and L3 of SEQ ID NO. 37-39; or the second binding region comprises mature heavy chain variable regions including CDR H1, H2, and H3 of SEQ ID NO. 13-15 and mature light chain variable regions including CDR L1, L2, and L3 of SEQ ID NO. 17-19; or the second binding region comprises mature heavy chain variable regions including CDR H1, H2, and H3 of SEQ ID NO. 49-51 and mature light chain variable regions including CDR L1, L2, and L3 of SEQ ID NO. 94-96 and mature light chain variable regions including CDR L1, L2, and L3 of SEQ ID NO. 97-99. The second binding region comprises mature light chain variable regions of CDRL1, L2 and L3 of SEQ ID NO. 53-55; or the second binding region comprises mature heavy chain variable regions of CDR H1, H2 and H3 of SEQ ID NO. 109-111 and mature light chain variable regions of CDR L1, L2 and L3 of SEQ ID NO. 113-115; or the second binding region comprises mature heavy chain variable regions of CDR H1, H2 and H3 of SEQ ID NO. 128-130 and mature light chain variable regions of CDR L1, L2 and L3 of SEQ ID NO. 132-134.Optionally, the first binding site comprises a mature heavy chain variable region including residues 20 to 135 of SEQ ID NO: 2 and a mature light chain variable region including residues 20-131 of SEQ ID NO: 7, and the second binding region comprises a mature heavy chain variable region including residues 20-137 of SEQ ID NO: 32 and a mature light chain variable region including residues 21-127 of SEQ ID NO: 36; or the second binding region 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; or the second binding region comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 48 and a mature light chain variable region including residues 23-127 of SEQ ID NO: 52; or the second binding region comprises a mature heavy chain variable region including residues 20-136 of SEQ ID NO: 108 and a mature light chain variable region including residues 20-136 of SEQ ID NO: 7. The first binding site comprises a mature light chain variable region consisting of residues 21-126 of SEQ ID NO: 112, or the second binding region comprises a mature heavy chain variable region consisting of residues 20-138 of SEQ ID NO: 127 and a mature light chain variable region consisting of residues 21-133 of SEQ ID NO: 131. Optionally, the first binding site comprises a mature heavy chain variable region consisting of residues 20-135 of SEQ ID NO: 2 and a mature light chain variable region consisting of residues 20-131 of SEQ ID NO: 7, and the second binding region comprises a single-stranded Fv fragment comprising SEQ ID NO: 22, 41, 57, 117, 122, 123, 124, 125, 136, or 139.

[0010] This 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 includes an Fc region. Optionally, the first binding site antagonizes the binding of EGFR to EGF, and the second binding site antagonizes GITR. Optionally, the first binding site comprises mature heavy chain variable regions including CDRH1, H2, and H3 of SEQ ID NO. 100-102 and mature light chain variable regions including CDR L1, L2, and L3 of SEQ ID NO. 103-105, and the second binding region comprises mature heavy chain variable regions including CDRH1, H2, and H3 of SEQ ID NO. 33-35 and mature light chain variable regions including CDRL1, L2, and L3 of SEQ ID NO. 37-39, or the second binding region comprises mature heavy chain variable regions including CDR H1, H2, and H3 of SEQ ID NO. 13-15 and mature light chain variable regions including CDRL1, L2, and L3 of SEQ ID NO. 17-19, or the second binding region comprises mature heavy chain variable regions including CDR H1, H2, and H3 of SEQ ID NO. 49-51 and mature light chain variable regions including CDR L1, H2, and H3 of SEQ ID NO. 53-55. The second binding region comprises mature light chain variable regions of L1, L2, and L3, respectively, or comprises mature heavy chain variable regions of CDRH1, H2, and H3 of SEQ ID NO. 109-111 and mature light chain variable regions of CDR L1, L2, and L3 of SEQ ID NO. 113-115, respectively; or the second binding region comprises mature heavy chain variable regions of CDR H1, H2, and H3 of SEQ ID NO. 128-130 and mature light chain variable regions of CDR L1, L2, and L3 of SEQ ID NO. 132-134, respectively.Optionally, the first binding site comprises a mature heavy chain variable region including residues 20 to 138 of SEQ ID NO: 29 and a mature light chain variable region including residues 21-127 of SEQ ID NO: 30, and the second binding region comprises a mature heavy chain variable region including residues 20-137 of SEQ ID NO: 32 and a mature light chain variable region including residues 21-127 of SEQ ID NO: 36; or the second binding region 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; or the second binding region comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 48 and a mature light chain variable region including residues 23-127 of SEQ ID NO: 52; or the second binding region comprises a mature heavy chain variable region including residues 20-136 of SEQ ID NO: 108 and a mature light chain variable region including residues 23-127 of SEQ ID NO: 30. The first binding site comprises a mature light chain variable region consisting of residues 21-126 of SEQ ID NO: 112, or the second binding region comprises a mature heavy chain variable region consisting of residues 20-138 of SEQ ID NO: 127 and a mature light chain variable region consisting of residues 21-133 of SEQ ID NO: 131. Optionally, the first binding site comprises a mature heavy chain variable region and a mature light chain variable region, wherein the mature heavy chain variable region comprises residues 20 to 138 of SEQ ID NO: 29, the mature light chain variable region comprises residues 21-127 of SEQ ID NO: 30, and the second binding region comprises a single-stranded Fv fragment comprising SEQ ID NO: 22, 41, 57, 117, 122, 123, 124, 125, 136, or 139.

[0011] The present invention further provides a monoclonal antibody comprising mature heavy chain variable regions comprising CDR H1, H2, and H3 of SEQ ID NO. 13-15 and mature light chain variable regions comprising CDR L1, L2, and L3 of SEQ ID NO. 17-19; or the monoclonal antibody comprising mature heavy chain variable regions comprising CDR H1, H2, and H3 of SEQ ID NO. 49-51 and mature light chain variable regions comprising CDR L1, L2, and L3 of SEQ ID NO. 53-55; or the monoclonal antibody comprising mature heavy chain variable regions comprising CDR H1, H2, and H3 of SEQ ID NO. 109-111 and mature light chain variable regions comprising CDR L1, L2, and L3 of SEQ ID NO. 113-115; or the monoclonal antibody comprising mature heavy chain variable regions comprising CDR H1, H2, and H3 of SEQ ID NO. 128-130 and mature light chain variable regions comprising CDR H1, H2, and H3 of SEQ ID NO. 128-130. The monoclonal antibody comprises mature light chain variable regions of CDRs L1, L2, and L3 of SEQ ID NO: 32-134; optionally, the monoclonal antibody comprises mature heavy chain variable regions comprising residues 20-137 of SEQ ID NO: 32 and mature light chain variable regions comprising residues 21-127 of SEQ ID NO: 36, or the monoclonal antibody comprises mature heavy chain variable regions comprising residues 20-138 of SEQ ID NO: 12 and mature light chain variable regions comprising residues 21-127 of SEQ ID NO: 16, or the monoclonal antibody comprises mature heavy chain variable regions comprising residues 20-138 of SEQ ID NO: 48 and mature light chain variable regions comprising residues 23-127 of SEQ ID NO: 52, or the monoclonal antibody comprises mature heavy chain variable regions comprising residues 20-136 of SEQ ID NO: 108 and mature light chain variable regions comprising residues 21-126 of SEQ ID NO: 112, or the monoclonal antibody comprises mature heavy chain variable regions comprising residues 20-136 of SEQ ID NO: 108 and mature light chain variable regions comprising residues 21-126 of SEQ ID NO: 112, or the monoclonal antibody comprises mature heavy chain variable regions comprising residues 20-137 of SEQ ID NO: 132-134. The mature heavy chain variable region consisting of residues 20-138 of SEQ ID NO: 127 and the mature light chain variable region consisting of residues 21-133 of SEQ ID NO: 131.

[0012] The present invention also 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 IgG Fc region. Optionally, the first binding site antagonizes the binding of PD-L1 to PD1, while the second binding site activates GITR, OX40, CD40, ICOS, or 4-1BB. Optionally, the first binding site comprises a mature heavy chain variable region including CDR H1, H2, and H3 of SEQ ID NO. 62-64, 75-77, or 85-87, and a mature light chain variable region including CDR L1, L2, and L3 of SEQ ID NO. 66-68, 79-81, or 89-91, respectively; and the second binding region comprises a mature heavy chain variable region including CDR H1, H2, and H3 of SEQ ID NO. 33-35, and a mature light chain variable region including CDR L1, L2, and L3 of SEQ ID NO. 37-39, or the second binding region comprises a mature heavy chain variable region including CDR H1, H2, and H3 of SEQ ID NO. 13-15, and a mature light chain variable region including CDR L1, L2, and L3 of SEQ ID NO. 17-19, or the second binding region comprises a mature heavy chain variable region including CDR H1, H2, and H3 of SEQ ID NO. 49-51, respectively. The second binding region comprises mature heavy chain variable regions of H1, H2 and H3 and mature light chain variable regions of CDRs L1, L2 and L3 of SEQ ID NO. 53-55, respectively; or the second binding region comprises mature heavy chain variable regions of CDRs H1, H2 and H3 of SEQ ID NO. 109-111 and mature light chain variable regions of CDRs L1, L2 and L3 of SEQ ID NO. 113-115, respectively; or the second binding region comprises mature heavy chain variable regions of CDRs H1, H2 and H3 of SEQ ID NO. 128-130 and mature light chain variable regions of CDRs L1, L2 and L3 of SEQ ID NO. 132-134, respectively.Optionally, the first binding site comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 61, residues 20-138 of SEQ ID NO: 74, and residues 19-137 of SEQ ID NO: 84, and a mature light chain variable region including residues 23-137 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 region comprises a mature heavy chain variable region including residues 20-137 of SEQ ID NO: 32 and a mature light chain variable region including residues 21-127 of SEQ ID NO: 36, or the second binding region 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, or the second binding region comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 48 and a mature light chain variable region including residues 20-137 of SEQ ID NO: 61, and a mature light chain variable region including residues 21-127 of SEQ ID NO: 74, or the second binding region comprises a mature heavy chain variable region including residues 20-138 of SEQ ID NO: 48 and a mature light chain variable region including residues 21-127 of SEQ ID NO: 61, SEQ ID NO: 74, SEQ ID NO: 84, SEQ ID NO: 65, SEQ ID NO: 74, SEQ ID NO: 85, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 85, SEQ ID NO: 66, SEQ ID NO: 65, SEQ ID NO: 74, SEQ ID NO: 65, SEQ ID NO: The second binding region comprises the mature light chain variable region of residues 23-127 of SEQ ID NO: 52, or the second binding region comprises the mature heavy chain variable region of residues 20-136 of SEQ ID NO: 108 and the mature light chain variable region of residues 21-126 of SEQ ID NO: 112, or the second binding region comprises the mature heavy chain variable region of residues 20-138 of SEQ ID NO: 127 and the mature light chain variable region of residues 21-133 of SEQ ID NO: 131. Optionally, the first binding site comprises a mature heavy chain variable region including 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 including residues 23-137 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 region comprises a single-stranded Fv fragment, the single-stranded Fv fragment comprising SEQ ID NO: 22, 41, 57, 117, 122, 123, 124, 125, 136, or 139.

[0013] Some of the aforementioned bispecific antibodies comprise 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, wherein the C-terminus of the first pair of heavy and light chain variable regions is fused to the N-terminus of the heavy and light chain constant regions, and the second pair of heavy and light chain variable regions forms an scFv fused to the C-terminus of the heavy chain constant region, and vice versa. In some bispecific antibodies, the C-terminus of the first pair of heavy and light chain variable regions is fused to the N-terminus of the heavy and light chain constant regions, and the second pair of heavy and light chain variable regions forms an scFv fused to the C-terminus of the heavy chain constant region. 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, veneered, or human, while the heavy and light chain constant regions are human. In some bispecific antibodies, the isotype of the heavy chain constant region is human IgG1, while the isotype of the light chain constant region is κ. Some bispecific antibodies contain two first binding sites and two second binding sites. In some bispecific antibodies, the heavy chain constant region has at least one mutation that reduces or increases FcRγ binding. In some bispecific antibodies, the heavy chain constant region has at least one mutation that increases binding to FcRn.

[0014] 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 co-stimulatory molecule. The first binding site is formed by a first pair of heavy and light chain variable regions, while the second binding site is formed by a second pair of heavy and light chain variable regions. The C-terminus of the first pair of heavy and light chain variable regions is fused to the N-terminus of the heavy and light chain constant regions, and the second pair of heavy and light chain variable regions forms an scFv fused to the C-terminus of the heavy chain constant region, and vice versa. Optionally, the first binding site is attached to the N-terminus of the heavy and light chain constant regions, and the second binding site is attached to the C-terminus of the heavy chain constant region.

[0015] The present invention further provides a monoclonal antibody that specifically binds to PD-L1, comprising mature heavy chain variable regions including CDR H1, H2, and H3 of SEQ ID NO. 62-64, 75-77, or 85-87, respectively, and mature light chain variable regions including CDR L1, L2, and L3 of SEQ ID NO. 66-68, 79-81, or 89-91, respectively. Optionally, the monoclonal antibody comprises a mature heavy chain variable region including 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 including 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.

[0016] The present invention further provides a pharmaceutical composition comprising any of the bispecific or monoclonal antibodies and a pharmaceutically acceptable carrier.

[0017] The present invention further provides a method for treating or preventing cancer, the method comprising an effective regimen of administering bispecific or monoclonal antibodies as defined above to a subject suffering from or at risk of cancer.

[0018] The present invention further provides a method for treating an infection, comprising an effective regimen of administering a bispecific antibody or monoclonal antibody as defined above to a subject suffering from or at risk of infection. Attached Figure Description

[0019] Figure 1A-D Illustrations of the expression vectors pHuM195-IgG1 (A), pBS824 (B), pFCm331 (C), and pFCm267 (D). Restriction enzyme sites are shown. Figures are not to scale.

[0020] Figure 2 The illustrated structure of the bispecific IgG antibody of the present invention.

[0021] Figure 3 Analysis of luciferase activity in Jurkat dual cells (JD / OX40) containing a bispecific antibody (BS824) that binds to CD33 and OX40 and expresses OX40. The mean relative luminescence units (RLU) of three replicate analyses are shown in the standard deviation bar.

[0022] Figure 4Analysis of luciferase activity in Jurkat dual cells (JD / GITR) containing a bispecific antibody (BS827) that binds to EGFR and GITR and expresses GITR. The mean relative luminescence units (RLU) of three replicate analyses are expressed as standard deviation bars.

[0023] Figure 5 FACS analysis of CD95 expression in Ramos cells equipped with a bispecific antibody (BS828) that binds CD33 and CD40. The X and Y axes indicate the expression levels of CD20 and CD95, respectively. The numbers in the upper right and lower right quadrants show the percentage of CD20-positive cells in each quadrant.

[0024] Figure 6 T cells were activated using a bispecific antibody (BS809) that binds to PD-L1 and GITR. The mean expression level of IL-2 is represented by standard deviation bars.

[0025] Figure 7 Analysis of luciferase activity in Jurkat dual cells (JD / OX40) containing a bispecific antibody (BS813) that binds to PD-L1 and OX40 and expresses OX40. The mean relative luminescence units (RLU) of three replicate analyses are expressed as standard deviation bars.

[0026] Figure 8 Analysis of luciferase activity in Jurkat dual cells (JD / GITR) containing a bispecific antibody (BS809) that binds to PD-L1 and GITR and expresses GITR. The mean relative luminescence units (RLU) of three replicate analyses are expressed as standard deviation bars.

[0027] Figure 9 Human T cells were activated using bispecific antibodies (BS813, BS841, and BS839) that bind to PD-L1 and OX40. The mean IL-2 expression level from three replicate analyses is represented by standard deviation bars.

[0028] Figure 10 SEB-treated human peripheral blood mononuclear cells were activated using bispecific antibodies (BS813, BS841, and BS839) that bind to PD-L1 and OX40. The mean IL-2 expression level of three replicate analyses is expressed as standard deviation bars.

[0029] Figure 11 Human T cells were activated using a bispecific antibody (BS859) that binds to both PD-L1 and ICOS. Mean expression levels of IL-2 and IL-10 are represented by standard deviation bars.

[0030] Figure 12Analysis of luciferase activity in Jurkat dual cells (JD / GITR) containing a bispecific antibody (BS840) that binds to PD-L1 and GITR and expresses GITR. The mean relative luminescence units (RLU) of three replicate analyses are expressed as standard deviation bars.

[0031] Figure 13 Ramos cells were activated using a bispecific antibody (BS846) that binds PD-L1 and CD40. Detailed Implementation

[0032] definition

[0033] The bispecific antibodies of this invention are typically provided in isolated form. This means that the bispecific antibody is usually 50% w / w pure from interfering proteins and other contaminants resulting from its production or purification, but does not preclude the possibility that the bispecific antibody may be bound to an excess of pharmaceutically acceptable carriers or other mediators for its use. Sometimes the bispecific antibody is at least 60%, 70%, 80%, 90%, 95%, or 99% w / w pure from interfering proteins and other contaminants resulting from its production or purification. Typically, the bispecific antibody is the major macromolecular component remaining after purification.

[0034] The specific binding of a bispecific antibody to its target antigen refers to at least 10 6 , 10 7 , 10 8 , 10 9 Or 10 10 M -1 Affinity varies depending on the target. Specific binding is high on a detectable scale, as opposed to nonspecific binding with at least one unrelated target. Specific binding can result from bond formation between specific functional groups or specific spatial fits (e.g., lock and key types), 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 distinct binding sites binds only to targets at those two binding sites.

[0035] The basic antibody structural unit is a tetramer of subunits. Each tetramer comprises two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50–70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. This variable region, when initially expressed, is linked to a cleavable signal peptide. Variable regions lacking a signal peptide are sometimes referred to as mature variable regions. Thus, for example, a light chain mature variable region refers to a light chain variable region lacking a light chain signal peptide. However, mentioning a variable region does not imply the presence of a signal sequence; in fact, the signal sequence is cleaved once the bispecific antibody of this invention is expressed and secreted. A pair of heavy and light chain variable regions defines the antibody binding region. The carboxyl-terminal portions of the light and heavy chains define the light and heavy chain constant regions, respectively. The heavy chain constant region is primarily responsible for 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 regions are divided into CH1, CH2, and CH3. The CH1 region is linked to the light chain constant region via disulfide bonds and non-covalent bonds. The hinge region provides flexibility between the antibody's binding and effector regions and also provides a site for intermolecular disulfide bond binding between the two heavy chain constant regions in the tetrameric subunit. The CH2 and CH3 regions are the main sites for effector function and FcRn binding.

[0036] The light chain is divided into κ or λ. The heavy chain is divided into γ, μ, α, δ, or ε, and antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked together by “J” regions of approximately 12 or more amino acids, and the heavy chain also includes “D” regions of approximately 10 or more amino acids. (See Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7, the full text of which is incorporated herein by reference for all purposes.)

[0037] The mature variable region of each light / heavy chain pair forms the antibody binding site. Therefore, a complete 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 can be identical or different, depending on the form (see, for example, Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). These variable regions all exhibit the same general structure, namely a relatively conserved framework region (FR) linked by three hypervariable regions, also known as complementarity-determining regions or CDRs. The CDRs of the two chains in each pair are aligned through the framework region, enabling them to bind to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The amino acid assignments within each domain are as defined in 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 system (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. Although Kabat numbering can be used for antibody constant regions, EU index numbering is more commonly used, as is the case in this application.

[0038] The term "epitope" refers to a site on an antigen that binds to the arm of a bispecific antibody. Epitopes can be formed from consecutive or discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folds (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Some antibodies bind to terminal-specific epitopes, meaning that antibodies preferentially bind to the same polypeptide with a free end, rather than to a polypeptide that fuses with another polypeptide, resulting in the loss of a free end. Epitopes typically contain at least three, more often at least five, or eight to ten amino acids in a distinctive spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0039] The term "antigen" or "target antigen" refers to a target molecule that binds to one of the binding sites of a bispecific antibody. Antigens can be proteins of any length (natural, synthetic, or recombinantly expressed), nucleic acids, carbohydrates, or other molecules. Antigens include receptors, ligands, antireceptors, and coating proteins.

[0040] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the competitive ability of one antibody to bind to a target antigen. An antibody's epitope can also be determined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have the same epitope. If certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have overlapping epitopes.

[0041] Competition between antibodies is determined by an assay in which the test antibody inhibits the specific binding of the reference antibody to a common antigen (see, for example, 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 2, 5, 10, 20, or 100 times) is detected in the competitive binding assay to inhibit the binding of the reference antibody by at least 50%, but preferably 75%, 90%, or 99%. Antibodies identified by the competitive assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, as well as antibodies that bind to neighboring epitopes sufficiently close to the epitope bound to the reference antibody to cause steric hindrance.

[0042] "Objects" include humans or other mammals receiving preventative or therapeutic treatment. Other non-human animals include animal models of human conditions (e.g., rodents, non-human primates) and veterinary objects.

[0043] To classify amino acid substitutions as conserved or non-conserved, amino acids are grouped as follows: Group I (hydrophobic side chains): methionine, alanine, valine, leucine, isoleucine; Group II (neutral hydrophilic side chains): cysteine, serine, threonine; Group III (acidic side chains): aspartic acid, glutamic acid; Group IV (basic side chains): asparagine, glutamine, histidine, lysine, arginine; Group V (residues affecting chain orientation): glycine, proline; and Group VI (aromatic side chains): tryptophan, tyrosine, phenylalanine. Conservative substitutions include substitutions between amino acids of the same class. Non-conservative substitutions involve replacing a member of one class with a member of another class.

[0044] The sequence identity percentage is determined by maximally matching antibody sequences using the Kabat numbering convention (for variable regions) or the EU numbering convention (for constant regions). After alignment, if the target antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared with the same region of the reference antibody, the sequence identity percentage between the target antibody region and the reference antibody region is calculated by dividing the number of positions of the same amino acids in both regions by the total number of positions compared between the two regions, ignoring gaps, and then multiplying by 100 to convert to a percentage.

[0045] A composition or method that "comprises" one or more of the listed elements may include other elements not specifically listed. For example, a composition containing an antibody may contain only the antibody or a combination of the antibody and other ingredients.

[0046] The term "antibody-dependent cytotoxicity" (ADCC) is a mechanism that induces cell death, dependent on the interaction between antibody-coated target cells (i.e., cells with bound antibodies) and lytically active immune cells (also known as effector cells). Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC is triggered by the interaction of the Fc region of the cell-bound antibody with Fcγ receptors (specifically FcγRI and FcγRIII) on immune effector cells such as neutrophils, macrophages, and natural killer cells. Depending on the type of effector cell involved, the target cell is eliminated through phagocytosis or lysis. The death of antibody-coated target cells is a consequence of effector cell activity.

[0047] The term "opsonization," also known as "antibody-dependent phagocytosis" or ADCP, refers to the process by which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (such as macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulins.

[0048] The term "complement-dependent cytotoxicity" or CDC (also known as CMC) refers to a mechanism that induces cell death in which one or more Fc effector regions of a target-binding antibody activate a series of enzymatic reactions that ultimately lead to the formation of pores on the target cell membrane. Typically, antigen-antibody complexes (e.g., antigen-antibody complexes on antibody-coated target cells) bind to and activate complement component C1q, which in turn activates a complement cascade, resulting in target cell death. Activation of complement can also lead to the deposition of complement components on the target cell surface, which promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0049] The pH-dependent binding of antibodies to FcRn receptors means that the antibody binds more strongly to this receptor at pH 6.0 than at pH 7.5. After internalization via endocytosis, the low-pH FcRn binding in the endosome rescues the IgG antibody from lysosomal catabolism and degradation. The rescued IgG antibody is then released from the FcRn at neutral pH and circulated back into the system. This pH-dependent FcRn binding is the molecular mechanism underlying the long serum half-life of IgG antibodies (and the bispecific antibody of this invention) (Ghetie et al., Annu. Rev. Immunol. 18:739-766, 2000). For example, human IgG antibodies bind to the human neonatal Fc receptor (FcRn) at pH 6.0, but only weakly at pH 7.5. The FcRn binding site in IgG antibodies is located at the junction of the CH2 and CH3 domains. Because the μ heavy chain does not bind to FcRn at pH 6.0 or 7.5, native IgM cannot utilize the FcRn-mediated pathway to rescue antibodies from lysosomal degradation, and therefore its half-life is usually shorter than that of native IgG antibodies.

[0050] Protein A is a 40-60 kDa surface protein initially discovered in the cell wall of Staphylococcus aureus. Protein A binds with specific high affinity to human IgG1, IgG2, and IgG4, as well as mouse IgG2a and IgG2b. It does not bind to human IgG3, IgA, or IgM. Protein A is used for affinity purification of antibodies.

[0051] Protein G is a 65 kDa (G148 protein G) and 58 kDa (C40 protein G) streptococcal cell surface protein. It contains a serum albumin-binding domain, which is not required for IgG binding and is frequently deleted. Protein G specifically binds to all human IgG isotypes but not to IgA or IgM. Protein G can also be used for antibody purification.

[0052] Detailed description

[0053] I. Overview

[0054] This invention provides bispecific antibodies, one arm of which binds to cancer-associated antigens such as CD33, EGFR, or PD-L1, and the second arm binds to co-stimulatory molecules such as OX40, CD40, GITR, ICOS, or 4-1BB. While an understanding of the mechanism is not required for carrying out this invention, it is believed that the bispecific antibodies of this invention bridge the gap between cancer cells expressing cancer-associated antigens and immune cells expressing co-stimulatory molecules, leading to the aggregation of the co-stimulatory molecules and selective activation of immune cells near the cancer cells. Thus, immune cells can exert immunotherapeutic effects against cancer cells with reduced toxicity to healthy tissues. Due to antagonistic interactions between receptors and ligands (e.g., EGFR-EGF interaction, PD-L1-PD1 interaction, or CD33-sialic acid interaction), bispecific antibodies can have additional activity against cells expressing cancer-associated antigens. Certain bispecific antibodies of this invention, binding to cancer cells (or pathogen-infected cells) and co-stimulatory molecules, can bridge the gap between these two cell types and trigger an immune response against cancer or infectious diseases through multivalent cross-linking of co-stimulatory molecules at cell-to-cell junctions. Some of the bispecific antibodies of the present invention can also enhance the immune response by inhibiting the function of checkpoint molecules (e.g., PD-L1) and triggering signal transduction pathways through multivalent cross-linking of co-stimulatory molecules (e.g., GITR, OX40, CD40, ICOS).

[0055] II. Target

[0056] The bispecific antibody of this invention has at least two arms, each arm specifically binding to a different target antigen. One class of proteins that can be used as targets are cancer-associated antigens. These antigens are expressed by cancer cells and are typically expressed at levels higher than in control-matched normal tissue (overexpression). Some examples of cancer-associated antigens are CD33, EGFR, and PD-L1. Exemplary human forms of these targets are designated SwissProt as P20138, P00533, and Q9NZQ7. CD33 binds to sialic acid and is primarily overexpressed in myeloid-origin cancers (e.g., acute myeloid leukemia). EGFR binds to EGF and is primarily overexpressed 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, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer. Another class of proteins are antigens expressed on the surface of cells infected by pathogens.

[0057] Another class of proteins are co-stimulatory molecules, including CD40, OX40, GITR, ICOS, and 4-1BB. Typical SwissProt designations for the human forms of these targets are P25942, P23510, Q9Y5U5, Q9Y6W8, and Q07011. Unless clearly indicated from the context, a specific target mentioned should be understood to refer to the human form. However, non-human forms, such as laboratory forms (e.g., mice, rats), non-human primates, companion animals, or farm animals, may also be used. III. Exemplary Antibodies for Each Target

[0058] Bispecific antibodies are formed from the heavy and light chain variable regions derived from component antibodies. Component antibodies can be rodent, chimeric, ornamented, humanized, primate, or human, etc. Component antibodies can be of the same or different types; for example, one can be humanized and the other human.

[0059] Other non-human monoclonal antibodies, such as those of mice, guinea pigs, primates, rabbits, or rats, can be produced by immunizing animals, for example, with the antigen or a fragment thereof or with cells carrying 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, through peptide synthesis, or through recombinant expression. Optionally, the antigen can be fused to or co-administered with a carrier protein. Optionally, the antigen can be administered with an adjuvant. Several types of adjuvants can be used as described below. Freund's complete adjuvant and subsequent incomplete adjuvant are preferred for immunizing experimental animals.

[0060] Humanized antibodies are genetically engineered antibodies in which a core sequence (CDR) derived from a non-human "donor" antibody is grafted into a human "recipient" antibody sequence (see, for example, Queen, U.S. Patent Nos. 5,530,101 and 5,585,089; Winter, U.S. Patent No. 5,225,539; Carter, U.S. Patent No. 6,407,213; Adair, U.S. Patent Nos. 5,859,205 and 6,881,557; Foote, U.S. Patent No. 6,881,557). The recipient antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a common sequence of human antibody sequences, or a germline region sequence. Therefore, a humanized antibody is an antibody having some or all of the CDRs, wholly or substantially derived from the donor antibody, and a variable region framework sequence and a constant region (if present), wholly or substantially derived from the human antibody sequence. Similarly, the humanized heavy chain has at least one, two, and usually all three CDRs, all or substantially derived from the donor antibody heavy chain, and, if present, a heavy chain variable region framework sequence and a heavy chain constant region, substantially derived from the human heavy chain variable region framework and constant region sequences. Similarly, the humanized light chain has at least one, two, and usually all three CDRs, all or substantially derived from the donor antibody light chain, and, if present, a light chain variable region framework sequence and a light chain constant region, substantially derived from the human light chain variable region framework and constant region sequences. Except for nanobodies and dAbs, humanized antibodies comprise humanized heavy chains and humanized light chains. The CDRs in a humanized antibody are substantially derived from the corresponding CDRs in a 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. When at least 85%, 90%, 95%, or 100% of the corresponding residues are identical as defined by Kabat, the variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from the human variable region framework sequence or the human constant region, respectively.

[0061] Although humanized antibodies typically contain all six CDRs from mouse antibodies (preferably as defined by Kabat), they can also be made from fewer than all of the CDRs (e.g., at least 3, 4, or 5 CDRs 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).

[0062] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) are combined with the constant regions of the human light and heavy chains. Such antibodies essentially or completely retain the binding specificity of mouse antibodies and are approximately two-thirds human sequences.

[0063] A decorated antibody is a humanized antibody that retains some, and typically all, of the CDRs and some nonhuman variable region framework residues of the nonhuman antibody, but replaces other variable region framework residues (e.g., exposed residues (Padlan, Mol. Immunol. 28:489, 1991)) that may contribute to B-cell or T-cell epitopes with residues from the corresponding positions in the human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially derived from the nonhuman antibody and the nonhuman antibody's variable region framework becomes more human-like through the substitutions.

[0064] Human antibodies can be isolated from humans or otherwise derived from the expression of human immunoglobulin genes (e.g., in transgenic mice, in vitro, or via phage display). Methods for generating human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666, on the use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al.). al., WO93 / 12227 (1993); U.S. Patents 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 148,1547-1553 (1994), Nature Biotechnology 14,826 (1996), Kucherlapati, WO 91 / 10741 (1991)) and phage display methods (see, for example, Dower et al., WO91 / 17271 and McCafferty et al., WO 92 / 01047, U.S. Patent Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332.

[0065] Screening can be performed to detect the specific binding of antibodies to antigens. Further screening can be conducted to detect the binding of antibodies to specific regions of antigens, competition with reference antibodies, and the activation or antagonism of antigen-carrying cells. As mentioned above, non-human antibodies can be converted into chimeric, decorated, or humanized forms.

[0066] 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 the Kabat CDR provided in the sequence listing shown in Table 1 below.

[0067] Table 1

[0068]

[0069]

[0070] This specification discloses exemplary single-chain Fv (scFv) antibodies that bind to CD40, OX40, GITR, ICOS, and 4-1BB. These scFv antibodies are listed in Table 2 below.

[0071] Table 2

[0072] target Antibody name SEQ ID NO OX40 HuOHX14DS.scFv 22 OX40 HuOHX14DS.scFv.ds 122 CD40 HuACS2.scFv 57 CD40 HuACS2.scFv.ds 123 GITR HuGAB11.scFv 41 GITR HuGAB11.scFv.ds 125 ICOS HuTAM14.scFv 117 ICOS HuTAM14.scFv.ds 124 4-1BB HuFOB5.scFv.LH.ds 136 4-1BB HuFOB5.scFv.HL.ds 139

[0073] Other antibodies with the same CDR, or those binding to the same epitope, or those competing with any of these antibodies for binding to their target protein, may also be used; the same CDR as defined by Kabat or as otherwise defined, such as Chothia, a complex of Chothia and Kabat, AbM, or Contact (see bioinf.org.uk / abs). Other methods of binding to any of the aforementioned targets may also be used instead of the antibodies described above. Antibodies binding to CD40, OX40, GITR, ICOS, and 4-1BB can stimulate their receptors, thereby activating immune cells expressing the receptors. Antibodies binding to cancer-associated antigens may or may not antagonize the interaction between the cancer-associated antigen and its ligand or antireceptor. Antagonism provides an additional cytotoxic mechanism against cancer cells but is not necessary for activating immune cells via the binding arm of bispecific antibodies that bind to co-stimulatory receptors.

[0074] Other antibodies targeting CD33 include lintozumab, lintozumab, and BI836858 (Heider Blood 2011; 118(15):4159-4168). Antibodies targeting EGFR include C225, the chimeric version of which is commercially available as cetuximab, panitumumab, matezumab, and necitumumab. Antibodies targeting PD-L1 include durvalumab, avelumab, atezolizumab, and MDX-1105 / BMS936559. Agonist antibodies targeting CD40 include CP-870,893 (Pfizer and VLST), dacetuzumab (Seattle Genetics), Chi Lob 7 / 4 (University of Southampton), and lucatumumab (Novartis) (Vonderheide et al., Clin Cancer Res 19, 1035-1043 (2013)). Antibodies against OX40 agonists 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 (Bellet et al., Clin Cancer Res 2017; 23(23_Suppl): Abstract nr37). Antibodies against GITR agonists include DTA-1 (Shimizu et al., Nat Immunol. 3, 135-142, 2002), INCAGN 01876 (Gonzalez et al. Cancer Res. 2016 Volume 76, Issue 14, Abstract 3220) and 29D33, 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), utomilumab (Pfizer), and AAGEN2373 (Agenus; Galand et al. 2019 J. Clin. Oncol. 37, no. 15 suppl. e14005).

[0075] Any of these antibodies, or other antibodies having the same CDR, or other antibodies binding to the same epitope, or other antibodies competing with any of these antibodies for binding to their target protein, can be incorporated into the bispecific antibody of the present invention. The same CDR is as defined by Kabat or as otherwise defined, such as Chothia, a complex of Chothia and Kabat, AbM, or Contact. Other methods of binding to any of the aforementioned targets can also be used instead of the antibodies described above.

[0076] This invention also provides monoclonal antibodies that specifically bind to human PD-L1, including HuPRO1, HuPRO2, and HuPRO5, and other antibodies having the same six CDRs (as defined in any conventional sense) or the same mature heavy and light chain variable region pairs as the monoclonal antibodies. Such antibodies inhibit the interaction between PD-L1 and PD1. This invention also provides monoclonal antibodies that specifically bind to human OX40, including HuOHX14DS, and other antibodies having the same six CDRs (as defined in any conventional sense) or the same mature heavy and light chain variable region pairs. This invention also provides monoclonal antibodies that specifically bind to human CD40, including HuACS2, and other antibodies having the same six CDRs (as defined in any conventional sense) or the same mature heavy and light chain variable region pairs. This invention also provides monoclonal antibodies that specifically bind to human ICOS, including HuTAM14, and other antibodies having the same six CDRs (as defined in any conventional sense) or the same mature heavy and light chain variable region pairs. The present invention also provides monoclonal antibodies that specifically bind to human 4-1BB, including HuFOB5 and other antibodies having the same six CDRs (as defined by any conventional definition) or the same mature heavy chain and light chain variable region pairs.

[0077] IV. Format of Bispecific Antibodies

[0078] More than 100 formats of bispecific antibodies 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 two targets. Preferred formats include two or more binding sites for each target.

[0079] Some formats have a tetrameric structure similar to normal antibodies, which have two binding regions, each targeting a single target. Each binding region is formed by a pair of heavy and light chain variable regions, which are linked to heavy and light chain constant regions, respectively. These bispecific antibodies differ from normal antibodies in that the two binding sites and the heavy and light chain pairs that form them are different. Therefore, such antibodies require the binding of two distinct pairs of heavy and light chains.

[0080] The "knobs-into-holes" approach (replacing a small amino acid with a large amino acid in the CH3 domain of one antibody ("knob"), and vice versa in another antibody ("hole")) has been used to reduce homodimer formation and heavy chain mismatches (Ridgway et al., Protein Eng 9:617-21, 1996; Atwell et al., J MolBiol 270:26-35, 1997; and U.S. Patent No. 7,695,936). Light chain mismatches in this format can be reduced through several strategies. One strategy is to use a common light chain variable region for two different heavy chain variable regions. However, this only applies to certain antibodies. Another approach is to express half-molecules containing both the knob and the hole in different bacteria. Another approach, known as CrossMab, involves exchanging the CH1 domain of one of the heavy chains with the constant CL domain of the corresponding light chain to induce proper pairing between engineered heavy and light chains (Schaefer et al., Proc Natl Acad Sci USA 108:11187-92, 2011; WO2009 / 080251; WO 2009 / 080252; WO2009 / 080253). Another approach involves introducing additional mutations into the VH-VL and CH1-CL interfaces (Lewis et al., Nat. Biotechnol., 32(2014), pp.191-198). These mutations induce the heavy chain to preferentially pair with the light chain. Another approach is to introduce a mutation that promotes protein A binding into one of the Fc regions and select heterodimers with moderate protein A binding from homodimers with high or low protein A binding by affinity chromatography (Tusdian et al, MAbs. 2016 May-June; 8(4):828-38).

[0081] Other bispecific antibodies avoid mismatch problems by combining multiple binding specificities within the same heavy and light chain pair. To this end, one approach called dual variable domains involves tandemly connecting two different heavy chain variable regions to the heavy chain constant region, and tandemly connecting two different light chain variable regions to the light chain constant region (Correia et al., MAbs May 1, 2013; 5(3):364–372). This antibody can be assembled into a tetramer by binding two identical heavy and light chain pairs. The assembled antibody includes two distinct binding sites for each target.

[0082] Another approach followed in embodiments of the invention introduces a second binding specificity by linking a single-chain Fv (scFv) to the C-terminus of the heavy chain constant region. This bispecific antibody includes a first binding site, as in standard antibodies, formed by variable regions of the heavy and light chains linked to the N-termini of the heavy and light chain constant regions. The C-terminus of the heavy chain is linked to the scFv, which provides the second binding site. The scFv is typically linked via a linker, and another linker links the heavy and light chain variable regions within the scFv. The scFv can be linked to the Fc region via a linker through either its light chain variable region or the end of its heavy chain variable region. When assembled by compounding two identical heavy and light chain pairs, this bispecific antibody includes two binding sites targeting each of two different specificities. The cancer-associated antigen or infected cell antigen and co-stimulatory antigen binding arms of this bispecific antibody can be linked in either direction. The arm linked to the N-terminus of the heavy and light chain constant regions is provided as a separate heavy and light chain variable region, while the arm to be linked to the C-terminus is provided as an scFv fragment. One advantage of this format is that the two distinct binding spaces are separated by the entire heavy chain constant region, which may promote intercellular bridging.

[0083] Another format links the scFv that specifically binds to the first target to the heavy chain constant region, and the scFv that specifically binds to the second target to the light chain constant region. This antibody assembles into a tetramer, comprising two copies of each binding site (Bs(scFv)4-IgG) (Zuo et al., Protein Eng13:361-367, 2000).

[0084] Other formats connect scFv binding regions on single strands without constant regions. For example, the BiTe format connects two scFv fragments via a connector (see, for example, Ross et al., PLoS ONE 12(8):e0183390, 2017). This format lacks effector functionality and tends to have a shorter half-life, but may have the advantages of accessibility and ease of fabrication due to its small size.

[0085] Many of the aforementioned formats include linker peptides between variable regions of the heavy and light chains or between variable and constant regions. Linkers are short peptides that impart flexibility and are typically dominated 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.

[0086] V. Selection of the Constant Region

[0087] Many formats of bispecific antibodies include at least a portion of the human constant region. The choice of the constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent phagocytosis, and / or complement-dependent cytotoxicity are required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. The light chain constant region can be λ or κ. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. Here, while ADCC, ADCP, and CDC can be used to provide an additional mechanism of action against cancer or infected cells bound by one arm of a bispecific antibody, it is not useful for stimulating co-stimulatory molecules in the other arm to activate immune cells.

[0088] One or more amino acids at the amino or carboxyl terminus of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be deleted or derived in a certain proportion or in all of the molecules. Amino acid substitutions can be made in constant regions to reduce or increase effector functions, such as complement-mediated cytotoxicity or ADCC (see, for example, Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, for example, 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 number). Increased FcRn binding facilitates a more intense competition between the hybrid protein of the present invention and endogenous IgG for binding to FcRn. Many mutations are also known to reduce any of ADCC, ADCP, or CDC. (See Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006). For example, substituting any amino acid residue at positions 234, 235, 236, and / or 237 reduces affinity for Fcγ receptors, particularly the FcγRI receptor (see, for example, US 6,624,821). Alternatively, amino acid residues at positions 234, 236, and / or 237 in human IgG2 are replaced with Ala, and amino acid residue at position 235 is replaced with Gln or Glu (see, for example, US 5,624,821). Other substitutions that reduce 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.

[0089] Human constant regions exhibit both allotropic and isoallotypic variation among individuals, meaning that the constant region can differ at one or more polymorphic locations in different individuals. The difference between allotropic and isoallotypic variation lies in the binding of serum that identifies isoallotypic regions to one or more non-polymorphic regions of other isotypes.

[0090] VI. Expression of recombinant antibodies

[0091] Bispecific antibodies are typically produced via recombinant expression. Depending on the bispecific format, one, two, or more antibody chains may need to be expressed. If multiple chains are expressed, they can be expressed using the same or different vectors. Recombinant polynucleotide constructs typically include expression control sequences operatively linked to the coding sequences of the antibody chains, including naturally related or heterologous expression control elements such as promoters. The expression control sequences can be promoter systems within vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector is integrated into a suitable host, the host is maintained under conditions suitable for high levels of expression of the nucleotide sequence and for the collection and purification of the bispecific antibodies.

[0092] These expression vectors can typically replicate in the host organism as an episome or as part of the host's chromosomal DNA. Often, expression vectors contain selection markers, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence.

[0093] Escherichia coli is a prokaryotic host used for expressing antibodies (especially antibody fragments). Microorganisms, such as yeast, can also be used for expression. Saccharomyces cerevisiae is a yeast host with suitable vectors containing expression control sequences, origins of replication, and termination sequences as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.

[0094] Mammalian cells can be used to express nucleotide fragments encoding immunoglobulins or fragments thereof. See Winnacker, *From Genes to Clones* (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting complete heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and antibody-free myeloma cells, including Sp2 / 0 and NSO. Cells can be non-human. Expression vectors for these cells can include expression control sequences such as origin of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)) and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148: 1149 (1992).

[0095] Alternatively, the antibody-coding sequence can be incorporated into the transgene to be introduced into the genome of the transgenic animal and subsequently expressed in the transgenic animal's milk (see, for example, U.S. Patent No. 5,741,957; U.S. Patent No. 5,304,489; and U.S. Patent No. 5,849,992). Suitable transgenes include coding sequences for light and / or heavy chains that are operatively linked to promoters and enhancers from mammary gland-specific genes, such as casein or β-lactoglobulin.

[0096] Vectors containing the DNA fragment of interest can be transferred into host cells using methods that depend on the cell host type. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipid transfection, gene gun methods, or virus-based transfection can be used for other cell hosts. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To produce transgenic animals, the transgene can be microinjected into fertilized oocytes, or it can be integrated into the genome of embryonic stem cells, with the nucleus of such cells transferred into enucleated oocytes.

[0097] After introducing vectors encoding the heavy and light chains of antibodies into cell cultures, cell libraries can be screened for antibody productivity and quality in serum-free media. The cell libraries with the highest yields can then be used for FACS-based single-cell cloning to generate monoclonal lines. Specific productivity of over 50 pg or 100 pg per cell per day can be used, corresponding to product titers greater than 7.5 g / L of culture. The turbidity, filtration properties, PAGE, IEF, UV scan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore can also be tested on the antibodies produced by the single-cell clones. The selected clones can then be stored in multiple vials and frozen for later use.

[0098] Once expressed, the bispecific antibody can be purified according to standard procedures in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, etc. (see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0099] Commercial antibody production methods can be employed, including codon optimization, promoter selection, transcription element selection, terminator selection, blood-free cell cloning, cell banking, use of selection markers to amplify copy number, CHO terminators, or increases in protein titer (e.g., US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; WO2004 / 050884; WO2008 / 012142; WO2008 / 012142; WO2005 / 019442; WO2008 / 107388; WO2009 / 027471; and US 5,888,809).

[0100] VII. Nucleic Acids

[0101] The present invention further provides nucleic acids encoding any of the aforementioned heavy and light chains. Optionally, such nucleic acids further encode signal peptides and can be expressed together with signal peptides linked to constant region coding sequences of the nucleic acid, and can be operatively linked to regulatory sequences to ensure expression of the coding sequences, such as promoters, enhancers, ribosome binding sites, transcription termination signals, etc. The nucleic acids encoding heavy and light chains can exist in isolated forms or can be cloned into one or more vectors. The nucleic acids can be synthesized, for example, by solid-state synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding heavy and light chains can be linked as a single continuous nucleic acid (e.g., within an expression vector), or they can be separate (e.g., each cloned into its own expression vector).

[0102] VIII. Treatment Methods and Pharmaceutical Compositions

[0103] The invented bispecific antibodies can be used to treat cancer, including any cancer in which one arm of the bispecific antibody binds to a target expressed or overexpressed in the cancer, such as those disclosed above. Bispecific antibodies can be used to treat solid tumors and hematologic malignancies. Hematologic malignancies include leukemia (e.g., T-cell large granular lymphocytic leukemia), lymphoma (Hodgkin's or non-Hodgkin's), or multiple myeloma. Solid tumors include skin (e.g., melanoma), ovaries, endometrium, kidneys, liver, pancreas, bladder, breast, ovary, prostate, rectum, colon, stomach, intestines, pancreas, lungs, thymus, thyroid, kidneys, and brain.

[0104] The bispecific antibodies of the present invention can also be used to treat pathogen infections when they have an arm that specifically binds to an antigen expressed in infected cells but not in matched uninfected cells. Such antigens may be encoded by the pathogen or may be expressed by cells in response to pathogen infection. Examples of such antigens expressed in infected cells are human immunodeficiency virus (HIV) glycoproteins gp41 and gp120, human T-cell leukemia virus type 1 (HTLV-1) Env protein, herpes simplex virus (HSV) glycoproteins gB and gH, influenza hemagglutinin (HA) and neuraminidase (NA), and respiratory syncytial virus (RSV) F protein. Examples of pathogen infections that can be treated with bispecific antibodies include viral, bacterial, protozoan, or fungal infections. Some examples of viral infections include HIV, hepatitis A, B, or C, herpesviruses (such as VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein-Barr virus), adenovirus, XMRV, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, 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 arbovirus encephalitis virus. Some examples of bacterial infections include Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum toxin, Anthrax, Plague, Leptospirosis, Lyme disease bacteria, Streptococcus, or Neisseria. Some examples of pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Plasmodium.

[0105] Bispecific antibodies are administered at an effective regimen, meaning the dosage, route of administration, and frequency of administration for delaying onset, reducing severity, inhibiting further deterioration, and / or improving at least one symptom or condition of the disease. If the subject already has the disease, the regimen may be called a therapeutically effective regimen. If the subject is at high risk of the disease relative to the general population but has not yet developed symptoms, the regimen may be called a preventatively effective regimen. In some cases, therapeutic or preventative efficacy can be observed in a single subject relative to a historical control or past experience with the same subject. In other cases, therapeutic or preventative efficacy can be demonstrated in a preclinical or clinical trial in a population of treated subjects relative to a control population of untreated subjects.

[0106] Preferably, the bispecific antibody exhibits at least additive (more preferably synergistic) activity against cancer or infected cells compared to its individual component antibodies. The synergistic effect is preferably assessed quantitatively, as discussed, for example, in Tallarida, Genes Cancer. November 2011; 2(11):1003–1008. Preferably, the bispecific antibody also exhibits increased activity compared to a mixture of its component antibodies (each component antibody being at an equimolar concentration of the bispecific antibody). Such activity can, for example, be measured as cytotoxicity or cytosuppressive activity against cancer cells or infected cells; said infected cells, in the presence of immune cells expressing a co-stimulatory molecule specifically bound by one arm of the bispecific antibody, also expressing an antigen specifically bound by the other arm of the bispecific antibody.

[0107] Exemplary doses of bispecific antibodies are 0.01-20, or 0.5-5, or 0.01-1, or 0.01-0.5, or 0.05-0.5 mg / kg body weight (e.g., 0.1, 0.5, 1, 2, 3, 4, or 5 mg / kg) or as a fixed dose of 10-1500 mg. Dosage depends on the patient's condition and response to previous treatment (if any), whether the treatment is prophylactic or therapeutic, whether the disease is acute or chronic, and other factors.

[0108] Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Intravenous or subcutaneous administration is preferred for systemic circulation. Intravenous administration can be achieved, for example, by infusion over a period of 30-90 minutes.

[0109] Dosing frequency depends on the bispecific antibody's half-life in circulation, the subject's condition, the route of administration, and other factors. This frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the subject's condition or progression of the disease being treated. For continuous treatment, exemplary frequencies for intravenous administration are between weekly and quarterly, although higher or lower frequencies are also possible. For subcutaneous administration, exemplary frequencies are daily to monthly, although higher or lower frequencies are also possible.

[0110] The dosage depends on whether the disease is acute or chronic, and also on the disease's response to treatment. For acute illness or acute exacerbations of chronic illness, 1 to 10 doses are usually sufficient. Sometimes, a single bolus dose (optionally in split doses) is sufficient to manage acute illness or acute exacerbations of chronic illness. For relapses of acute illness or acute exacerbations, treatment can be repeated. For chronic illness, bispecific antibodies can be administered periodically, such as weekly, bi-weekly, monthly, quarterly, or every six months for at least 1, 5, or 10 years, or continuously for the subject's lifetime.

[0111] The pharmaceutical composition is preferably suitable for parenteral administration to humans (e.g., according to FDA standards). Such pharmaceutical compositions are preferably sterile, substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., a single-dose dose). The pharmaceutical composition may be formulated using one or more pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Pharmaceutically acceptable means suitable for human administration are, for example, those approved by or potentially approved by the FDA. The formulation depends on the chosen route of administration. For injection, antibodies may be formulated in an aqueous solution, preferably in a physiologically compatible buffer (e.g., Hank's solution, Ringer's solution, or physiological saline or acetate buffer) (to reduce injection site discomfort). This solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the antibody may be in lyophilized form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to use.

[0112] Treatment using the bispecific antibody of the present invention can be combined with other treatments effective against the disease being treated. When used to treat cancer, the bispecific antibody of the present invention can be combined with chemotherapy, radiotherapy, stem cell therapy, surgery, or other biological agents, such as Herceptin targeting the HER2 antigen. TM (Trastuzumab), Avastin targeting VEGF TM (Bevacizumab) or antibodies against the EGF receptor, such as (Erbitux) TM (cetuximab) and Vectibix TM (Panitumab). Chemotherapy agents include chlorambucil, cyclophosphamide or melphalan, carboplatin, daunorubicin, doxorubicin, idarubicin and mitoxantrone, methotrexate, fludarabine and cytarabine, etoposide or topotecan, vincristine and vinblastine. For infections, treatment can be combined with antibiotics, antiviral agents, antifungal agents or antiprotozoal agents.

[0113] IX. Other Methods

[0114] The bispecific antibodies of this invention can also be used in diagnostic, prognostic, and laboratory methods. They can be used to measure the level of an antigen expressed in the circulation of cancer or cancer patients to determine whether that level is measurable or even elevated, thereby tracking and guiding cancer treatment, as cancers associated with measurable or elevated antigen levels are most susceptible to treatment with bispecific antibodies containing an arm that binds to the cancer. Bispecific antibodies can be used in ELISA assays, radioimmunoassays, or immunohistochemistry, etc. Bispecific antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in kit form containing all the reagents required to perform the assay.

[0115] For all purposes, all patent documents, websites, other publications, registry numbers, etc., cited above or below are incorporated herein by reference in their entirety to the same extent as if each individual item were specifically and individually indicated as incorporated herein by reference. If different versions of a sequence are associated with a registry number at different times, it refers to the version associated with the registry number on the effective filing date of this application. The effective filing date is the earlier of the actual filing date or the filing date of the priority application (if any) that mentions the registry number. Similarly, if different versions of publications, websites, etc., are published at different times, it refers to the most recently published version on the effective filing date of this application, unless otherwise stated. Unless otherwise expressly indicated, any feature, step, element, embodiment, or aspect of the invention may be used in combination with any other. Although the invention has been described in detail by way of illustration and example for clarity and understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

[0116] Example

[0117] Example 1: General Methods and Materials

[0118] Gene cloning, mutagenesis, plasmid construction, ELISA, and FACS are performed using standard laboratory techniques described below (e.g., Green and Sambrook (Molecular Cloning, A Laboratory Manual, 4th Edition, 2012, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Greenfield (Antibodies, A Laboratory Manual, 2nd Edition, 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) and as described in the supplier’s instructions.

[0119] Mouse hybridomas were generated at JN Biosciences (Mountain View, CA) using standard cell fusion techniques with the GenomONE CF EX cell fusion reagent (Cosmo Bio, Carlsbad, CA). These hybridomas produced monoclonal antibodies against the desired antigens. For mouse immunization, purified, soluble forms of recombinant antigens were used, typically fused to the Fc region of human IgG, or mouse cell lines expressing the recombinant antigens on their surfaces. The mouse monoclonal antibodies secreted in the hybridoma cell culture supernatant were screened to identify antibodies with the following characteristics: (1) specific binding to human antigens, (2) specific binding to cynomolgus monkey antigens, and (3) the desired biological function, such as blocking the interaction between the antigen and its ligand or stimulating cell activity through the binding of the antigen to the cell surface. The selected hybridoma cells were expanded 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 (MabSelectSuRe, GE Healthcare Life Sciences, Marlborough, MA) according to the manufacturer's instructions. The buffer for purifying the antibody was replaced with phosphate-buffered saline (PBS) via dialysis. The antibody concentration was determined by measuring the absorbance at 280 nm (OD 1.4 = 1 mg / ml).

[0120] The expression concentration of mouse IgG antibodies in the culture supernatant was measured using a sandwich ELISA. In a typical experiment, the ELISA plate was covered with goat anti-mouse IgG Fc-specific polyclonal antibody (Southen Biotech, 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 in ELISA buffer, was loaded onto the ELISA plate. Suitable mouse IgG / κ antibodies were used as standards. After incubating the ELISA plate at room temperature for 60 minutes and washing with wash buffer, the bound mouse antibodies were detected using HRP-conjugated goat anti-mouse κ chain polyclonal antibody (BethylLaboratories, Montgomery, TX). After incubating at room temperature for 30 minutes and washing with wash buffer, color development was initiated by adding ABTS substrate (Sigma-Aldrich, St. Louis, MO) and terminated with 2% oxalic acid. The absorbance was read at 405 nm.

[0121] As described above, the expression of chimeric and humanized IgG / κ antibodies in culture supernatant was measured by sandwich ELISA, the difference being that the ELISA plate was covered with goat anti-human IgG Fc specific polyclonal antibody (Jackson Immuno Research, WestGrove, PA), and the binding antibody was detected using HRP-conjugated goat anti-human κ chain polyclonal antibody (BethylLaboratories).

[0122] Sequencing of the heavy and light chain variable regions (VH and VL, respectively) of mouse monoclonal antibodies was performed using standard molecular biology techniques, such as the procedure described by Tsurushita et al. (Methods 36:69-83, 2005). Humanization of the VH and VL was performed according to the general procedure described by Tsurushita et al. (as above). First, a three-dimensional molecular model of the mouse antibody variable region to be humanized was constructed using suitable software. Second, the molecular model was used to identify framework amino acid residues important for the formation of the CDR (complementarity-determining region) structure or essential for antigen binding. Simultaneously, human VH and VL amino acid sequences derived from cDNA that are highly homologous to the mouse VH and VL amino acid sequences were selected. Finally, the CDR sequence, along with the identified framework amino acid residues important for the formation of antigen-binding sites, was transplanted from the mouse variable region into the corresponding selected human framework sequences.

[0123] Stable transfection into the Chinese hamster ovary cell line CHO-K1 was achieved via electroporation. Prior to transfection, the expression vector was linearized using an appropriate restriction enzyme. In a typical experiment, approximately 10 cells were transfected with 20 μg of the linearized plasmid. 7 CHO-K1 cells were suspended in SFM4CHO medium (GE Healthcare Life Sciences) and seeded at 100 μl / well in several 96-well plates after appropriate cell dilution. After 48 hours, stable transfectants were isolated by adding 100 μl / well of SFM4CHO medium containing 20 μg / ml puromycin. Approximately ten days after selection, antibody production in the culture supernatant of the transfectants was measured. As described above, stable CHO-K1 transfectants producing high levels of antibodies were identified by ELISA.

[0124] Stable CHO-K1 transfectants (e.g., the humanized and bispecific IgG antibodies of this invention) highly producing recombinant antibodies were amplified in SFM4CHO cells until cell viability was below 50%. After centrifugation and filtration, the culture supernatant was loaded onto a protein A column (HiTrapMAB SelectSuRe, GE Healthcare Life Sciences). The column was washed with PBS before eluting the antibody with 0.1M glycine-HCl (pH 3.0) containing 0.1M NaCl or 0.1M sodium acetate (pH 3.6). The antibody elution buffer was neutralized with 1M Tris-HCl (pH 8.0) and then replaced with PBS by dialysis. The antibody concentration (1 mg / ml = 1.4 OD) was determined by measuring the absorbance at 280 nm.

[0125] Example 2: Construction of a bispecific antibody (BS824) binding to CD33 and OX40

[0126] The mammalian expression vector pHuM195-IgG1 (Figure 1A), designed for the production of humanized anti-CD33 IgG1 / κ antibodies binding 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 components. Starting clockwise from the SalI site in Figure 1A, the vector contains heavy chain transcription units that initiate transcription of the antibody heavy chain gene from the human cytomegalovirus (CMV) major immediate early promoter and enhancer (CMV-P in the figure). Following the CMV promoter are exons encoding humanized M195 (HuM195)VH (SEQ ID NO:2) (flanked by SpeI and HindIII sites (HuM195-VH)) and a genome sequence containing exons CH1 (SEQ ID NO:3), the hinge (SEQ ID NO:4), CH2 (SEQ ID NO:5), and CH3 (SEQ ID NO:6) encoding the constant region of the human γ-1 heavy chain, along with inserted introns; after the EagI site is the polyadenylation site of the human γ-1 heavy chain gene. Following the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter and enhancer (CMV-P), followed by exons encoding humanized M195 (HuM195)VL (SEQ ID NO:7) (flanked by NheI and EcoRI sites (HuM195-VL), and an exon containing the human κ chain constant region exon (Ck) (SEQ ID NO:7). The sequence includes NO:8 and part of the preceding intron's genomic sequence, as well as the polyadenylation site of the human κ chain gene following the Ck exon. Following the light chain gene are the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase gene (puro) for anti-purulent activity, and a fragment containing the SV40 polyadenylation site (SV40-A). Finally, pHuM195-IgG1 contains the bacterial origin of replication (pUCori) and the β-lactamase gene (β-lactamase). The arrows in the diagram indicate the direction of transcription. The humanized IgG1 / κ monoclonal antibody (HuM195-IgG1) is expressed from pHuM195-IgG1 in mammalian cells.

[0127] As described in Example 1, mouse hybridomas producing the IgG / κ monoclonal antibody OHX14, which specifically binds to human and cynomolgus monkey OX40 (also known as TNFRSF4 and CD134), were isolated. As the immunogen, the mouse myeloma cell line NS0, stably expressing recombinant human OX40 on its surface, was used (OX40-FLAG-GPI; SEQ ID NO:11), consisting of the extracellular region of human OX40 fused with the FLAG peptide (SEQ ID NO:9) and the glycosylphosphatidylinositol (GPI) anchoring signal of human CD55. Sequencing and humanization of the VH and VL of OHX14 were performed according to the description of Tsurushita et al. (ibid.).

[0128] The amino acid sequence of humanized OHX14 (HuOHX14DS)VH is

[0129] MGRLTSSFLLLIVPAYVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS (SEQ ID NO: 12). According to the definition of Kabat et al. (Sequences of Proteins of Immunological Interests, 5th Edition, NIH Publication No. 91-3242, USD Department of Health and Human Services, 1991), the CDR1, 2, and 3 amino acid sequences 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 is synthesized as an exon, including 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 OHX14 (HuOHX14DS)VL is as follows:

[0131] MMSSAQFLGLLLLCFQGTRCDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO:16). According to the definition by Kabat et al. (ibid.), 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. The gene encoding HuOHX14DSVL was synthesized into exons, including the splice donor signal at the 3' end of the coding region, the NheI site at the 5' end of the fragment, and the EcoRI site at the 3' end of the fragment.

[0132] The expression vector pHuOHX14DS-IgG1.AA has the same structure as pHuM195-IgG1 (Fig. 1A), except that (i) the HuOHX14DS VH exon is located between the SpeI and HindIII sites, (ii) the HuOHX14DS VL exon is placed between the NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU number of Kabat et al., ibid.) in CH2 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 new vector phuOHX14DS-IgG1.AA expresses humanized anti-OX40 IgG1 / κ antibody (HuOHX14DS-IgG1.AA) in mammalian cells.

[0133] The mammalian expression vector pBS824 (Figure 1B), designed to express a bispecific antibody binding to human CD33 (SEQ ID NO:1) and human OX40 (SEQ ID NO:21), was constructed by modifying pHuM195-IgG1 as described below. The VL and VH coding regions of the humanized anti-human OX40 monoclonal antibody in pHuOHX14DS-IgG1.AA were converted to a single-chain Fv (scFv) form (HuOHX14DS.scFv; SEQ ID NO:22) in the order of VL, peptide linker, and VH from the N-terminus to the C-terminus. The N-terminus of HuOHX14DS.scFv was fused to the penultimate glycine residue in CH3 of pHuM195-IgG1, and the peptide linker separated them (CH3-HuOHX14D.scFv; SEQ ID NO:23). Furthermore, in CH2, two leucine residues at positions 234 and 235 (EU number) are replaced with alanine residues (L234A and L235A, respectively). The resulting vector pBS824 expresses a bispecific IgG antibody called BS824, which binds to human CD33 and OX40.

[0134] The amino acid sequence encoding the mature heavy chain in pBS824 is:

[0135] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS(SEQ ID NO:24).

[0136] The amino acid sequence of the mature light chain encoded in pBS824 is

[0137] DIQMTQSPSSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 25).

[0138] A schematic structure of the bispecific IgG antibody (e.g., BS824) of the present invention is shown below. Figure 2 As shown.

[0139] 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 their respective stable CHO-K1 transfectants using a protein A affinity column (as described above). SDS-PAGE analysis under reducing conditions showed that each of the three antibodies exhibited only two major bands: approximately 50 kDa heavy chain and 25 kDa light chain (for HuM195-IgG1 and HuOHX14DS-IgG1.AA) or approximately 75 kDa heavy chain and 25 kDa light chain (for BS824).

[0140] Example 3: Biological activity of a bispecific antibody (BS824) binding to CD33 and OX40

[0141] The bioactivity of BS824 binding to CD33 and OX40 was examined in Jurkat Dual reporter cells (InvivoGen, San Diego, CA) (JD / OX40) expressing human OX40 and in the mouse myeloma cell line NS0 (NS0 / CD33) expressing human CD33. It is known that binding to OX40 on the cell surface triggers 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 Lucia luciferase.

[0142] JD / OX40 cells were generated by electroporation of Jurkat Dual cells with the expression vector pFCm331 for human OX40 expression. The expression vector pFCm331 (Figure 1C) has the same structure as pHuM195-IgG1, except that (a) the SpeI-EagI fragment was replaced with a DNA fragment encoding OX40 (SEQ ID NO:21) and (b) the light chain gene was removed. Jurkat Dual cells (JD / OX40) expressing OX40 were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 7.5% CO2. CD33 expression was not detected in JD / OX40 cells by flow cytometry using HuM195-IgG1 and a secondary PE-labeled goat anti-human IgG antibody.

[0143] NS0 / CD33 was generated by electroporation of the expression vector pFCm267 for expressing human CD33 into NS0 cells. The expression vector pFCm267 (Figure 1D) has the same structure as pFCm331, except that (a) the puromycin N-acetyltransferase gene (puro) is replaced by Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt), and (b) the SpeI-EagI fragment is replaced by a DNA fragment encoding recombinant human CD33, which consists of the following from the N-terminus to the C-terminus: 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 anchoring signal (SEQ ID NO: 10) (CD33-FLAG-GPI). NS0 stable transfectants (i) 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) the CD33-FLAG-GPI expressed on the surface was maintained in DME medium containing 10% FBS in a 7.5% CO2 incubator at 37°C.

[0144] OX40, a member of the TNF receptor superfamily of immune co-stimulatory molecules, requires trimerization on the cell surface to trigger the NF-κB pathway of intracellular signal transduction (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 cross-linking of OX40 on the cell surface upregulates the expression and secretion of Lucia luciferase. Approximately 200,000 JD / OX40 cells were cultured for one day in 200 μl of RPMI 1640 medium in a 96-well plate at 37°C and 7.5% CO2, with or without approximately 200,000 NSO / CD33 cells. This medium contained 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 uptake) (Jackson ImmunoResearch) to provide the primary signal for T cell activation along with: (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. According to the supplier's protocol, the luciferase activity in the culture supernatant was measured in triplicate using QUANTI-Luc reagent (InvivoGen). The luminescence was measured using a Synergy HT microplate reader (BioTek, Winooski, VT). Results are as follows: Figure 3 As shown.

[0145] The mean relative luciferase units (RLU) 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 NS06, 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 was significantly increased in JD / OX40 cells only in the presence of both a bispecific antibody binding to CD33 and OX40 (BS824) and CD33-expressing cells (NS0 / CD33). These results indicate that only BS824 can establish a bridge between JD / OX40 and NSO / CD33 cells, but neither HuM195-IgG1 nor HuOHX14DS-IgG1.AA causes OX40 to accumulate on the surface of JD / OX40 cells near NSO / CD33 cells and activates JD / OX40 cells to increase luciferase expression.

[0146] Example 4: Construction of a bispecific antibody (BS827) binding to EGFR and GITR

[0147] The mammalian expression vector pCh225-IgG1 for expressing a chimeric IgG1 / κ antibody binding to human epidermal growth factor receptor (EGFR; SEQ ID NO:28) was generated 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 also synthesized as an exon, including 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 Ch225 VH and VL exons were cloned into the corresponding sites in the antibody expression vector. The resulting plasmid pCh225-IgG1, used to express the anti-EGFR antibody Ch225-IgG1, has the same structure as pHuM195-IgG1 (Figure 1A), except that the VH and VL coding regions are replaced by Ch225VH and VL, respectively.

[0148] Mouse hybridomas producing the IgG / κ 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. The extracellular region of human GITR fused to the Fc region of the human γ-1 heavy chain (hGITR-Fc; SEQ ID NO:31) was used as the immunogen. Sequencing and humanization of GAB11 VH and VL were performed as described by Tsurushita et al. (ibid.).

[0149] The amino acid sequence of humanized GAB11 (HuGAB11)VH is as follows:

[0150] MAVLGLLLCLVTFPSCVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (SEQ ID NO:32). According to the definition by Kabat et al. (ibid.), 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. The gene encoding HuGAB11 VH is synthesized as an exon, including 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.

[0151] The amino acid sequence of humanized GAB11 (HuGAB11) VL is as follows:

[0152] MRVLAELLGLLLFCFLGVRCDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIK (SEQ ID NO:36). According to the definition by Kabat et al. (ibid.), the amino acid sequences of CDR1, 2, and 3 of HuGAB11 VL are HASQNINVWLS (SEQ ID NO:37), KASNLHT (SEQ ID NO:38), and...

[0153] QQGQSYPLT (SEQ ID NO:39). The gene encoding HuGAB11 VL is synthesized as an exon, including 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.

[0154] The expression vector pHuGAB11-IgG1.AA has the same structure as pHuM195-IgG1 (Fig. 1A), except that (i) the HuGAB11 VH exon is located between the SpeI and HindIII sites, (ii) the HuGAB11 VL exon is placed between the NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU numbering of Kabat et al., ibid.) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., ibid.). The new vector pHuGAB11-IgG1.AA expresses humanized anti-GITR IgG1 / κ antibody (HuGAB11-IgG1.AA) in mammalian cells.

[0155] The mammalian expression vector pBS827, designed to express a bispecific antibody binding to human EGFR (SEQ ID NO:28) and human GITR (SEQ ID NO:40), was constructed by modifying pCh225-IgG1 as described below. The VL and VH coding regions of the humanized anti-human GITR monoclonal antibody in pHuGAB11-Ig1.AA were converted to scFv form (HuGAB11.scFv; SEQ ID NO:41) from the N-terminus to the C-terminus. The N-terminus of HuGAB11.scFv was fused to the penultimate glycine residue in CH3, with a polypeptide linker between them in pCh225-IgG1 (CH3-HuGAB11.scFv; SEQ ID NO:42). Furthermore, the two leucine residues at positions 234 and 235 (EU number) in CH2 were replaced with alanine residues (L234A and L235A, respectively). The resulting vector pBS827 expresses a bispecific IgG antibody called BS827, which binds to human EGFR and GITR.

[0156] The amino acid sequence of the mature heavy chain encoded in pBS827 is as follows:

[0157] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS(SEQ ID NO:43).

[0158] The amino acid sequence of the mature light chain encoded in pBS827 is

[0159] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:44).

[0160] The structural diagram of BS827 is as follows: Figure 2 As shown.

[0161] As described above, three expression vectors, pCh225-IgG1, pHuGAB11-IgG1.AA, and pBS827, were stably transfected into CHO-K1. Ch225-IgG1, HuGAB11-IgG1.AA, and BS827 were purified from the culture supernatant of their respective stable CHO-K1 transfectants using a protein A affinity column (as described above). SDS-PAGE analysis under reducing conditions showed that each of the three antibodies had only two major bands: approximately 50 kDa heavy chain and 25 kDa light chain (for Ch225-IgG1 and HuGAB11-IgG1.AA) or approximately 75 kDa heavy chain and 25 kDa light chain (for BS827).

[0162] Example 5: Biological activity of a bispecific antibody (BS827) binding to EGFR and GITR

[0163] The bioactivity of BS827 in binding to EGFR and GITR was examined using JurkatDual reporter cells (InvivoGen, JD / GITR) expressing human GITR and the mouse myeloma cell line NS0 (NS0 / EGFR) expressing human EGFR.

[0164] JD / GITR was generated by electroporation of Jurkat Dual cells with the stable transfection of the expression vector pFCm331 for human GITR expression. The expression vector pFCm331 (Figure 1C) has the same structure as pFCm331, except that the SpeI-EagI fragment is replaced with a DNA fragment encoding a synthetic signal peptide (SEQ ID NO:26) fused with mature human GITR (SEQ ID NO:45). GITR-expressing puromycin-resistant Jurkat Dual cells (JD / GITR) were maintained in RPMI 1640 medium at 37°C and 7.5% CO2. CD33 expression was not detected in JD / GITR cells by flow cytometry using Ch225-IgG1 and secondary PE-labeled goat anti-human IgG antibodies.

[0165] NS0 / EGFR was generated by electroporation of the expression vector pFCm507 stably transfected into NS0 cells. The expression vector pFCm507 has the same structure as pFCm331 (Figure 1C), except that the SpeI-EagI fragment is replaced by a DNA fragment encoding recombinant human EGFR, which consists of the following from the N-terminus to the C-terminus: 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 a human CD55 GPI anchoring signal (SEQ ID NO: 10) (EGFR-FLAG-GPI). Stable transfectants of puromycin-resistant NS0 cells expressing EGFR-FLAG-GPI were maintained in DME medium containing 10% FBS at 37°C and 7.5% CO2.

[0166] GITR, a member of the TNF receptor superfamily of immune co-stimulatory molecules, requires trimerization on the cell surface to trigger the NF-κB pathway of intracellular signal transduction (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 cross-linking of GITR on the cell surface upregulates the expression and secretion of Lucia luciferase. Approximately 200,000 JD / OX40 cells were cultured for one day in 200 μl of RPMI 1640 medium in a 96-well plate at 37°C and 7.5% CO2, in the presence of approximately 200,000 NSO or NSO / EGFR cells. This medium contained 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 uptake) (Jackson ImmunoResearch) to provide the primary signal for T cell activation along with: (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 triplicate using the QUANTI-Luc reagent (InvivoGen) according to the supplier's protocol. The luminescence was measured using a Synergy HT microplate reader (BioTek). Results are as follows: Figure 4 As shown.

[0167] The mean relative luciferase units (RLU) 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. Significantly increased luciferase activity in JD / GITR cells was only observed in the presence of both the bispecific antibody binding EGFR and GITR (BS827) and EGFR-expressing cells (NS0 / EGFR). The results showed that only BS827 could establish a bridge between JD / GITR and NSO / EGFR cells, but neither Ch225-IgG1 nor HuGAB11-IgG1.AA caused GITR to accumulate on the surface of JD / GITR cells near NSO / EGFR cells and to activate JD / GITR cells to increase luciferase expression.

[0168] Example 6: Bispecific antibody binding to CD33 and CD40 (BS828)

[0169] As described in Example 1, mouse hybridomas producing the IgG / κ monoclonal antibody ACS2 were isolated. This IgG / κ monoclonal antibody ACS2 binds to human and cynomolgus monkey CD40 (also known as TNFRSF5). The extracellular region of human CD40 fused to the Fc region of the human γ-1 heavy chain (hCD40-Fc; SEQ ID NO: 47) was used as the immunogen. Sequencing and humanization of ACS2 VH and VL were performed according to Tsurushita et al. (Methods 36:69-83, 2005).

[0170] The amino acid sequence of humanized ACS2 (HuACS2)VH is as follows:

[0171] MKLWLNWVFLLTLLHGIQCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS (SEQ ID NO:48). According to the definition by Kabat et al. (ibid.), 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. The gene encoding HuACS2 VH is synthesized as an exon, including 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.

[0172] The amino acid sequence of mature humanized ACS2 (HuACS2) VL is as follows:

[0173] MDFQVQIFSFLLISAVIISRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO:52). According to the definition by Kabat et al. (ibid.), the amino acid sequences of CDR1, 2, and 3 of HuACS2 VL are SASSVSYMH (SEQ ID NO:53), DTSKLAS (SEQ ID NO:54), and QQWSSNPLT (SEQ ID NO:55), respectively. The gene encoding HuACS2 VL is synthesized as an exon, including 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.

[0174] The expression vector pHuACS2-IgG1.AA has the same structure as pHuM195-IgG1 (Fig. 1A), except that (i) the HuACS2 VH exon is located between the SpeI and HindIII sites, (ii) the HuACS2 VL exon is placed between the NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU number) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., ibid.). The novel vector pHuGAB11-IgG1.AA expresses humanized anti-GITR IgG1 / κ antibody (HuGAB11-IgG1.AA) in mammalian cells. The novel vector pHuACS2-IgG1.AA expresses humanized anti-CD40 IgG1 / κ antibody (HuACS2-IgG1.AA) in mammalian cells.

[0175] The mammalian expression vector pBS828, designed to express a bispecific antibody binding to human CD33 (SEQ ID NO:1) and human CD40 (SEQ ID NO:56), was constructed by modifying pHuM195-IgG1 as described below. The VL and VH coding regions of the humanized anti-human CD40 monoclonal antibody in pHuACS2-Ig1.AA were converted to a single-chain Fv (scFv) form (HuACS2.scFv; SEQ ID NO:57) from the N-terminus to the C-terminus. The N-terminus of HuACS2.scFv was fused to the penultimate glycine residue in CH3, with a polypeptide linker between them in pHuM195-IgG1 (CH3-HuACS2.scFv; SEQ ID NO:58). Furthermore, the two leucine residues at positions 234 and 235 (EU number) in CH2 were replaced with alanine residues (L234A and L235A, respectively). The resulting vector pBS828 expresses a bispecific IgG antibody called BS828, which binds to human CD33 and CD40.

[0176] The amino acid sequence of the mature heavy chain encoded in pBS828 is as follows:

[0177] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS(SEQ ID NO:59).

[0178] The amino acid sequence of the mature light chain encoded in pBS828 is the same as the mature light chain sequence (SEQ ID NO:25) encoded in pBS824.

[0179] Figure 2 The schematic diagram of the structure of BS828 is as shown in

[0180] ​As described above, three expression vectors, pHuM195-IgG1, pHuACS2-IgG1.AA, and pBS828, were stably transfected into CHO-K1. HuM195-IgG1, HuACS2-IgG1.AA, and BS828 were purified from the culture supernatant of their respective stable CHO-K1 transfectants using a protein A affinity column (as described above). SDS-PAGE analysis under reducing conditions showed that each of the three antibodies exhibited only two major bands: approximately 50 kDa heavy chain and 25 kDa light chain (for HuM195-IgG1 and HuACS2-IgG1.AA) or approximately 75 kDa heavy chain and 25 kDa light chain (for BS828).

[0181] The human Burkitt's B lymphoma cell line, Ramos, expresses CD40 on its cell surface. It is known that cross-linking of 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 bioactivity of BS828 in upregulating CD95 expression in Ramos cells was examined in the presence and absence of the human promyelocytic leukemia cell line HL-60, which expresses CD33 on its cell surface.

[0182] Approximately 100,000 Ramos cells were cultured for one day in 200 μl of RPMI 1640 medium in a 96-well plate at 37°C and 7.5% CO2, with or without approximately 50,000 HL-60 cells. This RPMI 1640 medium comprised 10% FBS and (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. Ramos cell expression was detected using FITC-labeled mouse anti-human CD20 monoclonal antibody (Clone 2H7, BioLegend) and PE-labeled mouse anti-human CD95 antibody (Clone DX2, BioLegend) by flow cytometry. Ramos cells were CD20 positive, and HL-60 cells were CD20 negative. The results of FACS analysis are as follows: Figure 5 As shown.

[0183] The percentage of CD95-positive population in CD20-positive Ramos cells was 1.3% without antibodies, 0.8% with HuM195-IgG1, 1.4% with HuACS2-IgG1.AA, and 1.6% with BS828. When Ramos cells were cultured in the presence of HL-60 cells, the percentage of CD95-positive cell population in Ramos cells was 1.0% without antibodies, 1.1% with HuM195-IgG1, 0.7% with HuACS2-IgG1.AA, and 58.4% with BS828. CD95 expression in Ramos cells was significantly upregulated only in the presence of both a bispecific antibody binding to CD33 and CD40 (BS828) and CD33-expressing cells (HL-60 cells). These results indicate that only BS828 can establish a bridge between Ramos and HL-60 cells, but neither HuM195-IgG1 nor HuACS2-IgG1.AA causes CD40 polymer cross-linking on the surface of Ramos cells near HL-60 cells and leads to upregulation of CD95 in Ramos cells.

[0184] Example 7: Bispecific antibody binding PD-L1 and GITR (BS809)

[0185] 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. PD-L1 binding to PD-1 on T cells suppresses the immune response. PD-L1 is frequently expressed on cancer cells (Patelet 1. 2015 Mol. Cancer Ther. 14:847-856; Brody et al. 2017 Lung Cancer 112:200-215; Sun et al. 2018 Immunity 48:434-452). As described in Example 1, mouse hybridomas producing the IgG / κ monoclonal antibody PRO1 were isolated, which bound to human and cynomolgus monkey PD-L1 and blocked the interaction between PD-1 and PD-L1. As an immunogen, the extracellular region of human PD-L1 fused to the Fc region of the human γ-1 heavy chain (hPD-L1-Fc; SEQ ID NO: 60) was used. Sequencing and humanization of PRO1 VH and VL were performed as described by Tsurushita et al. (ibid.).

[0186] The amino acid sequence of humanized PRO1 (HuPRO1)VH is as follows:

[0187] MEWNWVVLFLLSLTAGVYAQVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSS (SEQ ID NO:61). According to the definition by Kabat et al. (ibid.), 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. The gene encoding HuPRO1 VH is synthesized as an exon, including 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 PRO1 (HuPRO1) VL is as follows:

[0189] MDFQVQIFSFLLISAVIMSRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK (SEQ ID NO:65). According to the definition by Kabat et al. (ibid.), the amino acid sequences of CDR1, 2, and 3 of HuPRO1 VL are SASSVSYMH (SEQ ID NO:66), DTSNLAS (SEQ ID NO:67), and HQRSSYPWT (SEQ ID NO:68), respectively. The gene encoding HuPRO1 VL is synthesized as an exon, including 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.

[0190] The expression vector pHuPRO1-IgG1.AA has the same structure as pHuM195-IgG1 (Fig. 1A), except that (i) the HuPRO1 VH exon is located between the SpeI and HindIII sites, (ii) the HuPRO1 VL exon is located between the NheI and EcoRI sites, and (iii) the HuACS2 VL exon is placed between the NheI and EcoRI sites, and the two leucine residues at positions 234 and 235 (EU number) in (iii) CH2 are replaced with alanine residues (L234A and L235A, respectively) to eliminate effector function (Hezareh et al., ibid.). The new vector pHuPRO1-IgG1.AA expresses humanized anti-GITR IgG1 / κ antibody (HuGAB11-IgG1.AA) in mammalian cells. The new vector pHuPRO1-IgG1.AA expresses humanized anti-PD-L1 IgG1 / κ antibody (HuPRO1-IgG1.AA) in mammalian cells.

[0191] The mammalian expression vector pBS809, designed to express a bispecific antibody binding to 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).

[0192] The amino acid sequence of the mature heavy chain encoded in pBS809 is as follows:

[0193] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS(SEQ ID NO:70).

[0194] The amino acid sequence of the mature light chain encoded in pBS809 is

[0195] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:71).

[0196] The structural diagram of BS809 is as follows: Figure 2 As shown.

[0197] As described above, three expression vectors, pHuPRO1-IgG1.AA, pHuGAB11-IgG1.AA, and pBS809, were stably transfected into CHO-K1. HuPRO1-IgG1.AA, HuGAB11-IgG1.AA, and BS809 were purified from the culture supernatant of their respective stable CHO-K1 transfectants using a protein A affinity column (as described above). SDS-PAGE analysis under reducing conditions showed that each of the three antibodies exhibited only two major bands: approximately 50 kDa heavy chain and 25 kDa light chain (for HuPRO1-IgG1.AA and HuGAB11-IgG1.AA) or approximately 75 kDa heavy chain and 25 kDa light chain (for BS809).

[0198] The bioactivity of BS809 in enhancing immune responses was analyzed by measuring IL-2 expression levels 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 grown for three days in RPMI-1640 containing 10% FBS and 5 μg / ml phytohemagglutinin-L (PHA-L) in a 7.5% CO2 incubator at 37°C 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 PBMC cells were cultured for 1 day in the wells of a 96-well plate at 37°C in a 7.5% CO2 incubator. The 96-well plate was pre-coated with 1 μg / ml of mouse anti-human CD3 monoclonal antibody OKT3. The culture was conducted with (i) no additional antibody (control), (ii) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1), (iii) 1 μg / ml HuGAB11-IgG1.AA (anti-GITR), (iv) 1 μg / ml HuPRO1-IgG1.AA and 1 μg / ml HuGAB11-IgG1.AA, and (v) 1 μg / ml BS809. IL-2 concentrations in the culture supernatant were measured using the Human IL-2 ELISA MAX Standard Kit (BioLegend, San Diego, CA). Results are as follows. Figure 6 As shown.

[0199] The mean IL-2 concentrations in PBMC-derived T cells from donor 3124 were (i) 2,776 pg / ml without antibody (control); (ii) 3,075 pg / ml, HuPRO1-IgG1.AA; (iii) 2,577 pg, HuGAB11-IgG1.AA; (iv) 2,747 pg / ml, a mixture of HuPRO1-IgG1.AA and HuGAB11-IgG1.AA; and (v) 4,826 pg / ml, BS809. IL-2 expression was significantly increased only in the presence of bispecific antibodies binding to both PD-L1 and GITR compared to IL-2 levels in untreated control cells. This is because BS809 bridges the gap between PD-L1-expressing cells and GITR-expressing T cells, leading to multimer cross-linking of CD40 on the surface near PD-L1-expressing cells, resulting in T cell activation and upregulation of IL-2 expression.

[0200] The mean IL-2 concentrations in PBMC-derived T cells from donor 4143 were (i) 658 pg / ml, without antibody; (ii) 1,453 pg / ml, HuPRO1-IgG1.AA; (iii) 659 pg / ml, HuGAB11-IgG1.AA; (iv) 2,136 pg / ml, a mixture of HuPRO1-IgG1.AA and HuGAB11-IgG1.AA; and (v) 4,196 pg / ml, BS809. IL-2 expression was increased in the presence of HuPRO1-IgG1.AA compared to IL-2 levels in untreated control cells, likely because the PD-L1-PD-1 immunosuppressive interaction was blocked by HuPRO1-IgG1.AA. Due to (a) its anti-PD-L1 antagonist activity and (b) its bridging ability between PD-L1-expressing cells and GITR-expressing T cells, which enables polymeric crosslinking of GITR on the surface, leading to effective T cell activation, IL-2 expression was further significantly increased in the presence of BS809.

[0201] Example 8: Bispecific antibody (BS813) binding to PD-L1 and OX40

[0202] The mammalian expression vector pBS813, designed to express a bispecific antibody binding to human PD-L1 (SEQ ID NO:69) and human OX40 (SEQ ID NO:21), was constructed by replacing the CH3 coding region in pHuPRO1-IgG1.AA with the coding region of CH3-HuOHX14DS.scFv (SEQ ID NO:23).

[0203] The amino acid sequence of the mature heavy chain encoded in pBS813 is as follows:

[0204] (SEQ ID NO:72).

[0205] The amino acid sequence of the mature light chain encoded in pBS813 is identical to the sequence of the mature light chain encoded in pBS809 (SEQ ID NO: 71).

[0206] The structural diagram of BS813 is as follows: Figure 2 As shown.

[0207] As described above, the three expression vectors, pHuPRO1-IgG1.AA, pHuOHX14DS-IgG1.AA, and pBS813, were stably transfected into CHO-Kl.

[0208] HuPRO1-IgG1.AA, HuOHX14DS-IgG1.AA, and BS813 were purified from the culture supernatant of their respective CHO-K1 stably transfected cells using a protein A affinity column (as described above). SDS-PAGE analysis under reducing conditions showed that each of the three antibodies exhibited only two major bands: approximately 50 kDa heavy chain and 25 kDa light chain (for HuPRO1-IgG1.AA and HuOHX14DS-IgG1.AA) or approximately 75 kDa heavy chain and 25 kDa light chain (for BS813). NS0 / PD-L1 cells expressed recombinant human PD-L1 on their surface, which was generated by electroporation of the expression vector pFCm370 into NS0 cells. The expression vector pFCm370 has the same structure as pFCm331, except that the SpeI-EagI fragment is replaced with a DNA fragment encoding recombinant human PD-L1, which consists of the following from the N-terminus to the C-terminus: a synthetic signal peptide (SEQ ID NO:26), an extracellular region of human PD-L1 (SEQ ID NO:73), a FLAG polypeptide (SEQ ID NO:9), and a GPI anchoring signal of human CD55 (SEQ ID NO:10).

[0209] (PD-L1-FLAG-GPI). NS0 stable transfectants (i) survived in DME medium containing 10% FBS, 1 μg / ml mycophenolic acid, HT medium supplement and 0.25 mg / ml xanthine and (ii) the PD-L1-FLAG-GPI expressed on the surface was maintained in DME medium containing 10% FBS in a 7.5% CO2 incubator at 37°C.

[0210] Approximately 200,000 JD / OX40 cells were cultured for one day in 96-well plates in RPMI 1640 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 uptake) (Jackson ImmunoResearch) to provide the primary signal for T cell activation, along with: no antibody (a and f), 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). 200,000 NSO cells were added to reactions a, b, c, d, and e. 200,000 NSO / PD-L1 cells were added to reactions f, g, h, i, and j. Luciferase activity in the culture supernatant was measured in triplicate using the QUANTI-Luc reagent (InvivoGen) according to the supplier's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek). Results are as follows. Figure 7 As shown.

[0211] The mean relative luciferase units (RLU) in JD / OX40 cells were 4683, NS0 cells (a); 6,193, HuPRO1-IgG1.AA and NS0 cells (b); 7,230, HuOHX14DS-IgG1.AA (c); 7,671, HuPRO1-IgG1.AA, HuOHX14DS-Ig1.AA and NS0 cells (d); 6,146, BS813 and NS0 cells (e); 3,842, NS0 / PD-L1 cells (f); 3,866, HuPRO1-IgG1.AA and NS0 / PD-L1 cells (g); 5,921, HuOHX14DS-IgG1.AA and NS0 / PD-L1 cells (h); 2,836, HuPRO1-IgG1.AA, HuOHX14DS-Ig1.AA and NS0 / PD-L1 cells (i); and 23,715, BS813 and NS0 / PD-L1 cells (j). Luciferase activity was significantly increased in JD / OX40 cells only in the presence of both the bispecific antibody binding to PD-L1 and OX40 (BS813) and PD-L1-expressing cells (NS0 / PD-L1). The results showed that only BS813 could establish a bridge between JD / OX40 and NSO / PD-L1 cells, but the binding of HuPRO1-IgG1.AA, HuOHX14DS-IgG1.AA and these two monospecific antibodies did not cause OX40 to accumulate on the surface of JD / OX40 cells near NSO / PD-L1 cells and activate JD / OX40 cells to increase luciferase expression.

[0212] Example 9: Generation of a bispecific antibody (BS841) binding to PD-L1 and OX40

[0213] As described in Example 1, mouse hybridomas producing the IgG / κ monoclonal antibody PRO2 were isolated, which (i) binds to human and cynomolgus monkey PD-L1 and (ii) blocks the interaction between PD-1 and PD-L1. Sequencing and humanization of the VH and VL of PRO2 were performed according to the description of Tsurushita et al. (ibid.).

[0214] The amino acid sequence of humanized PRO2 (HuPRO2)VH is

[0215] MGWNWIFLFLSGTAGVHCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSS (SEQ ID NO:74). According to the definition by Kabat et al. (ibid.), the CDR1, 2, and 3 amino acid sequences of HuPRO2 VH are SYGIN (SEQ ID NO:75), YIYPGSGGPVYNQKFKG (SEQ ID NO:76), and ENYRYWYFDV (SEQ ID NO:77), respectively. The gene encoding HuPRO2 VH is synthesized as an exon, including 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.

[0216] The amino acid sequence of humanized PRO2 (HuPRO2)VL is as follows:

[0217] MHFQVQIFSFLLISASVIMSRGDIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK (SEQ ID NO:78). According to the definition by Kabat et al. (ibid.), the CDR1, 2, and 3 amino acid sequences of HuPRO2 VL are SASSVNYMH (SEQ ID NO:79), STSNLAS (SEQ ID NO:80), and QQRSSYPLT (SEQ ID NO:81), respectively. The gene encoding HuPRO2 VL was synthesized into exons, including the splice donor signal at the 3' end of the coding region, the NheI site at the 5' end of the fragment, and the EcoRI site at the 3' end of the fragment.

[0218] The expression vector pHuPRO2-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuPRO2 VH exon is located between the SpeI and HindIII sites, (ii) the HuPRO2 VL exon is placed between the NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU number) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO:20) to eliminate effector function (Hezareh et al., ibid.). The new vector pHuPRO2-IgG1.AA expresses humanized anti-PD-L1IgG1 / κ antibody (HuPRO2-IgG1.AA) in mammalian cells.

[0219] The mammalian expression vector pBS841, designed to express bispecific antibodies binding to human PD-L1 (SEQ ID NO:69) and human OX40 (SEQ ID NO:21), was constructed by replacing the CH3 coding region in pHuPRO2-IgG1.AA with the coding region of CH3-HuOHX14DS.scFv (SEQ ID NO:23).

[0220] The amino acid sequence encoding the mature heavy chain in pBS841 is:

[0221] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS(SEQ ID NO:82).

[0222] The amino acid sequence of the mature light chain encoded in pBS841 is

[0223] DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:83).

[0224] Example 10: Generation of a bispecific antibody (BS839) binding to PD-L1 and OX40

[0225] As described in Example 1, mouse hybridomas producing the IgG / κ monoclonal antibody PRO5 were isolated, which (i) binds to human and cynomolgus monkey PD-L1 and (ii) blocks the interaction between PD-1 and PD-L1. Sequencing and humanization of the VH and VL of PRO5 were performed according to the description of Tsurushita et al. (ibid.).

[0226] The amino acid sequence of humanized PRO5 (HuPRO5)VH is

[0227] MMVLSLLYLLTALPGILSQVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSS (SEQ ID NO:84). According to the definition by Kabat et al. (ibid.), the CDR1, 2, and 3 amino acid sequences of HuPRO5 VH are SGYWN (SEQ ID NO:85), YISYTGSTYSNPSLKS (SEQ ID NO:86), and SQNWERAWFAY (SEQ ID NO:87), respectively. The gene encoding HuPRO5 VH is synthesized as an exon, including 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.

[0228] The amino acid sequence of humanized PRO5 (HuPRO5) VL is as follows:

[0229] MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK (SEQ ID NO:88). According to the definition by Kabat et al. (ibid.), the CDR1, 2, and 3 amino acid sequences of HuPRO5 VL are SASSVSYMH (SEQ ID NO:89), DTSKLAS (SEQ ID NO:90), and FQGSGYPFT (SEQ ID NO:91), respectively. The gene encoding HuPRO5 VL was synthesized into exons, including the splice donor signal at the 3' end of the coding region, the NheI site at the 5' end of the fragment, and the EcoRI site at the 3' end of the fragment.

[0230] The expression vector pHuPRO5-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 1A), except that (i) the HuPRO5 VH exon is located between the SpeI and HindIII sites, (ii) the HuPRO5 VL exon is placed between the NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU number) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO:20) to eliminate effector function (Hezareh et al., ibid.). The new vector pHuPRO5-IgG1.AA expresses humanized anti-PD-L1IgG1 / κ antibody (HuPRO5-IgG1.AA) in mammalian cells.

[0231] The mammalian expression vector pBS839, designed to express bispecific antibodies binding to human PD-L1 (SEQ ID NO:69) and human OX40 (SEQ ID NO:21), was constructed by replacing the CH3 coding region in pHuPRO5-IgG1.AA with the coding region of CH3-HuOHX14DS.scFv (SEQ ID NO:23).

[0232] The amino acid sequence encoding the mature heavy chain in pBS839 is:

[0233] QVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS(SEQ ID NO:92).

[0234] The amino acid sequence of the mature light chain encoded in pBS839 is

[0235] DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:93).

[0236] Example 11: Expression and characterization of bispecific antibodies BS839 and BS841 binding to PD-L1 and OX40

[0237] As described above, four expression vectors, pHuPRO2-IgG1.AA, pHuPRO5-IgG1.AA, pBS839, and pBS841, were stably transfected into CHO-K1. HuPRO2-IgG1.AA, HuPRO5-IgG1.AA, BS839, and BS841 were purified from the culture supernatant of their respective stable CHO-K1 transfectants using a protein A affinity column (as described above). SDS-PAGE analysis under reducing conditions was performed to determine the heavy and light chain sizes of these four antibodies.

[0238] The bioactivity of BS839 and BS841 in enhancing immune responses was analyzed by measuring the expression level of IL-2 in human T cells. As described above, human PBMCs were grown for three days in RPMI-1640 containing 10% FBS and 10 μg / ml PHA-L. Approximately 100,000 3-day-old PBMC cells were cultured for 1 day in the wells of a 96-well plate pre-coated with 1 μg / ml of mouse anti-human CD3 monoclonal antibody OKT3. The culture process was accompanied by the presence of (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 BS841, and (viii) 1 μg / ml BS839. The concentration of IL-2 in the culture supernatant was measured using the Human IL-2 ELISA MAX Standard Kit (BioLegend).

[0239] Example 12: Bispecific antibodies binding to cancer cells and co-stimulatory molecules

[0240] Monoclonal antibodies that bind to cancer cells were isolated using hybridoma or display technologies (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). As described above, the VH and VL regions of this isolated anticancer antibody, or its humanized form, are cloned into an expression vector, such as pHuM195-IgG1 (Figure 1A). Another monoclonal antibody that binds to an immunostimulatory 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 techniques. The VH and VL regions of this isolated anti-co-stimulatory molecule antibody, or its humanized form, are converted into a single-chain Fv (scFv) form (Nelson, mAbs 2: 77-83, 2010; Ahmad et al., Clin. Dev. Immunol. Article ID980250, 2012). The scFv form of an anti-co-stimulatory molecule antibody is fused to the C-terminus of an anti-cancer antibody heavy chain to generate a novel vector expressing a bispecific antibody that binds to both cancer cells and the co-stimulatory molecule. The biological activity of this generated bispecific antibody can (i) establish a bridge between cancer cells and immune cells and (ii) activate such immune cells through multimeric cross-linking of the co-stimulatory molecule; said biological activity is tested in appropriate disease models, such as tumor xenograft models in humanized mice using humanized immune cells (Day et al., Cell 163:39-53, 2015; Mortone et al., Cancer Res. 76:6153-6158, 2016).

[0241] Example 13: Bispecific antibodies binding pathogen-derived molecules and co-stimulatory molecules

[0242] Using the hybridoma or display techniques described above, monoclonal antibodies that bind to pathogens expressed by infectious agents on the surface of host cells are isolated. As described above, the VH and VL regions of this isolated antipathogenic antibody, or its humanized form, are cloned into an expression vector (e.g., pHuM195-IgG1 (Figure 1A)). Another monoclonal antibody that binds to an immune co-stimulatory molecule is isolated using the hybridoma or display techniques. As described above, the VH and VL regions of this isolated anti-co-stimulatory molecule antibody, or its humanized form, are converted to scFv form. As described above, in the expression vector of the antipathogenic IgG antibody, the scFv form of the anti-co-stimulatory antibody is fused to the C-terminus of the antipathogenic antibody heavy chain. The resulting novel vector expresses a bispecific IgG antibody capable of binding to cells infected by both the pathogen and the co-stimulatory molecule. The biological activity of such generated bispecific antibodies was tested in appropriate disease models (e.g., humanized mice with human immune cells infected by pathogens), which can (i) bridge pathogen-infected cells and immune cells, and (ii) activate such immune cells through polycrosslinking of co-stimulatory molecules (Shultz et al., Ann. NYAcad. Sci. 1245:50–54, 2011; Leung et al., Eur. J. Immunol. 43:2246–2254, 2013).

[0243] Example 14: Replacing scFv with different forms of receptor binding modules

[0244] In this invention, the scFv region of the bispecific antibody (which provides a second antigen-binding site for the IgG molecule) is replaced with a polypeptide capable of binding to a cell surface receptor, such as a growth factor, cytokine, chemokine, soluble form of the receptor, a single-domain antibody (e.g., VH, VL, or VHH (Holt et al., Trends Biotechnol. 21:484-490, 2003; Bannaset et al., Front. Immunol. 8: Article 1603, 2017)) or an antibody mimic (Yu et al., Annu. Rev. Anal. Chem. 10:293–320, 2017). The biological activity of this generated bispecific antibody in activating immune cells through multivalent cross-linking of co-stimulatory molecules is tested in appropriate animal disease models (e.g., the aforementioned models of cancer and infectious diseases).

[0245] Example 15: Fusion of scFv at the C-terminus of a light chain

[0246] The scFv region of the anti-co-stimulatory molecule is fused to the C-terminus of the light chain of an IgG antibody targeting checkpoint molecules, cancer cells, or pathogens. The resulting bispecific antibody, through multivalent cross-linking of the co-stimulatory molecule, is tested for its bioactivity in activating immune cells in appropriate animal disease models (e.g., the aforementioned cancer and infectious disease models).

[0247] Example 16: Activation of T cells via BS809

[0248] At 37°C and 7.5% CO2, approximately 100,000 JD / GITR cells were cultured in 96-well plates in 200 μl / well RPMI 1640 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 absorption) (Jackson ImmunoResearch). This culture was accompanied by: antibody-free (a and f), 1 μg / ml HuPRO1-IgG1.AA (… Figure 8 Anti-PD-L1 (b and g), 1 μg / ml HuGAB11-IgG1.AA ( Figure 8 Anti-GITR (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 added. 100,000 NSO cells were added to reactions a, b, c, d, and e. 100,000 NSO / PD-L1 cells were added to reactions f, g, h, i, and j. After one day of culture, luciferase activity in the culture supernatant was measured in triplicate using QUANTI-Luc reagent (InvivoGen) according to the supplier's protocol. Luminescence was measured using a SynergyHT microplate reader (BioTek, Winooski, VT). Results are as follows. Figure 8 As shown.

[0249] The mean relative luciferase units (RLU) in the supernatant of JD / GITR cell cultures were: (a) 479,187 for NS0 cells; (b) 400,158 for HuPRO1-IgG1.AA and NS0 cells; (c) 472,050 for HuGAB11-IgG1.AA and NS0 cells; (d) 531,275 for HuPRO1-IgG1.AA, HuGAB11-IgG1.AA and NS0 cells; and (e) 422,955 for BS809 and NS0 cells. (f) NS0 / PD-L1 cells: 405,780; (g) HuPRO1-IgG1.AA and NS0 / PD-L1 cells: 443,588; (h) HuGAB11-IgG1.AA and NS0 / PD-L1 cells: 568,268; (i) HuPRO1-IgG1.AA, HuGAB11-Ig1.AA and NS0 / PD-L1 cells: 569,172; and (j) BS809 and NS0 / PD-L1 cells: 1,035,453. Luciferase activity was significantly increased in JD / GITR cells only in the presence of both the bispecific antibody binding to PD-L1 and GITR (BS809) and PD-L1-expressing cells (NS0 / PD-L1). The results showed that only BS809 could establish a bridge between JD / GITR and NSO / PD-L1 cells, but the binding of HuPRO1-IgG1.AA, HuGAB11-IgG1.AA and these two monospecific antibodies did not lead to multimeric cross-linking of GITR on the surface of JD / GITR cells near the NSO / PD-L1 cell site, resulting in activation of JD / GITR cells to increase luciferase expression.

[0250] Example 17: Activation of PHA-L-treated PBMCs using BS813, BS841 and BS839

[0251] The bioactivity of BS813, BS841, and BS839 in enhancing immune responses was analyzed by measuring IL-2 expression in human T cells. Bispecific antibodies BS813, BS841, and BS839, each binding to PD-L1 and OX40 respectively, were purified from the culture supernatant of CHO-K1 cells stably transfected with pBS813, pBS841, and pBS839 using a protein A affinity column. As described above, human PBMCs were grown for three days (3-day-old PBMCs) in RPMI-1640 medium containing 10% FBS and 10 μg / ml PHA-L. Approximately 100,000 3-day-old PBMC cells were cultured for 1 day in 96-well plates at 200 μl / well in a 7.5% CO2 incubator at 37°C. The 96-well plates were pre-coated with 1 μg / ml of mouse anti-human CD3 monoclonal antibody OKT3. This culture process was accompanied by (a) no additional antibody, and (b) 1 μg / ml of HuPRO1-IgG1.AA. Figure 9 Anti-PD-L1#1) and 1 μg / ml HuOHX14DS-IgG1.AA Figure 9 (c) 1 μg / ml HuPRO2-IgG1.AA (anti-OX40), (c) 1 μg / ml Figure 9 Anti-PD-L1#2) and 1 μg / ml HuOHX14DS-IgG1.AA, (d) HuPRO5-IgG1.AA Figure 9 The presence of 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 was observed. The IL-2 concentration in the culture supernatant was measured using the Human IL-2 ELISA MAX Standard Kit (BioLegend). Results are shown in... Figure 9 .

[0252] The mean IL-2 concentrations in human PBMC-derived T cells were (a) 439 pg / ml, no antibody tested (control); (b) 324 pg / ml, a combination of HuPRO1-IgG1.AA and HuOHX14DS-IgG1.A; (c) 281 pg / ml, a combination of HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA; (d) 53 pg / ml, a combination of HuPRO5-IgG1.AA and HuOHX14DS-IgG1.AA; (e) 2,202 pg / ml, BS813; (f) 1,698 pg / ml, BS841; and (g) 1,259 pg / ml, BS839. Compared to control IL-2 levels or IL-2 levels in combinations of the two parental antibodies to each of BS813, BS841, and BS839, IL-2 expression was significantly increased only in the presence of one of the bispecific antibodies binding to PD-L1 and OX40 (BS813, BS841, and BS839). This is because PD-L1-expressing cells bridge with OX40-expressing T cells via each of BS813, BS841, and BS839, resulting in multimeric cross-linking of OX40 on the cell surface near PD-L1-expressing cells, leading to activation of OX40-expressing T cells and upregulation of IL-2 expression.

[0253] Example 18: Activation of SEB-processed PBMCs via BS813, BS841, and BS839

[0254] Superantigens such as SEB (Staphylococcus enterotoxin B) activate T cells by linking MHC class II molecules on antigen-presenting cells to the vβ element of the T cell receptor, leading to T cell activation and the production of cytokines including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ) (see, e.g., Krakauer et al., Toxins (Basel). Aug 2010; 2(8):1963–1983). SEB can activate up to 10% to 20% of T cells in human blood, depending on the proportion of T cells with vβ3, vβ12, vβ14, and vβ17 found in each specific blood donor. Therefore, SEB can be used for T cell-based cytokine secretion assays to monitor the activation of immune co-stimulatory molecules.

[0255] Approximately 400,000 PBMCs were cultured in 96-well plates in 200 μl / well of RPMI-1640 medium containing 10% FBS and 1 μg / ml SEB. This culture process was accompanied by (a) antibody-free (control) and (b) 1 μg / ml HuPRO1-IgG1.AA. Figure 10 Anti-PD-L1#1 and 1 μg / ml HuOHX14DS-IgG1.AA (in the sample) Figure 10 (c) 1 μg / ml HuPRO2-IgG1.AA (anti-OX40), (c) 1 μg / ml Figure 10 Anti-PD-L1#2) and 1 μg / ml HuOHX14DS-IgG1.AA, (d) HuPRO5-IgG1.AA Figure 10 The culture supernatants contained 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. After incubation at 37°C in a 7.5% CO2 incubator for one day, the IL-2 concentration in the culture supernatant was measured in triplicate using the Human IL-2 ELISA MAX Standard Kit (BioLegend). The results are shown in... Figure 10 .

[0256] The mean IL-2 concentrations were (a) 11.3 ng / ml, no antibody tested; (b) 10.5 ng / ml, a combination of HuPRO1-IgG1.AA and HuOHX14DS-IgG1.A; (c) 10.0 ng / ml, a combination of HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA; (d) 10.0 ng / ml, a combination of HuPRO5-IgG1.AA and HuOHX14DS-IgG1.AA; (e) 17.7 ng / ml, BS813; (f) 16.5 ng / ml, BS841; and (g) 15.8 ng / ml, BS839. Compared with the control group of PBMCs without antibody added to SEB treatment, IL-2 expression was significantly increased only in the presence of any one of the bispecific antibodies binding to both PD-L1 and OX40 (BS813, BS841, and BS839). The combination of two parental antibodies for each of BS813, BS841, and BS839 did not increase IL-2 expression above the control level. This result indicates that each of BS813, BS841, and BS839 can establish a bridge between PD-L1-expressing cells and OX40-expressing T cells, leading to multimeric cross-linking of OX40 on the cell surface near PD-L1-expressing cells, and resulting in activation of OX40-expressing T cells, which in turn upregulates IL-2 expression.

[0257] Example 19: Bispecific antibody with disulfide-linked single-chain Fv

[0258] Brinkman et al. previously reported that the introduction of disulfide bonds stabilized the binding of VH and VL in the Fv format (dsFv) by converting the amino acid residue at position 100 in VL to a cysteine ​​residue and another amino acid residue at position 44 in VH to a cysteine ​​residue (Eu number). (Proc. Natl. Acad. Sci. 90:7538-7542, 1993). They also reported stabilizing the Fv structure by introducing a pair of cysteine ​​residues at different VH and VL positions. (Brinkman et al. ibid.; Young et al. FEBS Lett. 377:135-139, 1995; Schmiedl et al. Protein Eng. 13:724-730, 2000).

[0259] The bispecific anti-PD-L1 / GITR antibody (BS809) of this invention is modified to stabilize the single-chain Fv structure by introducing two cysteine ​​residues (underlined) into the anti-GITR variable region, one of which is located at position 44 in VH and the other at position 100 in VL, introduced via site-directed mutagenesis of pBS809. Although the resulting expression vector pBS853 has the same structure as pBS809, the amino acid sequences of the anti-GITR single-chain Fv region of pBS809 and pBS853 are different. Vectors pBS809 and pBS853 carry the same light chain sequence (SEQ ID NO: 71). The amino acid sequence encoding the mature heavy chain in pBS853 is as follows:

[0260] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS(SEQ ID NO:106). Cysteine ​​substitutions are underlined.

[0261] The modified bispecific antibody encoded in pBS853 (BS853) was transiently expressed in HEK293 cells according to the method of Durocher et al. (Nucl. Acids Res. 30:e9, 2002). BS853 showed the same binding levels to PD-L1 and GITR as BS809 by ELISA.

[0262] Example 20: Bispecific antibody (BS859) binding PD-L1 and ICOS

[0263] ICOS (Inducible T-cell Costimulator), also known as CD278, is an immune costimulatory molecule belonging to the CD28 family. Polycrosslinking of ICOS on the surface of immune cells is required to initiate intracellular signal transduction, thereby enhancing the immune response (Wikenheiser et al., Front. Immunol. 7: Article 304, 2016).

[0264] As described in Example 1, rat hybridomas producing the IgG / κ monoclonal antibody TAM14, which specifically binds to human and cynomolgus monkey ICOS, were isolated, except that Sprague Dawley rats were used for immunization. The extracellular region of human ICOS (hICOS-Fc; SEQ ID NO: 107) fused to the Fc region of the human γ-1 heavy chain was used as the immunogen. Sequencing and humanization of TAM14 VH and VL were performed as described by Tsurushita et al. (ibid.).

[0265] The amino acid sequence of humanized TAM14 (HuTAM14)VH is:

[0266] MAVLVLLLCLVTFPSCALSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS (SEQ ID NO:108). According to the definition by Kabat et al. (ibid.), 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. The gene encoding HuTAM14 VH was synthesized into exons, including the splice donor signal at the 3' end of the coding region, the SpeI site at the 5' end of the fragment, and the HindIII site at the 3' end of the fragment.

[0267] The amino acid sequence of humanized TAM14 (HuTAM14) VL is

[0268] MRTSIQLLGLLLFWLHDAQCDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIK (SEQ ID NO:112). According to the definition by Kabat et al. (ibid.), 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. The gene encoding HuTAM14VL was synthesized into exons, including 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.

[0269] The expression vector pHuTAM14-IgG1.AA has the same structure as pHuM195-IgG1 (Fig. 1A), except that (i) the HuTAM14 VH exon is placed between the SpeI and HindIII sites, (ii) the HuTAM14 VL exon is placed between the NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU number of Kabat et al.) are replaced with alanine residues in CH2 (L234A and L235A, respectively) to eliminate effector function (Hezarehet al., J.Virol. 75:12161-12168, 2001). The new vector pHuTAM14-IgG1.AA expresses humanized anti-ICOS IgG1 / κ antibody (HuTAM14-IgG1.AA) in mammalian cells.

[0270] The mammalian expression vector designed to express a bispecific antibody binding to human PD-L1 (SEQ ID NO: 69) and human ICOS (SEQ ID NO: 116) was constructed by modifying pHuPRO1-IgG1.AA as described below. The VL and VH coding regions of the humanized anti-human ICOS monoclonal antibody in pHuTAM14-IgG1.AA were converted to a single-chain Fv (scFv) form (HuTAM14.scFv; SEQ ID NO: 117) in the order of VL, peptide linker, and VH from the N-terminus to the C-terminus. The N-terminus of HuTAM14.scFv was fused to the penultimate glycine residue in CH3 of pHuPRO1-IgG1.AA, and the peptide linker separated them (CH3-HuTAM14.scFv; SEQ ID NO: 118). Furthermore, in CH2, the two leucine residues at positions 234 and 235 (EU number) were replaced with alanine residues (L234A and L235A, respectively). The resulting vector pBS859 expresses a bispecific IgG antibody called BS859, which binds to human PD-L1 and ICOS.

[0271] The amino acid sequence encoding the mature heavy chain in pBS859 is:

[0272] (SEQ ID NO:119).

[0273] The amino acid sequence of the mature light chain encoded in pBS859 is identical to the mature light chain sequence (SEQ ID NO: 71) encoded in pBS809.

[0274] The schematic structure of the bispecific IgG antibody (e.g., BS859) of the present invention is shown below. Figure 2 As shown.

[0275] Each of the three expression vectors, pHuPRO1-IgG1.AA, pHuTAM14-IgG1.AA, and pBS859, was stably transfected into CHO-Kl cells as described above.

[0276] HuPRO1-IgG1.AA, HuTAM14-IgG1.AA, and BS859 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 that each of these three antibodies exhibited only two major bands: approximately 50 kDa heavy chain and 25 kDa light chain (for HuPRO1-IgG1.AA and HuTAM14-IgG1.AA) or approximately 75 kDa heavy chain and 25 kDa light chain (for BS859).

[0277] The bioactivity of BS859 in enhancing immune responses 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 grown for three days in RPMI-1640 containing 10% FBS and 10 μg / ml phytohemagglutinin-L (PHA-L) in a 7.5% CO2 incubator at 37°C to enrich T cells (3-day-old PBMCs).

[0278] Approximately 200,000 3-day-old PBMC cells were cultured in the wells of a 96-well plate at 37°C in a 7.5% CO2 incubator. The 96-well plate was pre-coated with 1 μg / ml of mouse anti-human CD3 monoclonal antibody OKT3 and 1 μg / ml of human PD-L1-Fc fusion protein (recombinant human B7-H1-Fc chimera, BioLegend, San Diego, CA). The culture process was accompanied by: (i) no additional antibody (control), (ii) 1 μg / ml of HuPRO1-IgG1.AA (anti-PD-L1), (iii) 1 μg / ml of HuTAM14-IgG1.AA (anti-ICOS), (iv) 1 μg / ml of HuPRO1-IgG1.AA and 1 μg / ml of HuTAM14-IgG1.AA, and (v) 1 μg / ml of 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 .

[0279] The mean IL-2 concentrations in PBMC-derived T cells from donor 3486 were (i) 3.3 ng / ml, without antibody (control); (ii) 13.3 ng / ml, HuPRO1-IgG1.AA; (iii) 3.6 ng, HuTAM14-IgG1.AA; (iv) 9.9 ng / ml, a mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA; and (v) 46.2 ng / ml, BS859.

[0280] The mean IL-10 concentrations in PBMC-derived T cells from donor 3486 were (i) 604 pg / ml, without antibody (control); (ii) 676 pg / ml, HuPRO1-IgG1.AA; (iii) 527 pg / ml, HuTAM14-IgG1.AA; (iv) 699 pg / ml, a mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA; and (v) 1,201 pg / ml, BS859.

[0281] The mean IL-2 concentrations in PBMC-derived T cells from donor 4239 were (i) 0.01 ng / ml, antibody-free (control); (ii) 0.15 ng / ml, HuPRO1-IgG1.AA; (iii) 0.01 ng / ml, HuTAM14-IgG1.AA; (iv) 0.06 ng / ml, a mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA; and (v) 13.6 ng / ml, BS859.

[0282] The mean IL-10 concentrations in PBMC-derived T cells from donor 4239 were (i) 238 pg / ml, antibody-free (control), (ii) 252 pg / ml, HuPRO1-IgG1.AA; (iii) 217 ​​pg / ml, HuTAM14-IgG1.AA; (iv) 220 pg / ml, a mixture of HuPRO1-IgG1.AA and HuTAM14-IgG1.AA; and (v) 537 pg / ml, BS859.

[0283] Compared with their respective untreated control groups, HuPRO1-IgG1.AA alone increased IL-2 expression in PHA-L-treated PBMCs from two different donors. BS859 further significantly increased IL-2 expression from both donors.

[0284] IL-10 expression in PHA-L-treated PBMCs from two different donors was significantly increased only in the presence of the bispecific antibody of the present invention (BS859) that binds both PD-L1 and ICOS.

[0285] Example 21: Bispecific antibody (BS840) binding PD-L1 and GITR

[0286] The mammalian expression vector pBS840, designed to express a bispecific antibody binding to 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 replaced by CH3-HuGAB11.scFv (SEQ ID NO:42). Vector pBS840 expresses a bispecific IgG antibody called BS840, which binds to both human PD-L1 and GITR.

[0287] The amino acid sequence encoding the mature heavy chain in pBS840 is:

[0288] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS(SEQ ID NO:120).

[0289] The amino acid sequence of the mature light chain encoded in pBS840 is identical to the light chain sequence (SEQ ID NO:83) encoded in pBS841.

[0290] As described above, the expression vector pBS840 was stably transfected into CHO-K1 cells. BS840 was purified from the culture supernatant of the resulting stably transfected CHO-K1 cells using a protein A affinity column (as described above). Using gel filtration on a Superose 6 size-restriction 10 / 300 column (GE Healthcare Life Sciences, Pittsburgh, PA), protein A purified BS840 showed a single main peak of the expected size (approximately 200 kDa). SDS-PAGE analysis under reducing conditions showed only two major bands: approximately 75 kDa heavy chain and 25 kDa light chain.

[0291] The biological activity of Jurkat Dual cells stably expressing human GITR (JD / GITR) as described above in enhancing the immune response of BS840 was investigated. Data are as follows: Figure 12 As shown. The mean relative luciferase units (RLU) in the JD / GITR cell culture supernatant were: (a) 354,057, NS0 cells; (b) 341,277, HuPRO2-IgG1.AA ( Figure 12 The anti-PD-L1#2; parental monospecific IgG antibody of BS840), HuGAB11-IgG1.AA ( Figure 12 (c) 326,577, BS840 and NS0 cells; (d) 282,554, NS0 / PD-L1 cells; (e) 386,137, HuPRO2-IgG1.AA, HuGAB11-Ig1.AA and NS0 / PD-L1 cells; and (f) 989,407, BS840 and NS0 / PD-L1 cells. Luciferase activity was significantly increased in JD / GITR cells only in the presence of both the bispecific antibody binding to PD-L1 and GITR (BS840) and PD-L1-expressing cells (NS0 / PD-L1).

[0292] Example 22: Bispecific antibody binding to PD-L1 and CD40 (BS846)

[0293] The mammalian expression vector pBS846, designed to express a bispecific antibody binding to 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 replaced by CH3-HuACS2.scFv (SEQ ID NO:58). Vector pBS846 expresses a bispecific IgG antibody called BS846, which binds to both human PD-L1 and CD40.

[0294] The amino acid sequence of the mature heavy chain encoded in pBS846 is as follows:

[0295] (SEQ ID NO:121).

[0296] The amino acid sequence of the mature light chain encoded in pBS846 is identical to the amino acid sequence of the light chain encoded in pBS809 (SEQ ID NO:71).

[0297] As described above, the expression vector pBS846 was stably transfected into CHO-K1 cells. BS846 was purified from the culture supernatant of their respective stable CHO-K1 transfectants using a protein A affinity column (as described above). Using gel filtration on a Superose 6 size-restriction 10 / 300 column (GE Healthcare Life Sciences, Pittsburgh, PA), protein A purified BS846 showed a single main peak of the expected size (approximately 200 kDa). SDS-PAGE analysis under reducing conditions showed only two major bands: approximately 75 kDa heavy chain and 25 kDa light chain.

[0298] As described above, the biological activity of BS846 in enhancing CD40-mediated immune responses was investigated using Ramos cells. (a) Antibody-free and NSO cells, (b) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1) and NSO cells, (c) 1 μg / ml HuACS2-IgG1.AA (anti-CD40) and NSO cells, (d) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA and NSO cells, (e) 1 μg / ml BS846 and NSO cells, (f) Antibody-free and NSO / PD-L1 cells, (g) 1 μg / ml HuPRO1-IgG1.AA and NSO / PD-L1 cells, (h) 1 μg / ml HuACS2-IgG1.AA and NSO / PD-L1 cells, (i) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml BS846 and NSO cells, (d) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml BS846 and NSO cells, (e) 1 μg / ml BS846 and NSO cells, (f) Antibody-free and NSO / PD-L1 cells, (g) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml BS846 and NSO / PD-L1 cells, (h) 1 μg / ml HuACS2-IgG1.AA and NSO / PD-L1 cells, (i) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml BS846 and NSO / PD-L1 cells, (d) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml BS846 and NSO / PD-L1 cells, (f) Antibody-free and NSO / PD-L1 cells, (g) 1 μg / ml HuPRO1-IgG1.AA, Ramos cells were cultured for three days in DME medium containing 10% FBS in the presence of HuACS2-IgG1.AA and NSO / PD-L1 cells, and (j) BS846 and NSO / PD-L1 cells at 1 μg / ml. CD95 expression in Ramos cells was analyzed by flow cytometry using FITC-labeled mouse anti-human CD20 monoclonal antibody (Clone 2H7, BioLegend) to detect CD95 expression, and PE-labeled mouse anti-human CD95 antibody (Clone DX2, BioLegend) to monitor CD95 expression.

[0299] The percentage of CD95-positive population in CD20-positive Ramos cells was as follows: 0.3% without antibodies and NSO cells (a); 0.3% with 1 μg / ml HuPRO1-IgG1.AA and NSO cells (b); 0.3% with 1 μg / ml HuACS2-IgG1.AA and NSO cells (c); 0.3% with 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA and NSO cells (d); 2.5% with 1 μg / ml BS846 and NSO cells (e); 0. 2%, antibody-free and NSO / PD-L1 cells (f); 0.3%, 1 μg / ml HuPRO1-IgG1.AA and NSO / PD-L1 cells (g); 0.3%, 1 μg / ml HuACS2-IgG1.AA and NSO / PD-L1 cells (h); 0.2%, 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA and NSO / PD-L1 cells (i); and 59.5%, 1 μg / ml BS846 and NSO / PD-L1 cells (j). Figure 13 Only BS846, which can bridge the gap between Ramos and NSO / PD-L1 cells, can significantly increase CD95 expression in Ramos cells. This is because BS846 can polymerize CD40 on the surface of Ramos cells at the junction with NSO / PD-L1 cells, and trigger CD40-mediated intracellular signal transduction to upregulate CD95 expression in Ramos cells.

[0300] Example 24: Disulfide-linked single-chain Fv

[0301] Each single-chain Fv (scFv) antibody against OX40 (HuOHX14DS.scFv; SEQ ID NO:22), CD40 (HuACS2.scFv; SEQ ID NO:57), and ICOS (HuTAM14.scFv; SEQ ID NO:117) was modified by site-directed mutagenesis to replace the amino acid residue at position 44 in VH with a cysteine ​​residue and another amino acid residue at position 100 in VL with a cysteine ​​residue (Eu number), thereby stabilizing the structure (Brinkman et al., Proc. Natl. Acad. Sci. 90:7538-7542, 1993).

[0302] The amino acid sequence of the mature HuOHX14DS.scFv (HuOHX14DS.scFv.ds) with two cysteine ​​substitutions is:

[0303] DIQMTQSPSSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQ C LEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS (SEQ ID NO: 122).

[0304] The amino acid sequence of the mature HuACS2.scFv (HuACS2.scFv.ds) with two cysteine ​​substitutions is:

[0305] EIVLTQSPATLSLSPGERATLCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQ C LEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS (SEQ ID NO: 123).

[0306] The amino acid sequence of mature HuTAM14.scFv (HuTAM14.scFv.ds) with two cysteine ​​substitutions is:

[0307] DIQMTQSPSSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGK CLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS (SEQ ID NO: 124).

[0308] The generation of the amino acid sequence of mature HuGAB11.scFv (HuGAB11.scFv.ds) with two cysteine ​​substitutions is described in Example 19. This amino acid sequence is...

[0309] DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (SEQ ID NO: 125).

[0310] In the amino acid sequence shown in Example 24, cysteine ​​substitutions are marked with an underline.

[0311] Example 25: Construction of bispecific antibodies (BS883 and BS884) binding to PD-L1 and 4-1BB

[0312] 4-1BB, also known as CD137 and TNFRSF9, is an immune costimulatory molecule belonging to the TNF receptor superfamily. Cross-linking of 4-1BB on the surface of immune cells is required to initiate intracellular signal transduction, thereby enhancing the immune response (Chester et al. 2016 Cancer Immunol. Immunother. 65:1243–1248).

[0313] As described in Example 1, mouse hybridomas producing the IgG / κ monoclonal antibody FOB5, which specifically binds to human and cynomolgus monkey 4-1BB, were isolated. The extracellular region of human 4-1BB fused to the Fc region of the human γ-1 heavy chain (h4-1BB-Fc; SEQ ID NO: 126) was used as the immunogen. Sequencing and humanization of FOB5VH and VL were performed as described by Tsurushita et al. (ibid.).

[0314] The amino acid sequence of humanized FOB5 (HuFOB5)VH is

[0315] MERHWIFLFLFSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQGLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS (SEQ ID NO:127). According to the definition by Kabat et al. (ibid.), 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. The gene encoding HuFOB5VH was synthesized into exons, including the splice donor signal at the 3' end of the coding region, the SpeI site at the 5' end of the fragment, and the HindIII site at the 3' end of the fragment.

[0316] The amino acid sequence of humanized FOB5 (HuFOB5) VL is

[0317] MDSQAQVLMLLLLWVSGTCGDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK (SEQ ID NO:131). According to the definition by Kabat et al. (ibid.), the amino acid sequences of CDR1, 2, and 3 of HuFOB5 VL 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 into exons, including the splice donor signal at the 3' end of the coding region, the NheI site at the 5' end of the fragment, and the EcoRI site at the 3' end of the fragment.

[0318] The expression vector pHuFOB5-IgG1.AA has the same structure as pHuM195-IgG1 (Fig. 1A), except that (i) the HuFOB5 VH exon is placed between the SpeI and HindIII sites, (ii) the HuFOB5 VL exon is placed between the NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU number of Kabat et al.) are replaced with alanine residues in CH2 (L234A and L235A, respectively) to eliminate effector function (Hezarehet al., J.Virol. 75:12161-12168, 2001). The new vector pHuFOB5-IgG1.AA expresses humanized anti-4-1BB IgG1 / κ antibody (HuFOB5-IgG1.AA) in mammalian cells.

[0319] The mammalian expression vector designed to express bispecific antibodies binding to human PD-L1 (SEQ ID NO: 69) and human 4-1BB (SEQ ID NO: 135) was constructed by modifying pHuPRO1-IgGl.AA as described below. The VL and VH coding regions of the humanized anti-human 4-1BB monoclonal antibody in pHuFOB5-IgG1.AA were converted to single-chain Fv (scFv) form in the order of VL, peptide linker, and VH from the N-terminus to the C-terminus. Furthermore, the glycine residue at position 44 of HuFOB5 VH was replaced with a cysteine ​​residue, and the glycine residue at position 100 of HuFOB5 VL was also replaced with a cysteine ​​residue to construct a disulfide-linked HuFOB5 scFv (HuFOB5.scFv.LH.ds; SEQ ID NO: 136). The N-terminus of HuFOB5.scFv is fused to the penultimate glycine residue in CH3 of pHuPRO1-IgG1.AA, and the peptide linker separates them (CH3-HuFOB5.scFv.LH.ds; SEQ ID NO: 137). The CH3 coding region in pHuPRO1-IgG1.AA is replaced by the coding region of CH3-FOB5.scFv.LH.ds. The resulting vector pBS883 expresses a bispecific IgG antibody called BS883, which binds to human PD-L1 and 4-1BB.

[0320] The amino acid sequence encoding the mature heavy chain in pBS883 is:

[0321] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFG C GTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQ C LEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS(SEQ ID NO:138). Cysteine substitutions are underlined.

[0322] The amino acid sequence of the mature light chain encoded in pBS883 is identical to the mature light chain sequence (SEQ ID NO:71) encoded in pBS809.

[0323] Another mammalian expression vector designed to express a bispecific antibody binding to human PD-L1 (SEQ ID NO: 69) and human 4-1BB (SEQ ID NO: 135) was constructed by modifying pHuPRO1-IgGl.AA as described below. The VH and VL coding regions of the humanized anti-human 4-1BB monoclonal antibody in pHuFOB5-IgG1.AA were converted to scFv form in the order of VH, peptide linker, and VL from the N-terminus to the C-terminus. Furthermore, the glycine residue at position 44 of HuFOB5 VH was replaced with a cysteine ​​residue, and the glycine residue at position 100 of HuFOB5 VL was also replaced with a cysteine ​​residue to construct a disulfide-linked HuFOB5 scFv (HuFOB5.scFv.HL.ds; SEQ ID NO: 139). The N-terminus of HuFOB5.scFv is fused to the penultimate glycine residue in CH3 of pHuPRO1-IgG1.AA, and the peptide linker separates them (CH3-FOB5.scFv.HL.ds; SEQ ID NO: 140). The CH3 coding region in pHuPRO1-IgG1.AA is replaced with the coding region of CH3-HuFOB5.scFv.HL.ds. The resulting vector pBS884 expresses a bispecific IgG antibody called BS884, which binds to human PD-L1 and 4-1BB.

[0324] The amino acid sequence encoding the mature heavy chain in pBS884 is:

[0325] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQ C LEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFG C GTKVEIK (SEQ ID NO:141). Cysteine ​​substitutions are underlined.

[0326] The amino acid sequence of the mature light chain encoded in pBS8834 is identical to the sequence of the mature light chain encoded in pBS809 (SEQ ID NO:71).

[0327] A schematic structure of the bispecific IgG antibodies (e.g., BS883 and BS884) of the present invention is shown in [illustration]. Figure 2 .

[0328] Example 26: Blocking the interaction between PD-L1 and PD-1

[0329] The activity of HuPRO1-IgG1.AA, HuPRO2-IgG1.AA, and HuPRO5-IgG1.AA in blocking the interaction between human PD-1 (SEQ ID NO:142) and human PD-L1 was analyzed by flow cytometry using NSO / PD-L1 cells and the PD-1-Fc fusion protein. To serve as a ligand for PD-L1 binding, the extracellular region of human PD-1 was fused with the Fc region of the human γ-1 heavy chain (hPD-1-Fc; SEQ ID NO:143), expressed in NSO cells, and purified by protein A affinity chromatography. To monitor binding to NSO / PD-L1 cells, hPD-1-Fc was labeled with fluorescein isothiocyanate (FITC) using a standard binding method. NSO / PD-L1 cells were cultured in FACS buffer (PBS containing 0.5% BSA and 0.05% sodium azide) at 4°C with subsaturated concentrations of FITC-labeled hPD-1-Fc and different concentrations of test antibodies (HuPRO1-IgG1.AA, HuPRO2-IgG1.AA, or HuPRO5-IgG1.AA) for 60 min. After washing and resuspending with FACS buffer, the cells were analyzed by flow cytometry. HuPRO1-IgG1.AA blocked the binding of hPD-1-Fc to NSO / PD-L1 cells at a half-maximum concentration (IC50). 50 The concentration of IgG1 was 106 ng / ml, while that of HuPRO2-IgG1.AA was 55 ng / ml and that of HuPRO5-IgG1.AA was 87 ng / ml.

[0330] sequence list

[0331] SEQ ID NO: 1

[0332] Amino acid sequence of human CD33

[0333] MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSA APTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0334] SEQ ID NO:2

[0335] The amino acid sequence of HuM195 VH

[0336] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS

[0337] SEQ ID NO:3

[0338] The amino acid sequence of the CHI region of the human γ-1 heavy chain encoded in pHuM195-IgG1

[0339] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0340] SEQ ID NO:4

[0341] The amino acid sequence encoding the hinge region of the human γ-1 heavy chain in pHuM195-IgG1

[0342] EPKSCDKTHTCPPCP

[0343] SEQ ID NO:5

[0344] The amino acid sequence of the CH2 region of the human γ-1 heavy chain encoded in pHuM195-IgG1

[0345] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0346] SEQ ID NO:6

[0347] The amino acid sequence of the CH3 region of the human γ-1 heavy chain encoded in pHuM195-IgG1

[0348] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0349] SEQ ID NO:7

[0350] Amino acid sequence of HuM195 VL

[0351] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK

[0352] SEQ ID NO:8

[0353] The amino acid sequence encoding the human κ constant region in pHuM195-IgG1

[0354] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0355] SEQ ID NO:9

[0356] Amino acid sequence of FLAG peptide

[0357] DYKDDDDK

[0358] SEQ ID NO:10

[0359] The amino acid sequence of the GPI anchoring signal

[0360] PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0361] SEQ ID NO:11

[0362] The amino acid sequence of mature OX40-FLAG-GPI

[0363] LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNC TLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRATGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0364] SEQ ID NO:12

[0365] The amino acid sequence of HuOHX14DS VH

[0366] MGRLTSSFLLLIVPAYVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0367] SEQ ID NO:13

[0368] The amino acid sequence of CDR1 of HuOHX14DS VH

[0369] SYIMH

[0370] SEQ ID NO:14

[0371] The amino acid sequence of CDR2 of HuOHX14DS VH

[0372] YINPYNSGTKYNEKFKG

[0373] SEQ ID NO:15

[0374] The amino acid sequence of CDR3 of HuOHX14DS VH

[0375] YYGSTFTMDY

[0376] SEQ ID NO:16

[0377] The amino acid sequence of HuOHX14DS VL

[0378] MMSSAQFLGLLLLCFQGTRCDIQMTQSPSSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIK

[0379] SEQ ID NO:17

[0380] The amino acid sequence of CDR1 of HuOHX14DS VL

[0381] RASQDIRTYLN

[0382] SEQ ID NO: 18

[0383] The amino acid sequence of CDR2 in HuOHX14DS VL

[0384] YTSRLHS

[0385] SEQ ID NO: 19

[0386] The amino acid sequence of CDR3 in HuOHX14DS VL

[0387] QQGNTLPWT

[0388] SEQ ID NO:20

[0389] Amino acid sequence of the CH2 region with L234A and L235 mutations

[0390] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0391] SEQ ID NO:21

[0392] Amino acid sequence of human OX40

[0393] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0394] SEQ ID NO:22

[0395] Amino acid sequence of HuOHX14DS.scFv

[0396] DIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0397] SEQ ID NO:23

[0398] CH3 encoded in pBS824 and HuOHX14DS.scFv

[0399] (CH3-HuOHX14DS.scFv) fused amino acid sequence

[0400] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0401] SEQ ID NO:24

[0402] Amino acid sequence of the mature heavy chain encoded in pBS824

[0403] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0404] SEQ ID NO:25

[0405] Amino acid sequence of the mature light chain encoded in pBS824

[0406] DIQMTQSPSSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0407] SEQ ID NO:26

[0408] amino acid sequence of synthetic signal peptide

[0409] MGWSWIFFFLLSGTASVLS

[0410] SEQ ID NO:27

[0411] The amino acid sequence encoding the extracellular region of human CD33 in pFCm267

[0412] MDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVH VTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVH

[0413] SEQ ID NO:28

[0414] Amino acid sequence of human epidermal growth factor receptor (EGFR)

[0415]

[0416] SEQ ID NO:29

[0417] Amino acid sequence of mouse 225 (Ch225) VH

[0418] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

[0419] SEQ ID NO:30

[0420] Amino acid sequence of mouse 225 (Ch225) VL

[0421] MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

[0422] SEQ ID NO:31

[0423] Amino acid sequence of hGITR-Fc

[0424] QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0425] SEQ ID NO:32

[0426] The amino acid sequence of HuGAB11 VH

[0427] MAVLGLLLCLVTFPSCVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0428] SEQ ID NO:33

[0429] The amino acid sequence of CDR1 in HuGAB11 VH

[0430] DYGVS

[0431] SEQ ID NO:34

[0432] The amino acid sequence of CDR2 of HuGAB11 VH

[0433] VIWGGGGTYYNSALKS

[0434] SEQ ID NO:35

[0435] The amino acid sequence of CDR3 in HuGAB11 VH

[0436] HPYGHFGMDY

[0437] SEQ ID NO:36

[0438] The amino acid sequence of HuGAB11 VL

[0439] MRVLAELLGLLLFCFLGVRCDIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIK

[0440] SEQ ID NO:37

[0441] The amino acid sequence of CDR1 in HuGAB11 VL

[0442] HASQNINVWLS

[0443] SEQ ID NO:38

[0444] The amino acid sequence of CDR2 in HuGAB11 VL

[0445] KASNLHT

[0446] SEQ ID NO:39

[0447] The amino acid sequence of CDR3 in HuGAB11 VL

[0448] QQGQSYPLT

[0449] SEQ ID NO:40

[0450] Amino acid sequence of human GITR

[0451] MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTF SGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0452] SEQ ID NO:41

[0453] The amino acid sequence of HuGAB11.scFv

[0454] DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGG GSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0455] SEQ ID NO:42

[0456] The amino acid sequence encoded in pBS827 that is the fusion of CH3 with HuGAB11.scFv (CH3-HuGAB11.scFv).

[0457] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0458] SEQ ID NO:43

[0459] The amino acid sequence of the mature heavy chain encoded in pBS827

[0460] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0461] SEQ ID NO:44

[0462] Amino acid sequence of the mature light chain encoded in pBS827

[0463] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0464] SEQ ID NO:45

[0465] amino acid sequence of mature human GITR

[0466] QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTCTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTD CTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0467] SEQ ID NO:46

[0468] The amino acid sequence encoding the extracellular region of human EGFR in pFCm507

[0469] LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0470] SEQ ID NO:47

[0471] Amino acid sequence of hCD40-Fc

[0472] EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0473] SEQ ID NO:48

[0474] Amino acid sequence of HuACS2 VH

[0475] MKLWLNWVFLLTLLHGIQCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0476] SEQ ID NO:49

[0477] ...... Amino acid sequence of CDR1 of HuACS2 VH

[0478] SYWLH

[0479] SEQ ID NO:50

[0480] Amino acid sequence of CDR2 of HuACS2 VH

[0481] RIDPNSGDTKYNEKFKS

[0482] SEQ ID NO:51

[0483] Amino acid sequence of CDR3 of HuACS2 VH

[0484] YYYGRSYFDY

[0485] SEQ ID NO:52

[0486] The amino acid sequence of HuACS2 VL

[0487] MDFQVQIFSFLLISAVIISRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIK

[0488] SEQ ID NO:53

[0489] The amino acid sequence of CDR1 in HuACS2 VL

[0490] SASSSVSYMH

[0491] SEQ ID NO:54

[0492] The amino acid sequence of CDR2 in HuACS2 VL

[0493] DTSKLAS

[0494] SEQ ID NO:55

[0495] The amino acid sequence of CDR3 in HuACS2 VL

[0496] QQWSSNPLT

[0497] SEQ ID NO:56

[0498] Amino acid sequence of human CD40

[0499] MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0500] SEQ ID NO:57

[0501] Amino acid sequence of HuACS2.scFv

[0502] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0503] SEQ ID NO:58

[0504] Amino acid sequence of the CH3 encoded in pBS828 fused with HuACS2.scFv (CH3-HuACS.scFv)

[0505] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0506] SEQ ID NO:59

[0507] Amino acid sequence of the mature heavy chain encoded in pBS82

[0508] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0509] SEQ ID NO:60

[0510] Amino acid sequence of hPD-L1-Fc

[0511] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0512] SEQ ID NO:61

[0513] Amino acid sequence of HuPRO1 VH

[0514] MEWNWVVLFLLSLTAGVYAQVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSS

[0515] SEQ ID NO:62

[0516] Amino acid sequence of CDR1 of HuPRO1 VH

[0517] SSYIS

[0518] SEQ ID NO:63

[0519] Amino acid sequence of CDR2 of HuPRO1 VH

[0520] WIYAGTGGTSYNQKFTG

[0521] SEQ ID NO:64

[0522] The amino acid sequence of CDR3 in HuPRO1 VH

[0523] HEGVYWYFDV

[0524] SEQ ID NO:65

[0525] The amino acid sequence of HuPRO1 VL

[0526] MDFQVQIFSFLLISAVIMSRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK

[0527] SEQ ID NO:66

[0528] The amino acid sequence of CDR1 in HuPRO1 VL

[0529] SASSSVSYMH

[0530] SEQ ID NO:67

[0531] The amino acid sequence of CDR2 in HuPRO1 VL

[0532] DTSNLAS

[0533] SEQ ID NO:68

[0534] The amino acid sequence of CDR3 in HuPRO1 VL

[0535] HQRSSYPWT

[0536] SEQ ID NO:69

[0537] Amino acid sequence of human PD-L1

[0538] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

[0539] SEQ ID NO:70

[0540] Amino acid sequence of the mature heavy chain encoded in pBS809

[0541] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0542] SEQ ID NO:71

[0543] The amino acid sequences of the mature light chains encoded in pBS809 and pBS813

[0544] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0545] SEQ ID NO:72

[0546] The amino acid sequence of the mature heavy chain encoded in pBS813

[0547] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0548] SEQ ID NO:73

[0549] Amino acid sequence of the extracellular region of human PD-L1

[0550] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTH

[0551] SEQ ID NO:74

[0552] The amino acid sequence of HuPRO2 VH

[0553] MGWNWIFLFLSGTAGVHCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSS

[0554] SEQ ID NO:75

[0555] The amino acid sequence of CDR1 in HuPRO2 VH

[0556] SYGIN

[0557] SEQ ID NO:76

[0558] The amino acid sequence of CDR2 in HuPRO2 VH

[0559] YIYPGSGGPVYNQKFKG

[0560] SEQ ID NO:77

[0561] The amino acid sequence of CDR3 of HuPRO2 VH

[0562] ENYRYWYFDV

[0563] SEQ ID NO:78

[0564] The amino acid sequence of HuPRO2 VL

[0565] MHFQVQIFSFLLISASVIMSRGDIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK

[0566] SEQ ID NO:79

[0567] The amino acid sequence of CDR1 in HuPRO2 VL

[0568] SASSSVNYMH

[0569] SEQ ID NO:80

[0570] The amino acid sequence of CDR2 in HuPRO2 VL

[0571] STSNLAS

[0572] SEQ ID NO:81

[0573] The amino acid sequence of CDR3 in HuPRO2 VL

[0574] QQRSSYPLT

[0575] SEQ ID NO:82

[0576] The amino acid sequence of the mature heavy chain encoded in pBS841

[0577] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0578] SEQ ID NO:83

[0579] Amino acid sequence of the mature light chain encoded in pBS841

[0580] DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0581] SEQ ID NO:84

[0582] The amino acid sequence of HuPRO5 VH

[0583] MMVLSLLYLLTALPGILSQVQLQESGPGLVKPSQTLSLTCTVSGDSISSGY

[0584] WNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLS

[0585] SVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSS

[0586] SEQ ID NO:85

[0587] The amino acid sequence of CDR1 in HuPRO5 VH

[0588] SGYWN

[0589] SEQ ID NO:86

[0590] The amino acid sequence of CDR2 in HuPRO5 VH

[0591] YISYTGSTYSNPSLKS

[0592] SEQ ID NO:87

[0593] The amino acid sequence of CDR3 in HuPRO5 VH

[0594] SQNWERAWFAY

[0595] SEQ ID NO:88

[0596] The amino acid sequence of HuPRO5 VL

[0597] MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK

[0598] SEQ ID NO:89

[0599] The amino acid sequence of CDR1 in HuPRO5 VL

[0600] SASSSVSYMH

[0601] SEQ ID NO:90

[0602] The amino acid sequence of CDR2 in HuPRO5 VL

[0603] DTSKLAS

[0604] SEQ ID NO:91

[0605] The amino acid sequence of CDR3 in HuPRO5 VL

[0606] FQGSGYPFT

[0607] SEQ ID NO:92

[0608] The amino acid sequence of the mature heavy chain encoded in pBS839

[0609] QVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0610] SEQ ID NO:93

[0611] Amino acid sequence of the mature light chain encoded in pBS839

[0612] DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0613] SEQ ID NO:94

[0614] The amino acid sequence of CDR1 of HuM195 VH

[0615] DYNMH

[0616] SEQ ID NO:95

[0617] The amino acid sequence of CDR2 in HuM195 VH

[0618] YIYPYNGGTGYNQKFKS

[0619] SEQ ID NO:96

[0620] The amino acid sequence of CDR3 in HuM195 VH

[0621] GRPAMDY

[0622] SEQ ID NO:97

[0623] The amino acid sequence of CDR1 in HuM195 VL

[0624] RASESVDNYGISFMN

[0625] SEQ ID NO:98

[0626] The amino acid sequence of CDR2 in HuM195 VL

[0627] AASNQGS

[0628] SEQ ID NO:99

[0629] The amino acid sequence of CDR3 in HuM195 VL

[0630] QQSKEVPWT

[0631] SEQ ID NO:100

[0632] The amino acid sequence of CDR1 at Ch225 VH

[0633] NYGVH

[0634] SEQ ID NO:101

[0635] The amino acid sequence of CDR2 in Ch225 VH

[0636] VIWSGGNTDYNTPFTS

[0637] SEQ ID NO:102

[0638] The amino acid sequence of CDR3 in Ch225 VH

[0639] ALTYYDYEFAY

[0640] SEQ ID NO:103

[0641] The amino acid sequence of CDR1 in Ch225 VL

[0642] RASQSIGTNIH

[0643] SEQ ID NO:104

[0644] The amino acid sequence of CDR2 in Ch225 VL

[0645] YASESIS

[0646] SEQ ID NO:105

[0647] The amino acid sequence of CDR3 in Ch225 VL

[0648] QQNNNWPTT

[0649] SEQ ID NO:106

[0650] The amino acid sequence of the mature heavy chain encoded in pBS853

[0651] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0652] SEQ ID NO:107

[0653] Amino acid sequence of hICOS-Fc

[0654] GEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0655] SEQ ID NO:108

[0656] Amino acid sequence of HuTAM14 VH

[0657] MAVLVLLLCLVTFPSCALSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0658] SEQ ID NO:109

[0659] Amino acid sequence of CDR1 of HuTAM14 VH

[0660] SNSVS

[0661] SEQ ID NO:110

[0662] Amino acid sequence of CDR2 of HuTAM14 VH

[0663] AIWSGGSTDYNSALKS

[0664] SEQ ID NO:111

[0665] Amino acid sequence of CDR3 of HuTAM14 VH

[0666] WEQPYYFDY

[0667] SEQ ID NO:112

[0668] The amino acid sequence of HuTAM14 VL

[0669] MRTSIQLLGLLLFWLHDAQCDIQMTQSPSSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIK

[0670] SEQ ID NO:113

[0671] The amino acid sequence of CDR1 in HuTAM14 VL

[0672] QASQNIYKYIA

[0673] SEQ ID NO:114

[0674] The amino acid sequence of CDR2 in HuTAM14 VL

[0675] YTSTLES

[0676] SEQ ID NO:115

[0677] The amino acid sequence of CDR3 in HuTAM14 VL

[0678] LQYVNLYT

[0679] SEQ ID NO:116

[0680] Amino acid sequence of human ICOS

[0681] MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

[0682] SEQ ID NO:117

[0683] The amino acid sequence of HuTAM14.scFv

[0684] DIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0685] SEQ ID NO:118

[0686] Amino acid sequence of CH3 encoded in pBS859 fused with HuTAM14.scFv (CH3-HuTAM14.scFv)

[0687] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0688] SEQ ID NO:119

[0689] Amino acid sequence of the mature heavy chain encoded in pBS859

[0690] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0691] SEQ ID NO:120

[0692] Amino acid sequence of the mature heavy chain encoded in pBS840

[0693] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0694] SEQ ID NO:121

[0695] Amino acid sequence of the mature heavy chain encoded in pBS846

[0696] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0697] SEQ ID NO:122

[0698] Amino acid sequence of HuOHX14DS.scFv.ds

[0699] DIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQCLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0700] SEQ ID NO:123

[0701] Amino acid sequence of HuACS2.scFv.ds

[0702] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQCLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0703] SEQ ID NO:124

[0704] Amino acid sequence of HuTAM14.scFv.ds

[0705] DIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKCLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0706] SEQ ID NO:125

[0707] Amino acid sequence of HuGAB11.scFv.ds

[0708] DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGCGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKCLEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0709] SEQ ID NO:126

[0710] Amino acid sequence of h4-1BB-Fc

[0711] FERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0712] SEQ ID NO:127

[0713] Amino acid sequence of HuFOB5 VH

[0714] MERHWIFLFLFSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQGLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0715] SEQ ID NO:128

[0716] The amino acid sequence of CDR1 in HuFOB5 VH

[0717] NYWMH

[0718] SEQ ID NO:129

[0719] The amino acid sequence of CDR2 of HuFOB5 VH

[0720] YINPSTGYTESNQKFKD

[0721] SEQ ID NO:130

[0722] The amino acid sequence of CDR3 in HuFOB5 VH

[0723] SYVGYYYAVDY

[0724] SEQ ID NO:131

[0725] The amino acid sequence of HuFOB5 VL

[0726] MDSQAQVLMLLLLWVSGTCGDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK

[0727] SEQ ID NO:132

[0728] The amino acid sequence of CDR1 in HuFOB5 VL

[0729] KSSQSLLYSNNEKNYLA

[0730] SEQ ID NO:133

[0731] The amino acid sequence of CDR2 in HuFOB5 VL

[0732] WASTRES

[0733] SEQ ID NO:134

[0734] The amino acid sequence of CDR3 in HuFOB5 VL

[0735] QQYYSYPYT

[0736] SEQ ID NO:135

[0737] The amino acid sequence of human 4-1BB

[0738] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFND QKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0739] SEQ ID NO:136

[0740] The amino acid sequence of HuFOB5.scFv.LH.ds

[0741] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGS GGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0742] SEQ ID NO:137

[0743] The amino acid sequence encoding the CH3-HuFOB5.scFv.LH.ds fusion (CH3-HuFOB5.scFv.LH.ds) in pBS883.

[0744] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0745] SEQ ID NO:138

[0746] Amino acid sequence of the mature heavy chain encoded in pBS883

[0747] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0748] SEQ ID NO:139

[0749] Amino acid sequence of HuFOB5.scFv.HL.ds

[0750] QVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0751] SEQ ID NO:140

[0752] The amino acid sequence of CH3 encoded in pBS884 fused with HuFOB5.scFv.HL.ds (CH3-HuFOB5.scFv.HL.ds)

[0753] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0754] SEQ ID NO:141

[0755] The amino acid sequence of the mature heavy chain encoded in pBS884

[0756] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0757] SEQ ID NO:142

[0758] Amino acid sequence of human PD-1

[0759] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0760] SEQ ID NO:143

[0761] amino acid sequence of hPD-1-Fc

[0762] LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM Sequence Listing <110> JN Biosciences Co., Ltd. Naoya Tsurushita J. Yun Cao <120> Bispecific antibodies that activate immune cells <130> PCT 057761-530874 <150> US 62 / 760,328 <151> 2018-11-13 <150> US 62 / 838,579 <151> 2019-04-25 <160> 143 <170> PatentIn version 3.5 <210> 1 <211> 364 <212> PRT <213> Homo sapiens <400> 1 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln 20 25 30 Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro 35 40 45 Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly 50 55 60 Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln 65 70 75 80 Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro 85 90 95 Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp 100 10​​​​ Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg 130 135 140 Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn 145 150 155 160 Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile 165 170 175 Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr 180 185 190 His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr 195 200 205 Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu 210 215 220 Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr 225 230 235 240 Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly 245 250 255 Val Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu Leu Ala 260 265 270 Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr His Arg Arg Lys 275 280 285 Ala Ala Arg Thr Ala Val Gly Arg Asn Asp Thr His Pro Thr Thr Gly 290 295 300 Ser Ala Ser Pro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr 305 310 315 320 Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu 325 330 335 Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys 340 345 350 Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln 355 360 <210> 2 <211> 135 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 2 Met Gly Trp Ser Trp Ile Phe Phe Phe Leu Leu Ser Gly Thr Ala Ser 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn 65 70 75 80 Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val<{ 100 105 110 Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ser<{ 130 135 <210> 3 <21{}> 98 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 It should be noted that there seems to be an error in the original text where <21{}> is present. It should probably be <211>. The above translation is based on the corrected understanding as much as possible. If there are other specific requirements or corrections, please let me know.Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 4 <211> 15 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 4 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 5 <211> 110 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 5 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 6 <211> 107 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 6 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 7 <211> 131 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 7 Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser 35 40 45 Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro 50 55 60 Gly Gly Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys 100 105 110 Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 115 120 125 Glu Ile Lys 130 <210> 8 <211> 107 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 8 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 9 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 9 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 10 <211> 37 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 10 Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser 1 5 10 15 Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr 20 25 30 Met Gly Leu Leu Thr 35 <210> 11 <211> 238 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 11 Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His 1 5 10 15 Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln 20 25 30 Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val 35 40 45 Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly 50 55 60 Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg 65 70 75 80 Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp 85 90 95 Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala 100 105 110 Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln 115 120 125 Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro 130 135 140 Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr 145 150 155 160 Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr 165 170 175 Arg Pro Val Glu Val Pro Gly Gly Arg Ala Thr Gly Gly Gly Asp Tyr 180 185 190 Lys Asp Asp Asp Asp Lys Gly Gly Gly Pro Asn Lys Gly Ser Gly Thr 195 200 205 Thr Ser Gly Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu 210 215 220 Thr Gly Leu Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr 225 230 235 <210> 12 <211> 138 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 12 Met Gly Arg Leu Thr Ser Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 [[ID=A1]] Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys Tyr Asn 65 70 7,5 80 Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ser Asp Lys Ser Thr Ser ​Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala His Tyr Tyr Gly Ser Thr Phe Thr Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 <210> 13 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 13 Ser Tyr Ile Met His 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 14 Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 15 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 15 Tyr Tyr Gly Ser Thr Phe Thr Met Asp Tyr 1 5 10 <210> 16 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 16 Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln 1 5 10 15 Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Ile Arg Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro 50 55 60 Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn 100 105 110 Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 <210> 17 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 17 Arg Ala Ser Gln Asp Ile Arg Thr Tyr Leu Asn 1 5 10 <210> 18 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 18 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 19 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 20 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 20 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 21 <211> 277 <212> PRT <213> Homo sapiens <400> 21 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270 Thr Leu Ala Lys Ile 275 <210> 22 <211> 241 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Thr Tyr 20 2:5 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Ile Met His Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr 165 170 175 Asn Ser Gly Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile 180 185 190 Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 195 200 205 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala His Tyr Tyr Gly Ser 210 215 220 Thr Phe Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 225 230 235 240 Ser [[ID=3‌8]]<210> 23 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 23 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 115 120 125 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 130 135 140 Asp Ile Arg Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val 145 150 155 160 Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro 165 170 175 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 180 185 190 Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly 195 200 205 Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 225 230 235 240 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 245 250 255 Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Ile 260 265 270 Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 275 280 285 Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys Tyr Asn Glu Lys Phe Lys 290 295 300 Gly Arg Val Thr Ile Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr Met 305 310 315 320 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 325 330 335 His Tyr Tyr Gly Ser Thr Phe Thr Met Asp Tyr Trp Gly Gln Gly Thr 340 345 350 Thr Val Thr Val Ser Ser 355 <210> 24 <211> 697 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 24 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 450 455 460 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 465 470 475 480 Ala Ser Gln Asp Ile Arg Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 485 490 495 Gly Lys Val Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser 500 505 510 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 515 520 525 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys 530 535 540 Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val 545 550 555 560 Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 565 570 575 Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 580 585 590 Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 595 600 605 Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 610 615 620 Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys Tyr Asn Glu 625 630 635 640 Lys Phe Lys Gly Arg Val Thr Ile Thr Ser Asp Lys Ser Thr Ser Thr 645 650 655 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 660 665 670 Tyr Cys Ala His Tyr Tyr Gly Ser Thr Phe Thr Met Asp Tyr Trp Gly 675 680 685 Gln Gly Thr Thr Val Thr Val Ser Ser 690 695 <210> 25 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gly Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 脯氨酸-精氨酸-谷氨酸-丙氨酸-赖氨酸-缬氨酸-谷氨酰胺-色氨酸-赖氨酸-缬氨酸-天冬酰胺-丙氨酸-亮氨酸-谷氨酰胺-丝氨酸 145 150 155 160 甘氨酸-天冬酰胺-丝氨酸-谷氨酰胺-谷氨酸-丝氨酸-缬氨酸-苏氨酸-谷氨酸-谷氨酰胺-天冬氨酸-丝氨酸-赖氨酸-天冬氨酸-丝氨酸-苏氨酸 165 170 175 酪氨酸-丝氨酸-亮氨酸-丝氨酸-丝氨酸-苏氨酸-亮氨酸-苏氨酸-亮氨酸-丝氨酸-赖氨酸-丙氨酸-天冬氨酸-酪氨酸-谷氨酸-赖氨酸 180 185 190 组氨酸-赖氨酸-缬氨酸-酪氨酸-丙氨酸-半胱氨酸-谷氨酸-缬氨酸-苏氨酸-组氨酸-谷氨酰胺-甘氨酸-亮氨酸-丝氨酸-丝氨酸-脯氨酸 195 200 205 缬氨酸-苏氨酸-赖氨酸-丝氨酸-苯丙氨酸-天冬酰胺-精氨酸-甘氨酸-谷氨酸-半胱氨酸 210 215 <210> 26 <211> 19 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 26 甲硫氨酸-甘氨酸-色氨酸-丝氨酸-色氨酸-异亮氨酸-苯丙氨酸-苯丙氨酸-苯丙氨酸-亮氨酸-亮氨酸-丝氨酸-甘氨酸-苏氨酸-丙氨酸-丝氨酸 1 5 10 15 缬氨酸-亮氨酸-丝氨酸 <210> 27<o002298><211> 243 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 27 甲硫氨酸-天冬氨酸-脯氨酸-天冬酰胺-苯丙氨酸-色氨酸-亮氨酸-谷氨酰胺-缬氨酸-谷氨酰胺-谷氨酸-丝氨酸-缬氨酸-苏氨酸-缬氨酸-谷氨酰胺 1 5 10 15 Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro 20 25 30 Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly 35 40 45 Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln 50 55 60 Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro 65 70 75 80 Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp 85 90 95 Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser 100 105 110 Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg 115 120 125 Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn 130 135 140 Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile 145 150 155 160 Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr 165 170 175 His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr 180 185 190 Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu 195 200 205 Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr 210 215 220 Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly 225 230 235 240 Val Val His <210> 28 <211> 1210 <212> PRT <213> Homo sapiens <400> 28 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795 800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875 880 Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910 Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe 1010 1015 1020 Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035 Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn 1040 1045 1050 Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg 1055 1060 1065 Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp 1070 1075 1080 Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro 1085 1090 1095 Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln 1100 1105 1110 Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro 1115 1120 1125 His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln 1130 1135 1140 Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala 1145 1150 1155 Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 1160 1165 1170 Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys 1175 1180 1185 Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln 1190 1195 1200 Ser Ser Glu Phe Ile Gly Ala 1205 1210 <210> 29 <211> 138 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 29 Met Ala Val Leu Ala Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln 20 25 30 Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr 65 70 75 80 Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr 100 105 110 Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 130 135 <210> 30 <211> 127 <212> PRT <213> Synthetic sequence <220><{0002524}<223> Synthetic <400> 30 Met Arg Ala Pro Ala Gln Phe Leu Gly Phe Leu Leu Phe Trp Ile Pro 1 5 10 15 Ala Ser Arg Ser Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser 20 25 30 Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser 35 40 45 Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro 50 55 60 Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser 65 70 75 80 Note: The 7-digit tag was written as <{0002524} in the original, and I've corrected it to in the translation for consistency. If this was an intentional variation in the original that should be preserved, let me know and I'll adjust accordingly. Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 85 90 95 Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn 100 105 110 Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 <210> 31 <211> 375 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 31 Gln Arg Pro Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu 1 5 10 15 Gly Thr Gly Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys 20 25 30 Cys Arg Asp Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met 35 40 45 Cys Val Gln Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys 50 55 60 Arg His His Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys 65 70 75 80 Phe Ser Phe Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser 85 90 95 Gly Gly His Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe 100 105 110 Gly Phe Leu Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys 115 120 125 Val Pro Gly Ser Pro Pro Ala Glu Pro Leu Gly Thr Gly Gly Gly Glu 130 135 140 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 32 <211> 137 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 32 Met Ala Val Leu Gly Leu Leu Leu Cys Leu Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys 20 25 30 Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu [[ID=X]]35 40 45 Thr Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr Asn Ser 65 70 75 80 Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln 85 90 95 Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr 100 105 110 Tyr Cys Ala Lys His Pro Tyr Gly His Phe Gly Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 33 Asp Tyr Gly Val Ser 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 34 Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 <210> 35 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 35 His Pro Tyr Gly His Phe Gly Met Asp Tyr 1 5 10 <210> 36 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 36 Met Arg Val Leu Ala Glu Leu Leu Gly Leu Leu Leu Phe Cys Phe Leu 1 5 10 15 Gly Val Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn 35 40 45 Ile Asn Val Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro 50 55 60 Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Gln 100 105 110 Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 <210> 37 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 37 His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser 1 5 10 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 38 Lys Ala Ser Asn Leu His Thr 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 39 Gln Gln Gly Gln Ser Tyr Pro Leu Thr 1 5 <210> 40 <211> 241 <212> PRT <213> Homo sapiens <400> 40 Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu 1 5 10 15 Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro 20 25 30 Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg 35 40 45 Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu 50 55 60 Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His 65 70 75 80 Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro 85 90 95 Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys 100 105 110 Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys 115 120 125 Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro 130 135 140 Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala 145 150 155 160 Glu Pro Leu Gly Trp Leu Thr Val Val Leu Leu Ala Val Ala Ala Cys 165 170 175 Val Leu Leu Leu Thr Ser Ala Gln Leu Gly Leu His Ile Trp Gln Leu 180 185 190 Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln Leu Leu Leu Glu Val 195 200 205 Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln Phe Pro Glu Glu Glu 210 215 220 Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg Leu Gly Asp Leu Trp 225 230 235 240 Val <210> 41 <211> 240 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 41 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Thr Leu Lys Glu 115 120 125 Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys 130 135 140 Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr Gly Val Ser Trp Ile Arg 145 150 155 160 Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly Val Ile Trp Gly Gly 165 170 175 Gly Gly Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser 180 185 190 Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp 195 200 205 Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Pro Tyr Gly His 210 215 220 Phe Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 225 230 235 240 <210> 42 <211> 357 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 42 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 115 120 125 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln 130 135 140 Asn Ile Asn Val Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val 145 150 155 160 Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro 165 170 175 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 180 185 190 Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly 195 200 205 Gln Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 225 230 235 240 Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr 245 250 255 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr Gly 260 265 270 Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly 275 280 285 Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 290 295 300 Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu Thr 305 310 315 320 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys 325 330 335 His Pro Tyr Gly His Phe Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr 340 345 350 Val Thr Val Ser Ser<able> 355 <210> 43 <211> 699 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 43 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 450 455 460 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 465 470 475 480 Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser Trp Tyr Gln 485 490 495 Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn 500 505 510 Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 515 520 525 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr 530 535 540 Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu Thr Phe Gly Gly Gly 545 550 555 560 Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu 580 585 590 Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe 595 600 605 Ser Leu Thr Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 610 615 620 Ala Leu Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr 625 630 635 640 Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 645 650 655 Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala 660 665 670 Thr Tyr Tyr Cys Ala Lys His Pro Tyr Gly His Phe Gly Met Asp Tyr 675 680 685 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 690 695 <210> 44 <211> 214 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 44 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 45 <211> 216 <212> PRT <213> Homo sapiens <400> 45 Gln Arg Pro Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu 1 5 10 15 Gly Thr Gly Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys 20 25 30 Cys Arg Asp Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met 35 40 45 Cys Val Gln Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys 50 55 60 Arg His His Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys 65 70 75 80 Phe Ser Phe Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser 85 90 95 Gly Gly His Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe 100 105 110 Gly Phe Leu Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys 115 120 125 Val Pro Gly Ser Pro Pro Ala Glu Pro Leu Gly Trp Leu Thr Val Val 130 135 140 Leu Leu Ala Val Ala Ala Cys Val Leu Leu Leu Thr Ser Ala Gln Leu 145 150 155 160 Gly Leu His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro Arg Glu 165 170 175 Thr Gln Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala Arg Ser 180 185 190 Cys Gln Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu Glu Lys 195 200 205 Gly Arg Leu Gly Asp Leu Trp Val 210 215 <210> 46 <211> 621 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 46 Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln 1 5 10 15 Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn 20 25 30 Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg 35 40 45 Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr 50 55 60 Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu 65 70 75 80 Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala 85 90 95 Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro 100 105 110 Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn 115 120 125 Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val 130 135 140 Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu 145 150 155 160 Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp 165 170 175 Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala 180 185 190 Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys 195 200 205 His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys 210 215 220 Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys 225 230 235 240 Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn 245 250 255 Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro 260 265 270 Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly 275 280 285 Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys 290 295 300 Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu 305 310 315 320 Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys 325 330 335 Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe 340 345 350 Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu 355 360 365 Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln 370 375 380 Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu 385 390 395 400 Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val 405 410 415 Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile 420 425 430 Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala 435 440 445 Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr 450 455 460 Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln 465 470 475 480 Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro 485 490 495 Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val 500 505 510 Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn 515 520 525 Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn 530 535 540 Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His 545 550 555 560 Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met 565 570 575 Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val 580 585 590 Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly 595 600 605 Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser 610 615 620 <210> 47 <211> 409 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 47 Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln 1 5 10 15 Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr 20 25 30 Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu 35 40 45 Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp 50 55 60 Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp 65 70 75 80 Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys 85 90 95 Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys 100 105 110 Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val 115 120 125 Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp 130 135 140 Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn 145 150 155 160 Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Thr Gly Gly 165 170 175 Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 180 185 190 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 195 200 205 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 210 215 220 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 225 230 235 240 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 245 250 255 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 260 265 270 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 275 280 285 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 290 295 300 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 305 310 315 320 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 325 330 335 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 340 345 350 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 355 360 365 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 370 375 380 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 385 390 395 400 Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 <210> 48 <211> 138 <212> PRT <213> Synthetic sequence <220> <I223> Synthetic <400> 48 Met Lys Leu Trp Leu Asn Trp Val Phe Leu Leu Thr Leu Leu His Gly 1 5 10 15 Ile Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Arg Ile Asp Pro Asn Ser Gly Asp Thr Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Ser Arg Ala Thr Ile Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Tyr Tyr Tyr Gly Arg Ser Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 <210> 49 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 49 Ser Tyr Trp Leu His 1 5 <210> 50 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 50 Arg Ile Asp Pro Asn Ser Gly Asp Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 51 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 51 Tyr Tyr Tyr Gly Arg Ser Tyr Phe Asp Tyr 1 5 10 <210> 52 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 52 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Val 1 5 10 15 Ile Ile Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 20 25 30 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 35 40 45 Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser 100 105 110 Ser Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 <210> 53 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 53 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 54 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 54 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 55 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 55 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <210> 56 <211> 277 <212> PRT <213> Human beings (Homo sapiens) <400> 56 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr 100 105 110 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly 115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215 220 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235 240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His 245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser 260 265 270 Val Gln Glu Arg Gln<000327​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser 115 120 125 Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys 130 135 140 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Leu His Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Asn Ser 165 170 175 Gly Asp Thr Lys Tyr Asn Glu Lys Phe Lys Ser Arg Ala Thr Ile Thr 180 185 190 Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg 195 200 205 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Tyr Gly Arg 210 215 220 Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 225 230 235 240 <210> 58 <211> 357 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 58 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 115 120 125 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 130 135 140 Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 145 150 155 160 Arg Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala 165 170 175 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 180 185 190 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser 195 200 205 Ser Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly 210 215 220 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 225 230 235 240 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val 245 250 255 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Leu 260 265 270 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg 275 280 285 Ile Asp Pro Asn Ser Gly Asp Thr Lys Tyr Asn Glu Lys Phe Lys Ser 290 295 300 Arg Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 305 310 315 320 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 325 330 335 Tyr Tyr Tyr Gly Arg Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 340 345 350 Val Thr Val Ser Ser 355 <210> 59 <211> 696 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 59 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro 450 455 460 Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser 465 470 475 480 Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly 485 490 495 Gln Ala Pro Arg Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly 500 505 510 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu 515 520 525 Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 530 535 540 Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu 545 550 555 560 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 565 570 575 Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 580 585 590 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser 595 600 605 Tyr Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 610 615 620 Ile Gly Arg Ile Asp Pro Asn Ser Gly Asp Thr Lys Tyr Asn Glu Lys 625 630 635 640 Phe Lys Ser Arg Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala 645 650 655 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 660 665 670 Cys Ala Arg Tyr Tyr Tyr Gly Arg Ser Tyr Phe Asp Tyr Trp Gly Gln 675 680 685 Gly Thr Thr Val Thr Val Ser Ser 690 695 <210> 60 <211> 458 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 60 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80[[ID=4l]] Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His Thr Gly 210 215 220 Gly Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 225 230 235 240 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 340 345 350 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 355 360 365 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 420 425 430 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 61 <211> 138 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 61 Met Glu Trp Asn Trp Val Val Leu Phe Leu Leu Ser Leu Thr Ala Gly 1 5 10 15 Val Tyr Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Ser Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Ala Trp Ile Tyr Ala Gly Thr Gly Gly Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Thr Gly Arg Ala Thr Ile Thr Val Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Glu Gly Val Tyr Trp Tyr Phe Asp Val Trp 115 120 125 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 <210> 62 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 62 Ser Ser Tyr Ile Ser 1 5 <210> 63 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 63 Trp Ile Tyr Ala Gly Thr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Thr 1 5 10 15 Gly <210> 64 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 64 His Glu Gly Val Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 65 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 65 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Val 1 5 10 15 Ile Met Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 20 25 30 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 35 40 45 Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Pro Trp Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Phe Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Arg Ser 100 105 110 Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 <210> 66 <211> 10 <212> PRT <213> Synthetic sequence(这里的“人造序列”翻译为“Synthetic sequence”更符合专利文本的习惯表达) <220> <223> Synthetic <400> 66 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 67 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 67 Asp Thr Ser Asn Leu Ala Ser 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 68 His Gln Arg Ser Ser Tyr Pro Trp Thr 1 5 <210> 69 <211> 290 <212> PRT <213> Human beings (Homo sapiens) <400> 69 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> 70 <211> 699 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 70 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Tyr Ala Gly Thr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Thr Gly Arg Ala Thr Ile Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Glu Gly Val Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 450 455 460 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 465 470 475 480 Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser Trp Tyr Gln 485 490 495 Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn 500 505 510 Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 515 520 525 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr 530 535 540 Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu Thr Phe Gly Gly Gly 545 550 555 560 Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu 580 585 590 Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe 595 600 605 Ser Leu Thr Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys 610 615 620 Ala Leu Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr 625 630 635 640 Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 645 650 655 Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala 660 665 670 Thr Tyr Tyr Cys Ala Lys His Pro Tyr Gly His Phe Gly Met Asp Tyr 675 680 685 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 690 695 <210> 71 <211> 213 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 71 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Phe Pro Ala Arg Phe Ser Gly Ser 50 55 600] Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Glq Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 72 <211> 700 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 72 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Ser 20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Tyr Ala Gly Thr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Thr Gly Arg Ala Thr Ile Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Glu Gly Val Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 450 455 460 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 465 470 475 480 Thr Cys Arg Ala Ser Gln Asp Ile Arg Thr Tyr Leu Asn Trp Tyr Gln 485 490 495 Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg 500 505 510 Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 515 520 525 Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr 530 535 540 Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly 545 550 555 560 Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 580 585 590 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 595 600 605 Thr Phe Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln 610 615 620 Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys 625 630 635 640 Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ser Asp Lys Ser 645 650 655 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 660 665 670 Ala Val Tyr Tyr Cys Ala His Tyr Tyr Gly Ser Thr Phe Thr Met Asp 675 680 685 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 690 695 700 <210> 73 <211> 222 <212> PRT <213> Homo sapiens <400> 73 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15[[ID=2​​​​​​​​​​​​​​​​​​​​​​​​​​115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 210 215 220 <210> 74 <211> 137 <212> PRT <​​​​​​​​​​​​​​​Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 35 40 45 Ser Tyr Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 50 55 60 Trp Ile Gly Tyr Ile Tyr Pro Gly Ser Gly Gly Pro Val Tyr Asn Gln 65 70 75 80 Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr 85 90 95 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Trp Tyr Phe Asp Val Trp Gly 115 120 125 Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 <210> 75 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 75 Ser Tyr Gly Ile Asn 1 5 <210> 76 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 76 Tyr Ile Tyr Pro Gly Ser Gly Gly Pro Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 77 <211> 10 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 77 Glu Asn Tyr Arg Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 78 <211> 128 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 78 Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met His Trp Phe Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg 100 105 110 Ser Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 <210> 79 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 79 Ser Ala Ser Ser Ser Val Asn Tyr Met His 1 5 10 <210> 80 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 80 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 81 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 81 Gln Gln Arg Ser Ser Tyr Pro Leu Thr 1 5 <210> 82 <211> 700 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 82 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Gly Ser Gly Gly Pro Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asn Tyr Arg Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser...

Claims

1. A bispecific antibody comprising a first binding site that specifically binds to PD-L1 and a second binding site that specifically binds to GITR, wherein the first binding site comprises a mature heavy chain variable region comprising CDRs H1, H2, and H3 of SEQ ID NOs: 62-64, respectively, and a mature light chain variable region comprising CDRs L1, L2, and L3 of SEQ ID NOs: 66-68, respectively; and the second binding site comprises a mature heavy chain variable region comprising CDRs H1, H2, and H3 of SEQ ID NOs: 33-35, respectively, and a mature light chain variable region comprising CDRs L1, L2, and L3 of SEQ ID NOs: 37-39, respectively. The bispecific antibody according to claim 1 , further comprising an Fc region.

3. The bispecific antibody according to claim 1 or 2, wherein the first binding site antagonizes the binding of PD-L1 to PD1, and the second binding site agonizes GITR.

4. The bispecific antibody of claim 1, wherein the first binding site comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 61 and a mature light chain variable region comprising residues 23-137 of SEQ ID NO: 65; and the second binding site comprises a mature heavy chain variable region comprising residues 20-137 of SEQ ID NO: 32 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:

36.

5. The bispecific antibody of claim 1, wherein the first binding site comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 61 and a mature light chain variable region comprising residues 23-137 of SEQ ID NO: 65; and the second binding site comprises a single chain Fv fragment comprising SEQ ID NO: 41 or 125.

6. The bispecific antibody of claim 1, wherein the C-termini of the mature heavy chain variable region and the mature light chain variable region of the first binding site are fused to the N-termini of the heavy chain and light chain constant regions, and the mature heavy chain variable region and the mature light chain variable region of the second binding site form a scFv fused to the C-terminus of the heavy chain constant region.

7. The bispecific antibody according to claim 6, wherein the light chain variable region of the scFv is fused to the C-terminus of the heavy chain constant region.

8. The bispecific antibody of claim 6, wherein the first binding site and the second binding site are humanized, veneered or human, and the heavy and light chain constant regions are human. 9 . The bispecific antibody of claim 6 , wherein the isotype of the heavy chain constant region is human IgG1, and the isotype of the light chain constant region is κ.

10. The bispecific antibody of claim 1, comprising two first binding sites and two second binding sites. The bispecific antibody of claim 6 , wherein the heavy chain constant region has at least one mutation that reduces FcγR binding.

12. The bispecific antibody of claim 6, wherein the heavy chain constant region has at least one mutation that increases binding to FcRn.

13. A monoclonal antibody that specifically binds to PD-L1, comprising a mature heavy chain variable region including CDRH1, H2 and H3 of SEQ ID NOs: 62-64, respectively, and a mature light chain variable region including CDRL1, L2 and L3 of SEQ ID NOs: 66-68, respectively.

14. The monoclonal antibody of claim 13, comprising a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 61 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO:

65.

15. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

16. Use of a bispecific antibody as defined in any one of claims 1 to 12 in the preparation of a medicament for treating cancer, wherein the cancer is a solid tumor.

17. Use of a bispecific antibody as defined in any one of claims 1 to 12 in the preparation of a medicament for treating an infection.

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