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 of which binds to a co-stimulatory molecule, such as OX40, CD40, GITR, ICOS, or 4-1BB. Bridging of a bispecific antibody between a cancer cell expressing a cancer-associated antigen and an immune cell expressing a costimulatory molecule results in aggregation of the costimulatory molecule and selective activation of the immune cell in a location proximate to the cancer cell. Therefore, the immune cells can exert an immunotherapy effect on cancer cells, and toxicity to healthy tissues is reduced.

Description

Divisional Application Instructions

[0001] This application is a divisional application of the invention patent application with application date of November 12, 2019, application number 201980086014.0, and invention name “Bispecific antibodies for activating immune cells”. CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] This application includes a 214 KB Sequence Listing written in XML file 619852.XML, which is incorporated by reference. Background Art

[0004] Antigen-specific immune responses are complex biological processes that are controlled by multiple layers of positive and negative regulators. T cells are initially stimulated by 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 provided by co-stimulatory molecules of the CD28 superfamily (e.g., 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 a cell type and developmental stage-related manner and are negatively regulated by checkpoint molecules (e.g., PD-1, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD96, and CD112R). The function of checkpoint molecules is to prevent the human immune system from having an undesirable overreaction. However, they also limit the ability of the immune system to effectively fight 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.

[0005] It has been reported that antagonistic monoclonal antibodies that block the function of checkpoint molecules can effectively enhance the immune system (Mercier et al., supra; Baumeister et al., supra). 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 inhibits the body's immune response. The ligands of the checkpoint molecules expressed on cancer cells interact with their respective receptors on immune cells and weaken the immune response in the body, 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 U.S. 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, monoclonal IgG antibodies directed against other checkpoint molecules that block interaction with their respective ligands, including TIGIT, TIM-3, LAG-3, and VISTA, have been investigated for their therapeutic efficacy in clinical studies (Marin-Acevedo et al., J. Hematol. Oncol. 11:39, 2018).

[0006] Anti-CTLA-4 monoclonal IgG antibody (ipilimumab) has been used as a cancer therapeutic drug, which prevents the interaction of CTLA-4 on T cells with 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. Agonist IgG antibodies against co-stimulatory molecules (such as ICOS, CD40, OX40, GITR, CD27 and 4-1BB) 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 multimeric cross-linking on the surface of immune cells to initiate intracellular signal transduction to enhance immune responses (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). Since IgG is bivalent for antigen binding, unless the IgG molecule bound to the cell surface is conjugated, for example, by binding to an Fcγ receptor on another cell, it cannot effectively trigger signal transduction through these co-stimulatory molecules. In a mouse xenograft tumor model, an anti-CD40 IgG2 antibody with an Fc mutation to enhance binding to FcγRIIB (also known as CD32B) exhibited anti-tumor activity superior to the parental IgG2 antibody (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 human μ heavy chains at the C-terminus of human γ heavy chains (U.S. Patent No. 10,053,517). Such an engineered hexameric IgG antibody effectively crosslinks co-stimulatory molecules on the cell surface to trigger intracellular signal transduction.

[0007] Bispecific antibodies are artificially engineered antibodies that can bind to two different antigens or two different epitopes on the same antigen. Various forms of bispecific antibodies have been produced (Lameris et al., Crit. Rev. Oncol. Hematol. 92: 153-165, 2014; Kontermann et al., Drug Dicov. Today 20: 838-847, 2015; Spiess et al., Mol. Immunol. 67: 95-106, 2015; Brinkmann et al., MAbs 9: 182-212, 2017; Sedykh et al., Drug Des. Dev. Ther. 12: 195-208, 2018). Some bispecific antibodies, such as blinatumomab, which binds to both CD3 and CD19, are designed to bring T cells into proximity with CD19-bearing cancer cells and trigger T cell-mediated cytotoxicity against cancer cells (Viardot et al., Cancer Treat. Rev. 65:87-95, 2018). Summary of the invention

[0008] The present invention provides a bispecific antibody comprising a first binding site that specifically binds CD33 and a second binding site that specifically binds GITR, OX40, CD40, ICOS or 4-1BB. Optionally, the bispecific antibody further comprises an IgG Fc region. Optionally, the second binding site excites OX40 or CD40. Optionally, the first binding site comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 94-96, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 97-99, respectively, and the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 33-35, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 37-39, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 17-19, respectively; or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 49-51, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. NO.53-55 of CDRL1, L2 and L3; or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.109-111, respectively, and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.113-115, respectively; or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.128-130, respectively, and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.132-134, respectively.Optionally, the first binding site comprises a mature heavy chain variable region comprising residues 20 to 135 of SEQ ID NO:2 and a mature light chain variable region comprising residues 20 to 131 of SEQ ID NO:7, and the second binding region comprises a mature heavy chain variable region comprising residues 20 to 137 of SEQ ID NO:32 and a mature light chain variable region comprising residues 21 to 127 of SEQ ID NO:36, or the second binding region comprises a mature heavy chain variable region comprising residues 20 to 138 of SEQ ID NO:12 and a mature light chain variable region comprising residues 21 to 127 of SEQ ID NO:16, or the second binding region comprises a mature heavy chain variable region comprising residues 20 to 138 of SEQ ID NO:48 and a mature light chain variable region comprising residues 23 to 127 of SEQ ID NO:52, or the second binding region comprises a mature heavy chain variable region comprising residues 20 to 136 of SEQ ID NO:108 and a mature light chain variable region comprising residues 21 to 127 of SEQ ID NO:16. NO: 112, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 127 and a mature light chain variable region comprising residues 21-133 of SEQ ID NO: 131. Alternatively, the first binding site comprises a mature heavy chain variable region comprising residues 20 to 135 of SEQ ID NO: 2 and a mature light chain variable region comprising residues 20-131 of SEQ ID NO: 7, and the second binding region comprises a single chain Fv fragment comprising SEQ ID NO: 22, 41, 57, 117, 122, 123, 124, 125, 136 or 139.

[0009] The present invention further provides a first binding site that specifically binds to EGFR, and a second binding site that specifically binds to GITR, OX40, CD40, ICOS or 4-1BB. Optionally, the bispecific antibody further comprises an Fc region. Optionally, the first binding site antagonizes the binding of EGFR to EGF, and the second binding site antagonizes GITR. Optionally, the first binding site comprises a mature heavy chain variable region comprising CDRH1, H2 and H3 of SEQ ID NOs. 100-102, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 103-105, respectively, and the second binding region comprises a mature heavy chain variable region comprising CDRH1, H2 and H3 of SEQ ID NOs. 33-35, respectively, and a mature light chain variable region comprising CDRL1, L2 and L3 of SEQ ID NOs. 37-39, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDRL1, L2 and L3 of SEQ ID NOs. 17-19, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 49-51, respectively, and a mature light chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 53-55, respectively. The second binding region comprises a mature light chain variable region comprising CDRH1, H2 and H3 of SEQ ID NO.109-111, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NO.113-115, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NO.128-130, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NO.132-134, respectively.Optionally, the first binding site comprises a mature heavy chain variable region comprising residues 20 to 138 of SEQ ID NO:29 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:30, and the second binding region 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, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:16, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:48 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO:52, or the second binding region comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO:108 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: NO: 112, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 127 and a mature light chain variable region comprising residues 21-133 of SEQ ID NO: 131. Alternatively, the first binding site comprises a mature heavy chain variable region comprising residues 20 to 138 of SEQ ID NO: 29 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 30, and the second binding region comprises a single chain Fv fragment comprising SEQ ID No: 22, 41, 57, 117, 122, 123, 124, 125, 136 or 139.

[0010] The present invention further provides a monoclonal antibody comprising a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 17-19, respectively, or the monoclonal antibody comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 49-51, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 53-55, respectively, or the monoclonal antibody comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 109-111, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 113-115, respectively, or the monoclonal antibody comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 128-130, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. NO.132-134 of CDR L1, L2 and L3; optionally, the monoclonal antibody 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, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:16, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:48 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO:52, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO:108 and a mature light chain variable region comprising residues 21-126 of SEQ ID NO:112, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:48 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO:52, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO:108 and a mature light chain variable region comprising residues 21-126 of SEQ ID NO:112, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:112 The mature heavy chain variable region comprises residues 20-138 of SEQ ID NO:127 and the mature light chain variable region comprises residues 21-133 of SEQ ID NO:131.

[0011] 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 excites 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, respectively, 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, respectively, and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.37-39, respectively, or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.13-15, respectively, and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.17-19, respectively, or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.13-15, respectively, and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.17-19, respectively, 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 a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NO.109-111 and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NO.113-115, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NO.128-130 and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NO.132-134, respectively.Optionally, the first binding site comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:61, residues 20-138 of SEQ ID NO:74, residues 19-137 of SEQ ID NO:84, and a mature light chain variable region comprising 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 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, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:16, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:48 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:49. NO:52, or the second binding region comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO:108 and a mature light chain variable region comprising residues 21-126 of SEQ ID NO:112, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:127 and a mature light chain variable region comprising residues 21-133 of SEQ ID NO:131. Optionally, the first binding site comprises a mature heavy chain variable region comprising 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 comprising 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-chain Fv fragment comprising SEQ ID NO:22, 41, 57, 117, 122, 123, 124, 125, 136 or 139.

[0012] Some of the above-mentioned 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, and the heavy and light chain constant regions are human. In some bispecific antibodies, the isotype of the heavy chain constant region is human IgG1, and the isotype of the light chain constant region is κ. Some bispecific antibodies comprise 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.

[0013] The present invention further provides a bispecific antibody comprising a first binding site that specifically binds to an antigen on a cancer cell, a pathogen-infected cell or an immune cell and a second binding site that specifically binds to a co-stimulatory molecule, wherein a first pair of heavy chain and light chain variable regions form a first binding site, and a second pair of heavy chain and light chain variable regions form a second binding site, wherein the C-terminus of the first pair of heavy chain and light chain variable regions is fused to the N-terminus of the heavy chain and light chain constant regions, and the second pair of heavy chain 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 connected to the N-terminus of the heavy chain and light chain constant regions, and the second binding site is connected to the C-terminus of the heavy chain constant region.

[0014] The present invention further provides a monoclonal antibody that specifically binds to PD-L1, comprising a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NO. 62-64, 75-77 or 85-87, respectively, and a mature light chain variable region comprising 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 comprising 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 comprising 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.

[0015] The present invention further provides a pharmaceutical composition comprising any of the bispecific or monoclonal antibodies and a pharmaceutically acceptable carrier.

[0016] The present invention further provides a method of treating or achieving prevention of cancer, the method comprising administering to a subject suffering from or at risk of cancer an effective regimen of a bispecific or monoclonal antibody as defined above.

[0017] The invention further provides a method of treating an infection comprising administering to a subject suffering from or at risk of infection an effective regimen of a bispecific antibody or monoclonal antibody as defined above. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A-D : Schematic representation of the expression vectors pHuM195-IgG1 (A), pBS824 (B), pFCm331 (C) and pFCm267 (D). The relevant restriction enzyme sites are shown. The figures are not drawn to scale.

[0019] Figure 2 : Schematic structure of the bispecific IgG antibody of the present invention.

[0020] Figure 3 : Analysis of luciferase activity in Jurkat Dual cells (JD / OX40) expressing OX40 and harboring a bispecific antibody that binds CD33 and OX40 (BS824). The average relative luminescence units (RLU) of three replicate analyses are shown with standard deviation bars.

[0021] Figure 4: Analysis of luciferase activity in Jurkat dual cells (JD / GITR) with a bispecific antibody binding to EGFR and GITR (BS827) and expressing GITR. The mean relative luminescence units (RLU) of three replicate analyses are represented with standard deviation bars.

[0022] Figure 5 : FACS analysis of CD95 expression in Ramos cells with a bispecific antibody (BS828) that binds CD33 and CD40. The X-axis and Y-axis indicate the expression levels of CD20 and CD95, respectively. The numbers in the upper right quadrant and the lower right quadrant show the percentage of CD20-positive cells in each quadrant.

[0023] Figure 6 : T cells were activated with a bispecific antibody (BS809) that binds to PD-L1 and GITR. The average expression level of IL-2 is indicated with standard deviation bars.

[0024] Figure 7 : Analysis of luciferase activity in Jurkat dual cells (JD / OX40) with a bispecific antibody that binds PD-L1 and OX40 (BS813) and expresses OX40. The mean relative luminescence units (RLU) of three replicate analyses are represented with standard deviation bars.

[0025] Figure 8 : Analysis of luciferase activity in Jurkat dual cells (JD / GITR) with a bispecific antibody that binds PD-L1 and GITR (BS809) and expresses GITR. The mean relative luminescence units (RLU) of three replicate analyses are represented with standard deviation bars.

[0026] Fig. 9 : Human T cells were activated with bispecific antibodies (BS813, BS841 and BS839) that bind to PD-L1 and OX40. The mean IL-2 expression levels of three replicate analyses are indicated with standard deviation bars.

[0027] Fig.10 : SEB-treated human peripheral blood mononuclear cells were activated with bispecific antibodies (BS813, BS841 and BS839) that bind to PD-L1 and OX40. The mean IL-2 expression levels of three replicate analyses are indicated with standard deviation bars.

[0028] Fig.11 : Human T cells were activated with a bispecific antibody (BS859) that binds to both PD-L1 and ICOS. The mean expression levels of IL-2 and IL-10 are indicated with standard deviation bars.

[0029] Fig.12: Analysis of luciferase activity in Jurkat dual cells (JD / GITR) with a bispecific antibody that binds PD-L1 and GITR (BS840) and expresses GITR. The average relative luminescence unit (RLU) of three replicates is represented with standard deviation bars.

[0030] Fig.13 : Ramos cells were activated with a bispecific antibody (BS846) that binds PD-L1 and CD40. DETAILED DESCRIPTION definition

[0031] The bispecific antibodies of the present invention are usually provided in an isolated form. This means that the bispecific antibodies are usually 50% w / w pure from interfering proteins and other contaminants generated by their production or purification, but does not exclude the possibility that the bispecific antibodies are combined with an excess of pharmaceutically acceptable carriers or other vehicles to facilitate their use. Sometimes the bispecific antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w / w pure from interfering proteins and other contaminants generated by their production or purification. Usually, the bispecific antibody is the main macromolecular component remaining after purification.

[0032] The specific binding of a bispecific antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 or 10 10 M -1 Affinity for different targets may vary. Specific binding is higher in detectable magnitude, distinguished from nonspecific binding that occurs with at least one unrelated target. Specific binding can be the result of bond formation between specific functional groups or specific spatial fits (e.g., lock and key type), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that a bispecific antibody with two different binding sites only binds to the targets of these two binding sites.

[0033] The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. When the variable region is initially expressed, it is linked to a cleavable signal peptide. Variable regions that do not contain 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 that does not contain a light chain signal peptide. However, reference to a variable region does not mean that a signal sequence must be present; in fact, once the bispecific antibody of the present invention is expressed and secreted, the signal sequence is cleaved. A pair of heavy and light chain variable regions defines the binding region of the antibody. 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 constant region is divided into CH1, CH2 and CH3. The CH1 region is bound to the light chain constant region by disulfide bonds and non-covalent bonds. The hinge region provides flexibility between the binding region and the effector region of the antibody, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions in the tetramer subunit. The CH2 and CH3 regions are the main sites for effector function and FcRn binding.

[0034] Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ or ε, and define the isotype of the antibody as IgG, IgM, IgA, IgD and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked together by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. (See generally Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7), which is incorporated herein by reference in its entirety for all purposes).

[0035] The mature variable region of each light / heavy chain pair forms an 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 may be identical or different, depending on the form (see, e.g., 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, i.e., a relatively conservative framework region (FR) connected by three hypervariable regions, which are also referred to as complementary determining regions or CDRs. The CDRs of the two chains of each pair are aligned by the framework region, enabling them to bind to a specific epitope. From the N-terminus to the C-terminus, both the light chain and the heavy chain comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The distribution of amino acids in each domain is defined according to Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989). Kabat also provides a widely used numbering convention (Kabat numbering), 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 (EU index) is more commonly used, as in the case of the present application.

[0036] The term "epitope" refers to a site on an antigen that binds to the arm of a bispecific antibody. An epitope can be formed by continuous or discontinuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are usually lost when treated with denaturing solvents. Some antibodies bind to terminal specific epitopes, which means that the antibody preferentially binds to the same polypeptide with a free end relative to a polypeptide fused to another polypeptide and resulting in the loss of the free end. An epitope usually contains at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0037] The term "antigen" or "target antigen" refers to the target molecule that is bound by one binding site of the bispecific antibody. Antigens can be proteins of any length (natural, synthetic or recombinantly expressed), nucleic acids or carbohydrates, as well as other molecules. Antigens include receptors, ligands, counter-receptors and coating proteins.

[0038] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the ability of one antibody to compete with another antibody for binding to a target antigen. The epitopes of an antibody 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 binding of one antibody reduce or eliminate binding of the other antibody, then the two antibodies have the same epitope. If certain amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody, then the two antibodies have overlapping epitopes.

[0039] Competition between antibodies is determined by an assay in which the test antibody inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). If an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits binding of the reference antibody by at least 50%, but preferably 75%, 90%, or 99%, as measured in a competitive binding assay, the test antibody competes with the reference antibody. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, as well as antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to allow steric hindrance to occur.

[0040] A "subject" includes a human or other mammal receiving prophylactic or therapeutic treatment. Other non-human animals include animal models of the human condition (eg, rodents, non-human primates) and veterinary subjects.

[0041] To classify amino acid substitutions as conservative or non-conservative, the 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 involve substitutions between amino acids of the same class. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another of these classes.

[0042] The percentage of sequence identity is determined by the maximum match of the antibody sequence using the Kabat numbering convention (for variable regions) or EU numbering (for constant regions). After alignment, if the subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, then the percentage of sequence identity between the subject antibody region and the reference antibody region is the number of positions occupied by the same amino acid in the subject antibody region and the reference antibody region divided by the total number of positions in the alignment of the two regions, excluding gaps, and then multiplied by 100 to convert to a percentage.

[0043] A composition or method "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition comprising an antibody may comprise only the antibody or a combination of the antibody and other ingredients.

[0044] The term "antibody-dependent cellular toxicity" or ADCC is a mechanism of inducing cell death that depends on the interaction of antibody-coated target cells (i.e., cells with bound antibodies) with immune cells (also referred to as effector cells) with lytic activity. 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 (particularly FcγRI and FcγRIII) on immune effector cells (such as neutrophils, macrophages, and natural killer cells). Depending on the type of mediated effector cell, the target cell is eliminated by phagocytosis or lysis. The death of the antibody-coated target cell is a result of the activity of the effector cells.

[0045] The term "opsonization," also known as "antibody-dependent cellular phagocytosis" or ADCP, refers to the process by which antibody-coated cells are internalized in whole or in part by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulins.

[0046] The term "complement dependent cytotoxicity" or CDC (also known as CMC) refers to a mechanism of inducing cell death in which one or more Fc effector regions of a target-binding antibody activate a series of enzymatic reactions that ultimately result in the formation of pores in the target cell membrane. Typically, an antigen-antibody complex (e.g., on an antibody-coated target cell) binds and activates complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the target cell surface, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0047] The pH-dependent binding of an antibody to the FcRn receptor means that the antibody binds to this receptor more firmly at pH 6.0 than at pH 7.5. After internalization by pinocytosis, the FcRn binding at low pH in the endosome rescues the IgG antibody from the catabolism degradation in the lysosome. The rescued IgG antibody is then released from the FcRn at a neutral pH and circulates into the circulation. This pH-dependent FcRn binding is the basis of the molecular mechanism of the long serum half-life of IgG antibodies (and the bispecific antibodies of the present invention) (Ghetie et al., Annu. Rev. Immunol. 18: 739-766, 2000). For example, human IgG antibodies bind to human neonatal Fc receptors (FcRn) at pH 6.0, but only weakly bind to FcRn 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 the antibody from lysosomal degradation and therefore generally has a shorter half-life than native IgG antibodies.

[0048] Protein A is a 40-60 kDa surface protein originally found in the cell wall of Staphylococcus aureus. Protein A binds specifically and with high affinity to human IgG1, IgG2, and IgG4, and mouse IgG2a and IgG2b. It does not bind to human IgG3 or IgA or IgM. Protein A is used for affinity purification of antibodies.

[0049] 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 that is not required for IgG binding and is often deleted. Protein G specifically binds all human IgG isotypes but does not bind IgA or IgM. Protein G can also be used for antibody purification. Detailed description I. Overview

[0050] The present invention provides bispecific antibodies, one arm of which is combined with cancer-associated antigens, such as CD33, EGFR or PD-L1, and the second arm is combined with costimulatory molecules, such as OX40, CD40, GITR, ICOS or 4-1BB. Although it is not necessary to understand the mechanism for implementing the present invention, it is believed that the bispecific antibodies of the present invention bridge between cancer cells expressing cancer-associated antigens and immune cells expressing costimulatory molecules, resulting in the aggregation of the costimulatory molecules and selective activation of immune cells near cancer cells. Therefore, immune cells can play an immunotherapeutic role on cancer cells, accompanied by reduced toxicity to healthy tissues. Due to the interaction of antagonistic receptors with ligands (such as the interaction of EGFR with EGF, the interaction of PD-L1 with PD1 or the interaction of CD33 with sialic acid), bispecific antibodies can have additional activity on cells expressing cancer-associated antigens. Certain bispecific antibodies of the present invention combined with cancer cells (or cells infected by pathogens) and costimulatory molecules can bridge between these two types of cells and trigger immune responses by multivalent cross-linking of costimulatory molecules at the junction between cells to fight cancer or infectious diseases. Certain bispecific antibodies of the present invention can also enhance immune responses 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). II. Target

[0051] The bispecific antibodies of the present invention have at least two arms, each of which specifically binds to a different target antigen. A class of proteins that can be used as targets are cancer-associated antigens. Such antigens are expressed by cancer, usually at higher expression levels than normal tissues matched to the control (overexpression). Some examples of cancer-associated antigens are CD33, EGFR, and PD-L1. Exemplary SwissProt numbers for the human forms of these targets are P20138, P00533, and Q9NZQ7. CD33 binds sialic acid and is mainly overexpressed in cancers of myeloid origin (e.g., acute myeloid leukemia). EGFR binds to EGF and is mainly 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 is an antigen expressed on the surface of cells infected by pathogens.

[0052] Another class of proteins is co-stimulatory molecules, including CD40, OX40, GITR, ICOS, and 4-1BB. Typical SwissProt numbers for human forms of these targets are P25942, P23510, Q9Y5U5, Q9Y6W8, and Q07011. Unless otherwise apparent from the context, reference to a specific target should be understood as referring 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

[0053] Bispecific antibodies are formed from heavy and light chain variable region pairs from component antibodies. The component antibodies can be rodent, chimeric, veneered, humanized, primate, or human, etc. The component antibodies can be of the same or different types; for example, one can be humanized and the other can be human.

[0054] Can be by, for example, with antigen or its fragment or the cell immunization animal with this antigen, to realize other non-human monoclonal antibody of production antigen, for example mouse, guinea pig, primate, rabbit or rat.See Harlow & Lane, "Antibodies, A Laboratory Manual" (Antibodies, A Laboratory Manual) (CSHP NY, 1988) (incorporated by reference for all purposes). Such antigen can be obtained from natural source, by peptide synthesis or by recombinant expression. Alternatively, antigen can be fused with carrier protein or compound administration. Alternatively, antigen can be administered together with adjuvant. Several types of adjuvant can be used as described below. Freund's complete adjuvant and subsequent incomplete adjuvant are preferably used for immunization experimental animals.

[0055] Humanized antibodies are genetically engineered antibodies in which CDRs from non-human "donor" antibodies are transplanted into human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539, Carter, U.S. Pat. No. 6,407,213, Adair, U.S. Pat. Nos. 5,859,205 and 6,881,557, Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all of the CDRs from a donor antibody, and variable region framework sequences and constant regions (if present) that are entirely or substantially derived from human antibody sequences. Similarly, humanized heavy chain has at least one, two and usually all three CDRs from the heavy chain of the donor antibody, and if present, heavy chain variable region framework sequences and heavy chain constant regions substantially from the framework of the human heavy chain variable region and constant region sequences. Similarly, humanized light chain has at least one, two and usually all three CDRs from the light chain of the donor antibody, and if present, light chain variable region framework sequences and light chain constant regions substantially from the framework of the human light chain variable region and constant region sequences. Except for nano antibodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. When at least 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) are identical between the corresponding CDRs, the CDRs in the humanized antibodies are substantially from the corresponding CDRs in the non-human antibodies. The variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues are identical as defined by Kabat.

[0056] Although humanized antibodies typically contain all six CDRs from a mouse antibody (preferably as defined by Kabat), they can also be made with less than all CDRs (e.g., at least 3, 4, or 5 CDRs from a mouse antibody) (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).

[0057] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of non-human antibodies (e.g., mice) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of mouse antibodies and are approximately two-thirds human sequences.

[0058] A veneered antibody is a humanized antibody that retains some and usually all CDRs and some non-human variable region framework residues of a non-human antibody, but replaces other variable region framework residues that may contribute to B-cell epitopes or T-cell epitopes (e.g., exposed residues (Padlan, Mol. Immunol. 28:489, 1991)) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are completely or substantially from a non-human antibody and the variable region framework of the non-human antibody becomes more human-like through substitutions.

[0059] 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 by phage display). Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, the use of transgenic mice that include human immunoglobulin genes (see, e.g., Lonberg et al., U.S. Pat. No. 4,634,666). al., WO 93 / 12227 (1993); U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991)) and phage display methods (see, e.g., Dower et al., WO 91 / 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).

[0060] Screening antibodies for specific binding to antigens. Antibodies can be further screened for binding to specific regions of the antigen, competition with reference antibodies, excitement or antagonism of cells carrying the antigen. As described above, non-human antibodies can be converted into chimeric, veneered or humanized forms.

[0061] The present specification discloses exemplary antibodies that specifically bind to CD33, EGFR, PD-L1, CD40, OX40, GITR and ICOS. These antibodies are characterized by mature heavy and light chain variable region sequences and Kabat CDRs provided in the sequence listing shown in Table 1 below.

[0062] Table 1

[0063] 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.

[0064] Table 2 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

[0065] Other antibodies with the same CDR, or other antibodies that bind to the same epitope, or other antibodies that compete with any of these antibodies for binding to their target protein, can also be used; the same CDR is defined as Kabat or as defined by other definitions, such as Chothia, Chothia and Kabat's complex, AbM or Contact (see the World Wide Web bioinf.org.uk / abs). Other ways of binding to any of the above targets can also be used to replace the above antibodies. Antibodies bound to CD40, OX40, GITR, ICOS and 4-1BB can excite their receptors, thereby activating immune cells expressing receptors. Antibodies bound to cancer-associated antigens may or may not antagonize the interaction of cancer-associated antigens with their ligands or counter-receptors. Antagonism provides an additional cytotoxic mechanism for cancer cells, but is not necessary for activating immune cells by the binding arm of a bispecific antibody bound to a co-stimulatory receptor.

[0066] Other antibodies against CD33 include gintuzumab, lintuzumab, and BI836858 (Heider Blood 2011; 118(15): 4159-4168). Antibodies against EGFR include C225, whose chimeric versions are marketed as cetuximab, panitumumab, matuzumab, and necitumumab. Antibodies against PD-L1 include durvalumab, avelumab, atezolizumab, and MDX-1105 / BMS936559. Agonist antibodies against CD40 include CP-870,893 (Pfizer and VLST), dacetuzumab (Seattle Genetics), Chi Lob 7 / 4 (University of Southampton), and lucatumumab (Novartis) (Vonderheide et al., Clin Cancer Res 19, 1035-1043 (2013)). Agonist OX40 antibodies include MOXR0916 (Infante et al., Journal of Clinical Oncology 2016 34:15_suppl 101-101), PF-04518600 (Long et al., Journal of Clinical Oncology 2016 34:15_suppl,e14518-e14518), MEDI0562 (Glisson et al., Annals of Oncology, Volume 27, Issue suppl_6, 1 October 2016, 1052PD), and MEDI6469 (Belle et al., Clin Cancer Res 2017; 23(23_Suppl):Abstract nr37). Agonist antibodies against GITR include DTA-1 (Shimizu et al., Nat Immunol. 3, 135-142, 2002), INCAGN01876 (Gonzalez et al. Cancer Res. 2016 Volume 76, Issue 14, Abstract 3220) and 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 AGEN2373 (Agenus; Galand et al. 2019 J. Clin. Oncol. 37, no. 15suppl. e14005).

[0067] Any of these antibodies or other antibodies with the same CDRs, or other antibodies that bind to the same epitope, or other antibodies that compete with any of these antibodies for binding to their target protein can be incorporated into the bispecific antibodies of the present invention. The same CDRs are defined as Kabat or as defined by other definitions, such as Chothia, a complex of Chothia and Kabat, AbM or Contact. Other means of binding to any of the above targets can also be used to replace the above antibodies.

[0068] The present invention also provides monoclonal antibodies that specifically bind to human PD-L1, including HuPRO1, HuPRO2 and HuPRO5, and other antibodies that have the same six CDRs (according to any conventional definition) or the same mature heavy chain and light chain variable region pairs as one of these antibodies. Such antibodies can inhibit the interaction between PD-L1 and PD1. The present invention also provides monoclonal antibodies that specifically bind to human OX40, including HuOHX14DS and other antibodies that have the same six CDRs (according to any conventional definition) or the same mature heavy chain and light chain variable region pairs. The present invention also provides monoclonal antibodies that specifically bind to human CD40, including HuACS2 and other antibodies that have the same six CDRs (according to any conventional definition) or the same mature heavy chain and light chain variable region pairs. The present invention also provides monoclonal antibodies that specifically bind to human ICOS, including HuTAM14 and other antibodies that have the same six CDRs (according to any conventional definition) or the same mature heavy chain 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 (according to any conventional definition) or the same mature heavy chain and light chain variable region pairs. IV. Formats of Bispecific Antibodies

[0069] 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 the two targets. Preferred formats include two or more binding sites for each target.

[0070] Some formats have a tetrameric structure similar to a normal antibody with two binding regions, each for one target. Each binding region is formed by a pair of heavy and light chain variable regions, which are connected to heavy and light chain constant regions, respectively. Such 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 combination of two different pairs of heavy and light chains.

[0071] The "knobs-into-holes" approach (replacing a small amino acid with a large amino acid in the CH3 domain of one antibody (the "knob") and the opposite in the other antibody (the "hole")) has been used to reduce homodimer formation and mispairing of heavy chains (Ridgway et al., Protein Eng 9:617-21, 1996; Atwell et al., J Mol Biol 270:26-35, 1997; and U.S. Pat. No. 7,695,936). Light chain mispairing in this format can be reduced by a variety of strategies. One strategy is to use a common light chain variable region for two different heavy chain variable regions. But this only works for some antibodies. Another approach is to express the half-molecules containing the knob and hole separately in different bacteria. Another method, called CrossMab, exchanges the CH1 domain of one of the heavy chains with the constant CL domain of the corresponding light chain to induce the correct pairing between the engineered heavy and light chains (Schaefer et al., Proc Natl Acad Sci USA 108: 11187-92, 2011; WO 2009 / 080251; WO 2009 / 080252; WO 2009 / 080253). Another method is to introduce additional mutations into the VH-VL and CH1-CL interfaces (Lewis et al., Nat. Biotechnol., 32 (2014), pp. 191-198). These mutations cause the heavy chain to preferentially pair with the light chain. Another approach is to introduce mutations that promote protein A binding into one of the Fc regions and select heterodimer pairings with intermediate protein A binding from homodimers with higher or lower protein A binding by affinity chromatography (Tusdian et al, MAbs. 2016 May-June;8(4):828-38).

[0072] Other bispecific antibodies avoid the mispairing problem by combining multiple binding specificities in the same heavy and light chain pair. To this end, one approach, called dual variable domains, is to link two different heavy chain variable regions in tandem to a heavy chain constant region and two different light chain variable regions in tandem to a light chain constant region (Correia et al., MAbs .2013 May 1;5(3):364-372). Such antibodies can be assembled into tetramers by combining two identical heavy and light chain pairs. The assembled antibodies contain two different binding sites for each target.

[0073] Another method followed in an embodiment of the present invention is to introduce a second binding specificity by connecting a single-chain Fv (scFv) to the C-terminal of the heavy chain constant region. This bispecific antibody includes a first binding site formed by the heavy chain and light chain variable region connected to the N-terminal of the heavy chain and light chain constant regions as in a standard antibody. The C-terminal of the heavy chain is connected to the scFv providing a second binding site. ScFv is usually connected by a joint, and another joint connects the heavy chain and light chain variable regions in the scFv. ScFv can be connected to the Fc region via a joint through its light chain variable region or the end of the heavy chain variable region. When two identical heavy chains and light chains are combined to assemble, this bispecific antibody includes two binding sites for each of two different specificities. The cancer-associated antigen or infected cell antigen and costimulatory antigen binding arms of this bispecific antibody can be connected in either direction. The arm connected to the N-terminal of the heavy chain and light chain constant regions is provided as a separate heavy chain and light chain variable region, and the arm to be connected to the C-terminal is provided as an scFv fragment. An advantage of this format is that the two different binding spaces are separated by the entire heavy chain constant region, which may facilitate cell-to-cell bridging.

[0074] Another format connects the scFv that specifically binds to the first target to the heavy chain constant region, and connects the scFv that specifically binds to the other target to the light chain constant region. This antibody assembles into a tetramer, including two copies of each binding site (Bs(scFv)4-IgG) (Zuo et al., Protein Eng 13:361-367, 2000).

[0075] Other formats connect scFv binding regions on a single chain without a constant region. For example, the BiTe format connects two scFv fragments via a linker (see, e.g., Ross et al., PLoS ONE 12(8): e0183390, 2017). This format lacks effector function and tends to have a shorter half-life, but may have advantages in accessibility and ease of manufacturing due to its small size.

[0076] Many of the above formats include linker peptides between the heavy and light chain variable regions or between the variable and constant regions. The linker is a short peptide that confers flexibility, usually mainly occupied 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 multimers thereof. V. Selection of constant regions

[0077] Many formats of bispecific antibodies include at least a portion of a human constant region. The selection of constant regions depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and / or complement-dependent cellular toxicity are needed. 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 cell-mediated effector functions that are stronger than human IgG2 and IgG4. Here, although ADCC, ADCP and CDC can be used to provide additional mechanisms of action for cancer or infected cells bound by one arm of a bispecific antibody, it is useless for stimulating co-stimulatory molecules to activate immune cells on the other arm.

[0078] One or more amino acids at the amino or carboxyl terminus of the light chain and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in a certain proportion or all of the molecules. Amino acid substitutions can be made in the constant region to reduce or increase effector functions, such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (see, e.g., Hintone et al., J. Biol. Chem. 279:6213, 2004). For example, there are many known mutations in IgG Fc that increase FcRn binding. Exemplary substitutions include Gln at position 250 and / or Leu at position 428, Ser or Asn at position 434, Tyr at position 252, Thr at position 254, Glu at position 256, and Ala at position 434 (EU numbering). Increased FcRn binding is advantageous in allowing the hybrid protein of the present invention to compete more strongly with endogenous IgG for binding to FcRn. It is also known that many mutations can 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, substitution of any amino acid residue at position 234, 235, 236 and / or 237 reduces affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., US 6,624,821). Alternatively, the amino acid residues at positions 234, 236 and / or 237 in human IgG2 are substituted with Ala, and the amino acid residue at position 235 is substituted with Gln or Glu (see, e.g., 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.

[0079] Human constant regions show allotype variation and isoallotypic variation between different individuals, i.e., the constant regions of different individuals may differ at one or more polymorphic positions. Allotypes differ from isotypes in that sera that recognize isotypes bind to non-polymorphic regions of one or more other isotypes. VI. Expression of Recombinant Antibodies

[0080] Bispecific antibodies are usually produced by 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 by the same or different vectors. The recombinant polynucleotide construct usually includes an expression control sequence operably connected to the coding sequence of the antibody chain, including naturally related or heterologous expression control elements, such as promoters. The expression control sequence can be a promoter system in a vector capable of transforming or transfecting a eukaryotic or prokaryotic host cell. Once the vector is integrated into a suitable host, the host is maintained under conditions suitable for the high-level expression of the nucleotide sequence and the collection and purification of the bispecific antibody.

[0081] These expression vectors are typically replicable in the host organism as an episome or as an integral part of the host chromosomal DNA. Typically, the expression vector comprises a selection marker, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence.

[0082] Escherichia coli is a prokaryotic host for expressing antibodies (particularly antibody fragments). Microorganisms, such as yeast, can also be used for expression. Saccharomyces cerevisiae is a yeast host with a suitable vector having expression control sequences, replication origins, termination sequences, etc. as required. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, etc.

[0083] 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 myeloma cells that do not produce antibodies, including Sp2 / 0 and NS0. Cells can be non-human. The expression vectors of these cells can include expression control sequences, such as replication origins, 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 papilloma virus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0084] Alternatively, the antibody coding sequence can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequently expressed in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes (e.g., casein or beta lactoglobulin).

[0085] The vector containing the DNA fragment of interest can be transferred into a host cell by a method depending on the cell host type. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistic methods or viral-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. In order to produce transgenic animals, transgenic microinjection can be used in fertilized oocytes, or it can be integrated into the genome of embryonic stem cells, and the nucleus of such cells is transferred into enucleated oocytes.

[0086] After the vector encoding the heavy and light chains of the antibody is introduced into the cell culture, the cell bank can be screened for the productivity and quality of the antibody in the serum-free medium. The cell bank with the highest yield can then be subjected to single cell cloning based on FACS to generate a monoclonal system. The specific productivity (Specificproductivity) higher than 50pg or 100pg per cell can be used every day, which corresponds to a product titer greater than 7.5g / L culture. The turbidity, filtration characteristics, PAGE, IEF, UV scanning, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry and binding assays of the antibodies produced by single cell cloning can also be tested, such as ELISA or Biacore. Selected clones can then be stored in multiple vials and frozen for subsequent use.

[0087] Once expressed, the bispecific antibodies can be purified according to standard procedures in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0088] Methods for commercial production of antibodies can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, blood-free single cell cloning, cell banking, use of selectable markers to expand copy number, CHO terminator, or increase in protein titer (e.g., US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; WO2004 / 050884; W02008 / 012142; W02008 / 012142; W02005 / 019442; W02008 / 107388; W02009 / 027471; and US 5,888,809). VII. Nucleic Acids

[0089] The present invention further provides nucleic acid encoding any of the above-mentioned heavy chains and light chains. Alternatively, such nucleic acid further encodes a signal peptide and can be expressed together with the signal peptide of the constant region coding sequence connected to the nucleic acid, and can be operably connected with a regulatory sequence to ensure the expression of the coding sequence, and the coding sequence is, for example, a promoter, an enhancer, a ribosome binding site, a transcription termination signal, etc. The nucleic acid encoding the heavy chain and light chain can exist in a separated form or can be cloned into one or more vectors. Nucleic acid can be synthesized by, for example, solid-state synthesis or PCR of overlapping oligonucleotides. The nucleic acid encoding the heavy chain and light chain can be connected as a continuous nucleic acid (for example, in an expression vector), or can be separated (for example, each cloned into its own expression vector). VIII. Methods of treatment and pharmaceutical compositions

[0090] The bispecific antibodies of the invention 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 hematological malignancies. Hematological malignancies include leukemias (e.g., T-cell large granular lymphocytic leukemia), lymphomas (Hodgkin or non-Hodgkin), or multiple myeloma. Solid tumors include skin (e.g., melanoma), ovaries, endometrium, kidneys, liver, pancreas, bladder, breasts, ovaries, prostates, rectums, colons, stomachs, intestines, pancreas, lungs, thymus, thyroid, kidneys, and brains.

[0091] When the bispecific antibody of the present invention has an arm specifically bound to an antigen expressed in infected cells but not expressed in matching uninfected cells, the bispecific antibody of the present invention can also be used to treat pathogen infection. Such antigens can be encoded by pathogens or can be expressed by cells in response to pathogen infection. Examples of such antigens expressed in infected cells are human immunodeficiency virus (HIV) glycoprotein gp41 and gp120, human T-cell leukemia virus type 1 (HTLV-1) Env protein, herpes simplex virus (HSV) glycoprotein 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), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein-Barr virus), adenovirus, XMRV, influenza virus, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, MLV-related virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus. Some examples of bacterial infections include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum, Anthrax, Plague, Leptospirosis, Lyme disease bacteria, Streptococcus or Neisseria. Some examples of pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Plasmodium.

[0092] The bispecific antibody is administered with an effective regime, meaning a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and / or improves at least one symptom or condition of the disease. If the subject already has a disease, the regimen may be referred to as a therapeutically effective regimen. If the subject is at high risk of the disease relative to the general population, but symptoms have not yet occurred, the regimen may be referred to as a preventive effective regimen. In some cases, treatment or preventive efficacy may be observed in a single subject relative to historical controls or past experience of the same subject. In other cases, treatment or preventive efficacy may be demonstrated in a population consisting of treated subjects relative to a control population consisting of untreated subjects in preclinical or clinical trials.

[0093] Preferably, the bispecific antibody exhibits at least additive (more preferably synergistic) activity against cancer or infected cells compared to its individual component antibodies. Synergy is preferably assessed quantitatively, such as discussed 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 is at an equimolar concentration to the bispecific antibody). Such activity can, for example, be measured as cytotoxicity or cell inhibition against cancer cells or infected cells; the infected cells express an antigen specifically bound by another arm of the bispecific antibody in the presence of an immune cell expressing a co-stimulatory molecule specifically bound by one arm of the bispecific antibody.

[0094] Exemplary dosages of the 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 10-1500 mg as a fixed dose. The dosage depends on the patient's condition and response to previous treatment (if any), whether the treatment is preventive or therapeutic, and whether the disease is acute or chronic, among other factors.

[0095] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Preferably, the systemic circulation is entered by intravenous or subcutaneous administration. Intravenous administration can be, for example, by infusion over a period of, for example, 30-90 minutes.

[0096] The frequency of administration depends on the half-life of the bispecific antibody in the circulation, the condition of the subject, the route of administration, and other factors. The frequency can be daily, weekly, monthly, quarterly, or irregular intervals in response to changes in the condition of the subject or the progression of the disease being treated. In a continuous course of treatment, an exemplary frequency of intravenous administration is between weekly and quarterly, although it is also possible to administer at a higher or lower frequency. For subcutaneous administration, an exemplary frequency of administration is daily to monthly, although it is also possible to administer at a higher or lower frequency.

[0097] The number of doses administered depends on whether the disease is acute or chronic, and also on the response of the disease to treatment. For acute diseases or acute exacerbations of chronic diseases, 1 to 10 doses are usually sufficient. Sometimes, a single bolus dose (optionally in a separated form) is sufficient to cope with acute diseases or acute exacerbations of chronic diseases. For recurrence of acute diseases or acute exacerbations, treatment can be repeated. For chronic diseases, bispecific antibodies can be administered regularly, such as weekly, biweekly, monthly, quarterly, and every six months for at least 1, 5, or 10 years, or for the lifetime of the subject.

[0098] 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 can be provided in unit dosage form (i.e., a single dose). The pharmaceutical composition can be formulated using one or more pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Pharmaceutically acceptable means suitable for human administration are, for example, approved by the FDA or approved by the FDA. The formulation depends on the selected route of administration. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers (e.g., Hank's solution, Ringer's solution, or saline or acetate buffer) (to reduce discomfort at the injection site). The solution may contain formulatory agents, such as suspending agents, stabilizers, and / or dispersants. Alternatively, the antibody may be in a lyophilized form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) before use.

[0099] Treatment with the bispecific antibodies of the invention can be combined with other treatments that are effective for the disease being treated. When used to treat cancer, the bispecific antibodies of the invention can be combined with chemotherapy, radiotherapy, stem cell therapy, surgery, or other biological agents, such as Herceptin directed against the HER2 antigen. TM (trastuzumab), Avastin targeting VEGF TM (bevacizumab) or antibodies to EGF receptors, such as (Erbitux TM , cetuximab) and Vectibix TM (panitumumab). Chemotherapeutic 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, antivirals, antifungals or antiprotozoal agents, etc. IX. Other methods

[0100] The bispecific antibodies of the present invention can also be used for diagnosis, prognosis and laboratory methods. They can be used to measure the level of antigen expressed in the circulation of cancer or cancer patients to determine whether the level is measurable or even elevated, thereby tracking and guiding the treatment of cancer, because cancers associated with measurable or elevated antigen levels are most susceptible to the treatment of bispecific antibodies comprising arms that bind to cancer. Bispecific antibodies can be used for 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 the form of a kit, which contains all reagents required for the assay.

[0101] For all purposes, all patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated herein by reference as a whole, to the same extent as each individual project is specifically and individually indicated as being incorporated herein by reference. If different versions of a sequence are associated with an accession number at different times, it is meant that the version associated with the accession number is meant on the effective application date of the present application. The effective application date refers to the earlier date in the application date of the actual application date or the priority application (if any) that mentions the accession number. Similarly, if different versions of publications, websites, etc. are published at different times, it is meant, unless otherwise stated, the version published most recently on the effective application date of the present application. Unless otherwise clearly stated, any feature, step, element, embodiment, or aspect of the present invention may be used in combination with any other. Although the present invention has been described in detail by way of illustration and example for the purpose of clarity and understanding, it is apparent that certain changes and modifications may be made within the scope of the appended claims. Example

[0102] Example 1: General methods and materials

[0103] Gene cloning, mutagenesis, plasmid construction, ELISA, and FACS were 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.

[0104] Mouse hybridomas were produced at JN Biosciences (Mountain View, CA) using GenomONE CF EX cell fusion reagent (Cosmo Bio, Carlsbad, CA) according to standard cell fusion techniques. The mouse hybridomas produced monoclonal antibodies against the desired antigens. For mouse immunization, purified soluble forms of recombinant antigens were used, usually fused with human IgG Fc regions, or mouse cell lines expressing recombinant antigens on the surface were used. Mouse monoclonal antibodies secreted in hybridoma cell culture supernatants were screened for a series of properties, identifying antibodies with the following characteristics: (1) specific binding to human antigens, (2) specific binding to cynomolgus monkey antigens, and (3) desired biological functions, such as blocking the interaction of antigens with their ligands or stimulating cell activity by binding of antigens on the cell surface. The selected hybridoma cells were amplified in serum-free medium, such as Hybridoma SFM (Thermo Fisher Scientific, Waltham, MA). Mouse monoclonal IgG antibodies were purified using protein A affinity columns (MabSelect SuRe, GE Healthcare Life Sciences, Marlborough, MA) according to the manufacturer's instructions. The purified antibody was buffer exchanged into phosphate buffered saline (PBS) by dialysis. Antibody concentration was determined by measuring absorbance at 280 nm (OD 1.4 = 1 mg / ml).

[0105] The expression concentration of mouse IgG antibody in the culture supernatant was measured by sandwich ELISA. In a typical experiment, ELISA plates were covered with goat anti-mouse IgG Fc specific polyclonal antibodies (SouthenBiotech, Birmingham, AL), washed with wash buffer (PBS containing 0.05% Tween 20), and blocked with ELISA buffer (PBS containing 2% skim milk and 0.05% Tween20). 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 the ELISA plate was cultured for 60 minutes at room temperature and washed with wash buffer, the bound mouse antibodies were detected using HRP-coupled goat anti-mouse κ chain polyclonal antibodies (BethylLaboratories, Montgomery, TX). After 30 minutes of culture at room temperature and washing with wash buffer, color development was started by adding ABTS substrate (Sigma-Aldrich, St.Louis, MO), and terminated with 2% oxalic acid. The absorbance was read at 405nm.

[0106] The expression of chimeric and humanized IgG / κ antibodies in culture supernatants was measured by sandwich ELISA as described above, except that the ELISA plates were coated with a goat anti-human IgG Fc-specific polyclonal antibody (Jackson ImmunoResearch, West Grove, PA) and bound antibodies were detected with an HRP-conjugated goat anti-human κ chain polyclonal antibody (Bethyl Laboratories).

[0107] The heavy and light chain variable regions (VH and VL, respectively) of mouse monoclonal antibodies were sequenced using standard molecular biology techniques (e.g., the steps described by Tsurushita et al. (Methods 36: 69-83, 2005)). The humanization of VH and VL was performed according to the general procedures described by Tsurushita et al. (as above). First, a three-dimensional molecular model of the variable region of the mouse antibody to be humanized was constructed using appropriate software. Secondly, the molecular model was used to identify the framework amino acid residues that are important for forming the CDR (complementary determining region) structure or necessary for antigen binding. At the same time, cDNA-derived human VH and VL amino acid sequences that have a high degree of homology to the mouse VH and VL amino acid sequences, respectively, were selected. Finally, the CDR sequences, together with the identified framework amino acid residues that are important for forming the antigen binding site, were transplanted from the mouse variable region into the corresponding selected human framework sequence.

[0108] Stable transfection was performed into the Chinese Hamster Ovary cell line CHO-K1 by electroporation. Prior to transfection, the expression vector was linearized using appropriate restriction enzymes. In a typical experiment, approximately 10 cells were transfected with 20 μg of linearized plasmid. 7 CHO-K1 cells were suspended in SFM4CHO medium (GE Healthcare Life Sciences) and seeded in several 96-well plates at 100 μl / well after appropriate dilution of the cells. After 48 hours, SFM4CHO medium containing 20 μg / ml puromycin was added at 100 μl / well to isolate stable transfectants. About ten days after the start of selection, the culture supernatant of the transfectants was measured for antibody production. As described above, CHO-K1 stable transfectants that produced high levels of antibodies were identified by ELISA.

[0109] CHO-K1 stable transfectants (e.g., humanized and bispecific IgG antibodies of the present invention) that highly produce recombinant antibodies are amplified in SFM4CHO until the cell viability is less than 50%. After centrifugation and filtration, the culture supernatant is loaded onto a protein A column (HiTrapMAB Select SuRe, GE Healthcare Life Sciences). 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 column is washed with PBS. The buffer for eluting the antibody is neutralized with 1M Tris-HCl (pH 8.0) and then replaced with PBS by dialysis. The antibody concentration (1mg / ml=1.4OD) is determined by measuring the absorbance at 280nm.

[0110] Example 2: Construction of a bispecific antibody (BS824) that binds CD33 and OX40

[0111] The mammalian expression vector pHuM195-IgG1 ( FIG. 1A ), designed for the production of a humanized anti-CD33 IgG1 / κ antibody that binds to human CD33 (also known as Siglec-3; SEQ ID NO: 1) (Co et al., J. Immunol. 148: 1149-1154, 1992), contains the following genetic elements. Starting clockwise from the SalI site in FIG. 1A , the vector contains the heavy chain transcription unit, which begins with the human cytomegalovirus (CMV) major immediate early promoter and enhancer (CMV-P in the figure) to initiate transcription of the antibody heavy chain gene. The CMV promoter is followed by exons encoding humanized M195 (HuM195) VH (SEQ ID NO: 2) flanked by SpeI and HindIII sites (HuM195-VH) and a genomic sequence comprising exons encoding the constant region of the human γ-1 heavy chain CH1 (SEQ ID NO: 3), hinge (Hinge; SEQ ID NO: 4), CH2 (SEQ ID NO: 5) and CH3 (SEQ ID NO: 6), accompanied by inserted introns; after the EagI site is the polyadenylation site of the human γ-1 heavy chain gene. After the heavy chain gene sequence, the light chain transcription unit starts 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), exons encoding the constant region of the human κ chain (Ck) (SEQ ID NO: 8). NO:8) and part of the genomic sequence of the intron in front of it, and the polyadenylation site of the human kappa chain gene after the Ck exon. The light chain gene is followed by the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase gene (puro) for resistance to puromycin, and a fragment containing the SV40 polyadenylation site (SV40-A). Finally, pHuM195-IgG1 contains a bacterial origin of replication (pUCori) and a beta-lactamase gene (beta-lactamase). The arrows in the figure indicate the direction of transcription. Humanized IgG1 / κ monoclonal antibody (HuM195-IgG1) is expressed in mammalian cells from pHuM195-IgG1.

[0112] As described in Example 1, mouse hybridomas producing the IgG / κ monoclonal antibody OHX14 that specifically binds to human and cynomolgus monkey OX40 (also known as TNFRSF4 and CD134) were isolated. As an immunogen, a mouse myeloma cell line NS0 stably expressing recombinant human OX40 on the surface, which consists of the extracellular region of human OX40 fused to the FLAG polypeptide (SEQ ID NO: 9) and the glycosylphosphatidylinositol (GPI) anchor signal of human CD55 (SEQ ID NO: 10) was used (OX40-FLAG-GPI; SEQ ID NO: 11). Sequencing and humanization of VH and VL of OHX14 were performed as described by Tsurushita et al. (supra).

[0113] The amino acid sequence of humanized OHX14 (HuOHX14DS) VH is 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, US Department of Health and Human Services, 1991), the amino acid sequences of CDR1, 2 and 3 of HuOHX14DS VH are SYIMH (SEQ ID NO: 13), YINPYNSGTKYNEKFKG (SEQ ID NO: 14) and YYGSTFTMDY (SEQ ID NO: 15), respectively. The gene encoding HuOHX14DS VH was synthesized as an exon, 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.

[0114] The amino acid sequence of humanized OHX14 (HuOHX14DS) VL is MMSSAQFLGLLLLCFQGTRCDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIK (SEQ ID NO: 16). According to the definition of Kabat et al. (supra), 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). The gene encoding HuOHX14DS VL was 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.

[0115] 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 SpeI and HindIII sites, (ii) the HuOHX14DS VL exon is placed between NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 in CH2 (EU numbering of Kabat et al., supra) 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.

[0116] The mammalian expression vector pBS824 ( FIG. 1B ), designed for the expression of a bispecific antibody that binds to human CD33 (SEQ ID NO: 1) and human OX40 (SEQ ID NO: 21), was constructed by modifying pHuM195-IgG1 as follows. The VL and VH coding regions of the humanized anti-human OX40 monoclonal antibody in pHuOHX14DS-IgG1.AA were converted into a single-chain Fv (scFv) format in the order of VL, polypeptide linker, and VH from the N-terminus to the C-terminus (HuOHX14DS.scFv; SEQ ID NO: 22). The N-terminus of HuOHX14DS.scFv was fused to the penultimate glycine residue in CH3 of pHuM195-IgG1, and a polypeptide linker separated them (CH3-HuOHX14D.scFv; SEQ ID NO: 23). Furthermore, in CH2, the two leucine residues at positions 234 and 235 (EU numbering) were replaced by alanine residues (L234A and L235A, respectively).The resulting vector pBS824 expresses a bispecific IgG antibody called BS824 that binds to human CD33 and OX40.

[0117] The amino acid sequence of the mature heavy chain encoded in pBS824 is QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS(SEQ ID NO: 24).

[0118] The amino acid sequence of the mature light chain encoded in pBS824 is DIQMTQSPSSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 25).

[0119] The schematic structure of the bispecific IgG antibody of the present invention (e.g. BS824) is as follows Figure 2 shown.

[0120] 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 supernatants 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 the three antibodies had only two major bands: about 50 kD heavy chain and 25 kD light chain (for HuM195-IgG1 and HuOHX14DS-IgG1.AA) or about 75 kD heavy chain and 25 kD light chain (for BS824).

[0121] Example 3: Biological activity of a bispecific antibody (BS824) binding to CD33 and OX40

[0122] The biological activity of BS824 binding to CD33 and OX40 was examined using Jurkat Dual reporter cells (InvivoGen, San Diego, CA) (JD / OX40) expressing human OX40 on the surface and mouse myeloma cell line NS0 expressing human CD33 on the surface (NS0 / CD33). It is known that the binding of 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.

[0123] JD / OX40 was produced by stably transfecting the vector pFCm331 for expressing human OX40 into Jurkat Dual cells by electroporation. 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. Puromycin-resistant Jurkat Dual cells (JD / OX40) expressing OX40 on the surface were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C in a 7.5% CO2 incubator. No CD33 expression was detected in JD / OX40 cells by flow cytometry using HuM195-IgG1 and a secondary PE-labeled goat anti-human IgG antibody.

[0124] NS0 / CD33 was generated by stably transfecting the expression vector pFCm267 for expressing human CD33 into NS0 cells by electroporation. The expression vector pFCm267 ( FIG. 1D ) has the same structure as pFCm331, except that (a) the puromycin N-acetyltransferase gene (puro) was replaced by E. coli xanthine-guanine phosphoribosyltransferase (gpt), and (b) the SpeI-EagI fragment was replaced by a DNA fragment encoding recombinant human CD33, which consisted of the following from N-terminus to 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 anchor 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) expressed CD33-FLAG-GPI on their surface in DME medium containing 10% FBS at 37°C in a 7.5% CO2 incubator.

[0125] OX40, a member of the TNF receptor superfamily of immune co-stimulatory molecules, needs to be trimerized 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 surface OX40 upregulated the expression and secretion of Lucia luciferase. Approximately 200,000 JD / OX40 cells were cultured in 96-well plates for one day at 37° C. in a 7.5% CO 2 incubator in the presence or absence of approximately 200,000 NS0 / CD33 cells in 200 μl of 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) to provide the main signal for activating T cells together with the following: (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. Luciferase activity in culture supernatants was measured in triplicate using QUANTI-Luc reagent (InvivoGen) according to the supplier's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek, Winooski, VT). Results are shown in Table 1. Figure 3 shown.

[0126] The average relative luciferase units (RLU) in JD / OX40 cells were 6,458, no antibody; 5,997, HuM195-IgG1; 7,361, HuOHX14DS-IgG1.AA; 5,009, BS824; 6,918, NS06, CD33; 6,240, HuM195-IgG1 and NS0 / CD33; 8,698, HuOHX14DS-IgG1.AA and NS0 / CD33; and 21,684, BS824 and NS0 / CD33. Luciferase activity in JD / OX40 cells was significantly increased only in the presence of both a bispecific antibody that binds CD33 and OX40 (BS824) and cells expressing CD33 (NS0 / CD33). This result indicates that only BS824 can establish a bridge between JD / OX40 and NS0 / CD33 cells, but neither HuM195-IgG1 nor HuOHX14DS-IgG1.AA causes OX40 to aggregate on the surface of JD / OX40 cells close to NS0 / CD33 cells and leads to the activation of JD / OX40 cells to increase the expression of luciferase.

[0127] Example 4: Construction of a bispecific antibody (BS827) binding to EGFR and GITR

[0128] The mammalian expression vector pCh225-IgG1 for expressing a chimeric IgGl / κ antibody that binds 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, which contained 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, a 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 for expressing the anti-EGFR antibody Ch225-IgG1 had the same structure as pHuM195-IgG1 ( FIG. 1A ), except that the VH and VL coding regions were replaced by Ch225VH and VL, respectively.

[0129] The mouse hybridoma producing the IgG / κ monoclonal antibody GAB11 that specifically binds to human and cynomolgus GITR (also known as TNFRSF18 and CD357) was isolated as described in Example 1. As an immunogen, the extracellular region of human GITR fused to the Fc region of human γ-1 heavy chain (hGITR-Fc; SEQ ID NO: 31) was used. Sequencing and humanization of GAB11 VH and VL were performed as described by Tsurushita et al. (supra).

[0130] The amino acid sequence of humanized GAB11 (HuGAB11) VH is MAVLGLLLCLVTFPSCVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (SEQ ID NO: 32). According to the definition of Kabat et al. (supra), 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 was 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.

[0131] The amino acid sequence of humanized GAB11 (HuGAB11) VL is MRVLAELLGLLLFCFLGVRCDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIK (SEQ ID NO: 36). According to the definition of Kabat et al. (supra), the amino acid sequences of CDR1, 2 and 3 of HuGAB11 VL are HASQNINVWLS (SEQ ID NO: 37), KASNLHT (SEQ ID NO: 38) and QQGQSYPLT (SEQ ID NO: 39). The gene encoding HuGAB11 VL was 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.

[0132] 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 SpeI and HindIII sites, (ii) the HuGAB11 VL exon is placed between NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 in CH2 (EU numbering of Kabat et al., supra) are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., supra). The new vector pHuGAB11-IgG1.AA expresses humanized anti-GITR IgG1 / κ antibody (HuGAB11-IgG1.AA) in mammalian cells.

[0133] The mammalian expression vector pBS827, designed for the expression of a bispecific antibody that binds 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 into scFv format (HuGAB11.scFv; SEQ ID NO: 41) in the order of VL, polypeptide linker, and VH 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). In addition, the two leucine residues at positions 234 and 235 (EU numbering) in CH2 were replaced with alanine residues (L234A and L235A, respectively). The resulting vector, pBS827, expresses a bispecific IgG antibody, termed BS827, that binds to human EGFR and GITR.

[0134] The amino acid sequence of the mature heavy chain encoded in pBS827 is QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS(SEQ ID NO:43).

[0135] The amino acid sequence of the mature light chain encoded in pBS827 is DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:44).

[0136] The structural diagram of BS827 is as follows Figure 2 shown.

[0137] 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 CHO-K1 stable 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: about 50 kD heavy chain and 25 kD light chain (for Ch225-IgG1 and HuGAB11-IgG1.AA) or about 75 kD heavy chain and 25 kD light chain (for BS827).

[0138] Example 5: Biological activity of a bispecific antibody (BS827) that binds EGFR and GITR

[0139] The biological activity of BS827 binding to EGFR and GITR was examined using Jurkat Dual reporter cells (InvivoGen,) expressing human GITR on the surface (JD / GITR) and the mouse myeloma cell line NS0 expressing human EGFR on the surface (NS0 / EGFR).

[0140] JD / GITR was produced by stably transfecting the vector pFCm331 for expressing human GITR into Jurkat Dual cells through electroporation. 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 to mature human GITR (SEQ ID NO: 45). At 37°C, in a 7.5% CO2 incubator, the puromycin-resistant Jurkat Dual cells (JD / GITR) expressing GITR on the surface were maintained in RPMI 1640 culture medium. Using Ch225-IgG1 and secondary PE-labeled goat anti-human IgG antibodies, CD33 expression was not detected in JD / GITR cells by flow cytometry.

[0141] NS0 / EGFR was generated by stably transfecting the expression vector pFCm507 into NS0 cells by electroporation. 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 N-terminus to C-terminus: a synthetic signal peptide (SEQ ID NO: 26), an extracellular region of human EGFR (SEQ ID NO: 46), a FLAG polypeptide (SEQ ID NO: 9), and a GPI anchor signal of human CD55 (SEQ ID NO: 10) (EGFR-FLAG-GPI). Puromycin-resistant NS0 stable transfectants expressing EGFR-FLAG-GPI on the surface were maintained in DME medium containing 10% FBS at 37°C in a 7.5% CO2 incubator.

[0142] GITR, a member of the TNF receptor superfamily of immune co-stimulatory molecules, needs to be trimerized 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 surface GITR upregulated the expression and secretion of Lucia luciferase. Approximately 200,000 JD / OX40 cells were cultured in 200 μl of RPMI 1640 medium in a 96-well plate at 37° C. in a 7.5% CO2 incubator for one day in the presence of approximately 200,000 NS0 or NS0 / EGFR cells, the medium containing 10% FBS, 0.5 μg / ml mouse anti-human CD3 IgG antibody OKT3 (BioLegend) and 5 μg / ml goat anti-mouse IgG antibody (human IgG absorption) (Jackson ImmunoResearch) to provide the main signal for activating T cells together 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 culture supernatants was measured in triplicate using QUANTI-Luc reagent (InvivoGen) according to the supplier's protocol. Luminescence was measured using a Synergy HT microplate reader (BioTek). Figure 4 shown.

[0143] The average relative luciferase units (RLU) in JD / GITR cells were 18,086, NS0 cells alone; 17,183, Ch225-IgG1 and NS0 cells; 18,504, HuGAB11-IgG1.AA and NS0 cells; 17,490, BS827 and NS0 cells; 18,662, NS0 / EGFR cells alone; 20,748, Ch225-IgG1 and NS0 / EGFR cells; 18,391, HuGAB11-IgG1.AA and NS0 / EGFR cells; and 47,523, BS827 and NS0 / EGFR cells. Luciferase activity in JD / GITR cells was significantly increased only in the presence of both a bispecific antibody that binds EGFR and GITR (BS827) and cells expressing EGFR (NS0 / EGFR). The results showed that only BS827 could establish a bridge between JD / GITR and NS0 / EGFR cells, but neither Ch225-IgG1 nor HuGAB11-IgG1.AA could cause GITR to aggregate on the surface of JD / GITR cells close to NS0 / EGFR cells and lead to the activation of JD / GITR cells to increase the expression of luciferase.

[0144] Example 6: Bispecific antibody binding to CD33 and CD40 (BS828)

[0145] As described in Example 1, a mouse hybridoma producing the IgG / κ monoclonal antibody ACS2, which binds to human and cynomolgus CD40 (also known as TNFRSF5), was isolated. As an immunogen, the extracellular region of human CD40 fused to the Fc region of human gamma-1 heavy chain (hCD40-Fc; SEQ ID NO: 47) was used. Sequencing and humanization of ACS2 VH and VL were performed as described by Tsurushita et al. (Methods 36: 69-83, 2005).

[0146] The amino acid sequence of humanized ACS2 (HuACS2) VH is MKLWLNWVFLLTLLHGIQCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS (SEQ ID NO: 48). According to the definition of Kabat et al. (supra), 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 was 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.

[0147] The amino acid sequence of mature humanized ACS2 (HuACS2) VL is MDFQVQIFSFLLISAVIISRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO: 52). According to the definition of Kabat et al. (supra), the amino acid sequences of CDR1, 2 and 3 of HuACS2 VL are SASSSVSYMH (SEQ ID NO:53), DTSKLAS (SEQ ID NO:54) and QQWSSNPLT (SEQ ID NO: 55). The gene encoding HuACS2 VL was 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.

[0148] 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 numbering) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., supra). The new vector pHuGAB11-IgG1.AA expresses a humanized anti-GITR IgG1 / κ antibody (HuGAB11-IgG1.AA) in mammalian cells. The new vector pHuACS2-IgG1.AA expresses a humanized anti-CD40 IgG1 / κ antibody (HuACS2-IgG1.AA) in mammalian cells.

[0149] The mammalian expression vector pBS828, designed for the expression of 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 into a single-chain Fv (scFv) format in the order of VL, polypeptide linker, and VH from the N-terminus to the C-terminus (HuACS2.scFv; SEQ ID NO: 57). 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). In addition, the two leucine residues at positions 234 and 235 (EU numbering) in CH2 were replaced with alanine residues (L234A and L235A, respectively). The resulting vector, pBS828, expresses a bispecific IgG antibody, termed BS828, which binds human CD33 and CD40.

[0150] The amino acid sequence of the mature heavy chain encoded in pBS828 is QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS(SEQ ID NO:59).

[0151] 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.

[0152] The structural schematic diagram of BS828 is as Figure 2 shown.

[0153] 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 CHO-K1 stable 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: about 50 kD heavy chain and 25 kD light chain (for HuM195-IgG1 and HuACS2-IgG1.AA) or about 75 kD heavy chain and 25 kD light chain (for BS828).

[0154] The human Burkitt's B lymphoma cell line Ramos expresses CD40 on its surface. It is known that cross-linking of CD40 with soluble trimeric CD154 (also known as CD40L and TNFSF5, a natural CD40 ligand) on the surface of Ramos cells induces elevated expression of CD95 (Henriquez et al., J. Immunol. 162: 3298-3307, 1999). The biological activity of BS828 in upregulating CD95 expression in Ramos cells was examined in the presence or absence of HL-60, a human promyelocytic leukemia cell line expressing CD33 on its surface.

[0155] About 100,000 Ramos cells were cultured in 200 μl of RPMI 1640 medium containing 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 in a 96-well plate at 37° C., 7.5% CO2 incubator for one day. The expression of CD95 in Ramos cells was analyzed by flow cytometry using FITC-labeled mouse anti-human CD20 monoclonal antibody (Clone 2H7, BioLegend) and PE-labeled mouse anti-human CD95 antibody (Clone DX2, BioLegend). Ramos cells are CD20 positive, and HL-60 cells are CD20 negative. The results of FACS analysis are shown in Figure 5 shown.

[0156] The percentage of CD95-positive population in CD20-positive Ramos cells was 1.3% in the absence of antibody, 0.8% in HuM195-IgG1, 1.4% in HuACS2-IgG1.AA, and 1.6% in 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% in the absence of antibody, 1.1% in HuM195-IgG1, 0.7% in HuACS2-IgG1.AA, and 58.4% in BS828. The expression of CD95 in Ramos cells was significantly upregulated only in the presence of both a bispecific antibody that binds CD33 and CD40 (BS828) and cells expressing CD33 (HL-60 cells). This result indicated that only BS828 could establish a bridge between Ramos and HL-60 cells, but neither HuM195-IgG1 nor HuACS2-IgG1.AA caused multimeric cross-linking of CD40 on the surface of Ramos cells close to HL-60 cells and led to upregulation of CD95 in Ramos cells.

[0157] Example 7: Bispecific Antibody Binding to PD-L1 and GITR (BS809)

[0158] PD-L1 (also known as B7H1 and CD274) is a type I transmembrane protein that acts as a ligand for the checkpoint molecule PD-1. Binding of PD-L1 to PD-1 on T cells can inhibit immune responses. PD-L1 is often expressed on cancer cells (Patelet l. 2015 Mol. Cancer Ther. 14: 847-856; Brody et al. 2017 Lung Cancer 112: 200-215; Sun et al. 2018 Immunity 48: 434-452). As described in Example 1, a mouse hybridoma producing the IgG / κ monoclonal antibody PRO1 was isolated, which binds to human and cynomolgus monkey PD-L1 and blocks the interaction between PD-1 and PD-L1. As an immunogen, the extracellular region of human PD-L1 fused to the Fc region of 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. (supra).

[0159] The amino acid sequence of humanized PRO1 (HuPRO1) VH is MEWNWVVLFLLSLTAGVYAQVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSS (SEQ ID NO: 61). According to the definition of Kabat et al. (supra), 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 was 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.

[0160] The amino acid sequence of humanized PRO1 (HuPRO1) VL is MDFQVQIFSFLLISAVIMSRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK (SEQ ID NO: 65). According to the definition of Kabat et al. (supra), the amino acid sequences of CDR1, 2 and 3 of HuPRO1 VL are SASSSVSYMH (SEQ ID NO:66), DTSNLAS (SEQ ID NO:67) and HQRSSYPWT (SEQ ID NO: 68). The gene encoding HuPRO1 VL was 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.

[0161] 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 (iii) the two leucine residues at positions 234 and 235 (EU numbering) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., supra). 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.

[0162] The mammalian expression vector pBS809, designed for expressing a bispecific antibody that binds to human PD-L1 (SEQ ID NO: 69) and human GITR (SEQ ID NO: 45), was constructed by the method described below: the CH3 coding region in pHuPRO1-IgG1.AA was replaced with the coding region of CH3-HuGAB11.scFv (SEQ ID NO: 42).

[0163] The amino acid sequence of the mature heavy chain encoded in pBS809 is QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS(SEQ ID NO:70).

[0164] The amino acid sequence of the mature light chain encoded in pBS809 is EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:71).

[0165] The structural diagram of BS809 is as follows Figure 2 shown.

[0166] As mentioned 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 CHO-K1 stable transfectants with protein A affinity column (as mentioned above). SDS-PAGE analysis under reducing conditions showed that each of the three antibodies had only two major bands: about 50kD heavy chain and 25kD light chain (for HuPRO1-IgG1.AA and HuGAB11-IgG1.AA) or about 75kD heavy chain and 25kD light chain (for BS809).

[0167] The biological activity of BS809 in enhancing immune response was analyzed by measuring the expression level of IL-2 in human T cells (Malek, Annu. Rev. Immunol. 26: 453-79, 2008; Liao et al., Immunity 38: 13-25, 2013). Human peripheral blood mononuclear cells (PBMCs) from two donors (3124 and 4143) were obtained from iQ Biosciences (Berkeley, CA). PBMCs were grown in RPMI-1640 containing 10% FBS and 5 μg / ml phytohemagglutinin-L (PHA-L) at 37°C in a 7.5% CO2 incubator for three days to enrich T cells (3-day-old PBMCs). The expression of PD-1, PD-L1 and GITR in 3-day-old PBMCs was confirmed by flow cytometry. Approximately 100,000 3-day-old PBMC cells were cultured in the wells of a 96-well plate pre-coated with 1 μg / ml of mouse anti-human CD3 monoclonal antibody OKT3 at 37°C in a 7.5% CO2 incubator for 1 day; 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 HuGAB11-IgG1.AA (anti-GITR), (iv) 1 μg / ml of HuPRO1-IgG1.AA and 1 μg / ml of HuGAB11-IgG1.AA, and (v) 1 μg / ml of BS809. The IL-2 concentration in the culture supernatant was measured using the Human IL-2 ELISA MAX Standard Kit (BioLegend, San Diego, CA). The results are shown in Figure 2. Figure 6 shown.

[0168] The mean IL-2 concentration in PBMC-derived T cells from donor 3124 was (i) 2,776pg / ml, no antibody (control); (ii) 3,075pg / ml, HuPRO1-IgGl.AA; (iii) 2,577pg, HuGAB11-IgG1.AA; (iv) 2,747pg / ml, a mixture of HuPRO1-IgG1.AA and HuGAB11-IgG1.AA; and (v) 4,826pg / ml, BS809. Compared with the IL-2 levels in untreated control cells, IL-2 expression was significantly increased only in the presence of a bispecific antibody that binds both PD-L1 and GITR. This is because BS809 builds a bridge between cells expressing PD-L1 and T cells expressing GITR, resulting in multimeric cross-linking of CD40 on the surface near cells expressing PD-L1, and leading to the activation of T cells to upregulate the expression of IL-2.

[0169] The mean IL-2 concentration in PBMC-derived T cells from donor 4143 was (i) 658pg / ml, no antibody; (ii) 1,453pg / ml, HuPRO1-IgG1.AA; (iii) 659pg / ml, HuGAB11-IgG1.AA; (iv) 2,136pg / ml, a mixture of HuPRO1-IgG1.AA and HuGAB11-IgG1.AA; and (v) 4,196pg / ml, BS809. Compared with IL-2 levels in untreated control cells, IL-2 expression increased in the presence of HuPRO1-IgG1.AA, most likely because the interaction of PD-L1 with PD-1 to inhibit immune responses was blocked by HuPRO1-IgG1.AA. IL-2 expression was further significantly increased in the presence of BS809 due to (a) its anti-PD-L1 antagonist activity and (b) its bridging ability between PD-L1-expressing cells and GITR-expressing T cells for multimer cross-linking of GITR on the surface, leading to effective activation of T cells.

[0170] Example 8: Bispecific antibody binding to PD-L1 and OX40 (BS813)

[0171] The mammalian expression vector pBS813, designed for expressing a bispecific antibody that binds to human PD-L1 (SEQ ID NO: 69) and human OX40 (SEQ ID NO: 21), was constructed by the method described below: the CH3 coding region in pHuPRO1-IgG1.AA was replaced with the coding region of CH3-HuOHX14DS.scFv (SEQ ID NO: 23).

[0172] The amino acid sequence of the mature heavy chain encoded in pBS813 is (SEQ ID NO:72).

[0173] The amino acid sequence of the mature light chain encoded in pBS813 is identical to the mature light chain sequence encoded in pBS809 (SEQ ID NO: 71).

[0174] The structural diagram of BS813 is as follows Figure 2 shown.

[0175] As described above, three expression vectors, pHuPRO1-IgG1.AA, pHuOHX14DS-IgG1.AA and pBS813, were each stably transfected into CHO-K1. HuPRO1-IgG1.AA, HuOHX14DS-IgG1.AA and BS813 were purified from the culture supernatants 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 the three antibodies had only two major bands: about 50kD heavy chain and 25kD light chain (for HuPRO1-IgG1.AA and HuOHX14DS-IgG1.AA) or about 75kD heavy chain and 25kD light chain (for BS813). NS0 / PD-L1 cells express recombinant human PD-L1 on the surface, which was generated by stably transfecting the expression vector pFCm370 into NS0 cells through electroporation. 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 N-terminus to C-terminus: a synthetic signal peptide (SEQ ID NO: 26), the extracellular region of human PD-L1 (SEQ ID NO: 73), a FLAG polypeptide (SEQ ID NO: 9), and a GPI anchor signal of human CD55 (SEQ ID NO: 10) (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) maintained PD-L1-FLAG-GPI expressed on their surface in DME medium containing 10% FBS at 37°C in a 7.5% CO2 incubator.

[0176] Approximately 200,000 JD / OX40 cells were cultured for one day in 96-well plates at 37° C. in a 7.5% CO 2 incubator 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 absorption) (Jackson ImmunoResearch) to provide the main signal for activating T cells together with the following: 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 NS0 cells were added to reactions a, b, c, d, and e. 200,000 NS0 / PD-L1 cells were added to reactions f, g, h, i, and j. Luciferase activity in culture supernatants was measured in triplicate using QUANTI-Luc reagent (InvivoGen) according to the supplier's protocol. Luminescence was measured using a SynergyHT microplate reader (BioTek). Results are shown in Figure 7 shown.

[0177] The average relative luciferase units (RLU) in JD / OX40 cells were 4683, NS0 cells (a); 6,193, HuPRO1-IgGl.AA and NS0 cells (b); 7,230, HuOHX14DS-IgGl.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 in JD / OX40 cells was significantly increased only in the presence of both a bispecific antibody that binds PD-L1 and OX40 (BS813) and cells expressing PD-L1 (NS0 / PD-L1). The results showed that only BS813 could establish a bridge between JD / OX40 and NS0 / PD-L1 cells, but HuPRO1-IgG1.AA, HuOHX14DS-IgG1.AA and the combination of these two monospecific antibodies did not cause OX40 to aggregate on the surface of JD / OX40 cells close to NS0 / PD-L1 cells and lead to the activation of JD / OX40 cells to increase the expression of luciferase.

[0178] Example 9: Generation of a bispecific antibody (BS841) that binds PD-L1 and OX40

[0179] As described in Example 1, a mouse hybridoma producing an IgG / κ monoclonal antibody PRO2 was isolated that (i) binds to human and cynomolgus monkey PD-L1 and (ii) blocks the interaction between PD-1 and PD-L1. The sequencing and humanization of PRO2 VH and VL were performed as described by Tsurushita et al. (supra).

[0180] The amino acid sequence of humanized PRO2 (HuPRO2) VH is MGWNWIFLFLSGTAGVHCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSS (SEQ ID NO: 74). According to the definition of Kabat et al. (supra), 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.

[0181] The amino acid sequence of humanized PRO2 (HuPRO2) VL is MHFQVQIFSFLLISASVIMSRGDIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK (SEQ ID NO: 78). According to the definition of Kabat et al. (supra), the CDR1, 2 and 3 amino acid sequences of HuPRO2 VL are SASSSVNYMH (SEQ ID NO: 79), STSNLAS (SEQ ID NO: 80) and QQRSSYPLT (SEQ ID NO: 81), respectively. The gene encoding HuPRO2 VL was 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.

[0182] The expression vector pHuPRO2-IgG1.AA has the same structure as pHuM195-IgG1 ( FIG. 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 numbering) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., supra). The new vector pHuPRO2-IgG1.AA expresses humanized anti-PD-L1 IgG1 / κ antibody (HuPRO2-IgG1.AA) in mammalian cells.

[0183] The mammalian expression vector pBS841, designed for the expression of a bispecific antibody that binds 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).

[0184] The amino acid sequence of the mature heavy chain encoded in pBS841 is QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS(SEQ ID NO:82).

[0185] The amino acid sequence of the mature light chain encoded in pBS841 is DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:83).

[0186] Example 10: Generation of a bispecific antibody (BS839) that binds PD-L1 and OX40

[0187] As described in Example 1, a mouse hybridoma producing an IgG / κ monoclonal antibody PRO5 was isolated that (i) binds to human and cynomolgus monkey PD-L1 and (ii) blocks the interaction between PD-1 and PD-L1. Sequencing and humanization of PRO5 VH and VL were performed as described by Tsurushita et al. (supra).

[0188] The amino acid sequence of humanized PRO5 (HuPRO5) VH is MMVLSLLYLLTALPGILSQVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSS (SEQ ID NO: 84). According to the definition of Kabat et al. (supra), 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 was 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.

[0189] The amino acid sequence of humanized PRO5 (HuPRO5) VL is MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK (SEQ ID NO: 88). According to the definition of Kabat et al. (supra), the CDR1, 2 and 3 amino acid sequences of HuPRO5 VL are SASSSVSYMH (SEQ ID NO: 89), DTSKLAS (SEQ ID NO: 90) and FQGSGYPFT (SEQ ID NO: 91), respectively. The gene encoding HuPRO5 VL was 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 pHuPRO5-IgG1.AA has the same structure as pHuM195-IgG1 ( FIG. 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 numbering) in CH2 are replaced with alanine residues (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et al., supra). The new vector pHuPRO5-IgG1.AA expresses humanized anti-PD-L1 IgG1 / κ antibody (HuPRO5-IgG1.AA) in mammalian cells.

[0191] Mammalian expression vector pBS839, designed for expressing a bispecific antibody that binds to human PD-L1 (SEQ ID NO: 69) and human OX40 (SEQ ID NO: 21), is expressed by The coding region of CH3-HuOHX14DS.scFv (SEQ ID NO: 23) was used to replace the CH3 coding region in pHuPRO5-IgG1.AA.

[0192] The amino acid sequence of the mature heavy chain encoded in pBS839 is QVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS(SEQ ID NO:92).

[0193] The amino acid sequence of the mature light chain encoded in pBS839 is DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:93).

[0194] Example 11: Expression and characterization of bispecific antibodies BS839 and BS841 that bind to PD-L1 and OX40

[0195] 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 CHO-K1 stable transfectants using a protein A affinity column (as described above). SDS-PAGE analysis under reducing conditions was performed to detect the size of the heavy and light chains of the four antibodies.

[0196] The biological activity of BS839 and BS841 in enhancing immune responses was analyzed by measuring the expression level of IL-2 in human T cells. Human PBMCs were grown in RPMI-1640 containing 10% FBS and 10 μg / ml PHA-L for three days as described above. Approximately 100,000 3-day-old PBMC cells were cultured in the wells of a 96-well plate pre-coated with 1 μg / ml mouse anti-human CD3 monoclonal antibody OKT3 for 1 day at 37°C in a 7.5% CO2 incubator, and the culture process was accompanied by: (i) no additional antibodies, (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 IL-2 concentration in the culture supernatant was measured using Human IL-2 ELISA MAX Standard Kit (BioLegend).

[0197] Example 12: Bispecific Antibodies Binding to Cancer Cells and Co-stimulatory Molecules

[0198] Monoclonal antibodies that bind to cancer cells are 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 anti-cancer antibody or its humanized form are cloned into an expression vector, such as pHuM195-IgG1 (Figure 1A). Another monoclonal antibody that binds to an immune co-stimulatory molecule (Bakdash et al., Front. Immunol. 4: Article 53, 2013; Mahoney et al., Nat. Rev. Drug Discov. 14: 561-584, 2015) is isolated using hybridoma or display technology. The VH and VL regions of 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 ID 980250, 2012). The scFv form of the anti-co-stimulatory molecule antibody is fused to the C-terminus of the anti-cancer antibody heavy chain to produce a new vector expressing a bispecific antibody that binds to cancer cells and co-stimulatory molecules. The biological activity of the bispecific antibody produced can (i) establish a bridge between cancer cells and immune cells and (ii) activate such immune cells by multimeric cross-linking of co-stimulatory molecules; the biological activity is tested in an appropriate disease model, for example, a tumor xenograft model of humanized mice with human immune cells (Day et al., Cell 163: 39-53, 2015; Mortonet al., Cancer Res. 76: 6153-6158, 2016).

[0199] Example 13: Bispecific Antibodies Binding Pathogen-Derived Molecules and Co-stimulatory Molecules

[0200] Using hybridoma or display technology as described above, a monoclonal antibody that binds to a pathogen expressed by an infectious agent on the surface of a host cell is isolated. As described above, the VH and VL regions of this isolated anti-pathogen antibody or its humanized form are cloned into an expression vector (e.g., pHuM195-IgG1 (FIG. 1A)). Another monoclonal antibody that binds to an immune co-stimulatory molecule is isolated using hybridoma or display technology. As described above, the VH and VL regions of this isolated anti-co-stimulatory molecule antibody or its humanized form are converted into scFv forms. As described above, in the expression vector of the anti-pathogen IgG antibody, the scFv form of the anti-co-stimulatory antibody is fused to the C-terminus of the anti-pathogen antibody heavy chain. The resulting new vector expresses a bispecific IgG antibody that can bind to cells infected by pathogens and co-stimulatory molecules. The biological activity of the generated bispecific antibodies is tested in appropriate disease models (e.g., humanized mice with human immune cells infected with pathogens), which can (i) establish a bridge between pathogen-infected cells and immune cells, and (ii) activate such immune cells by multimeric cross-linking of co-stimulatory molecules (Shultz et al., Ann. NY Acad. Sci. 1245:50-54, 2011; Leung et al., Eur. J. Immunol. 43:2246-2254, 2013).

[0201] Example 14: Replacement of scFv with a different form of receptor binding module

[0202] The scFv region of the bispecific antibody of the present invention (which provides a second antigen binding site for IgG molecules) is replaced with a polypeptide capable of binding to a cell surface receptor, such as a growth factor, a cytokine, a chemokine, a soluble form of a receptor, a single domain antibody (e.g., VH, VL or VHH ((Holt et al., Trends Biotechnol. 21:484-490, 2003; Bannas et al, Front. Immunol. 8:Article 1603, 2017)) or an antibody mimetic (Yu et al., Annu. Rev. Anal. Chem. 10:293-320, 2017). The generated bispecific antibodies are tested for their biological activity of activating immune cells in appropriate animal disease models (e.g., the above-mentioned models of cancer and infectious diseases) through multivalent cross-linking of co-stimulatory molecules.

[0203] Example 15: Fusion of scFv at the C-terminus of the light chain

[0204] The scFv region of the anti-co-stimulatory molecule is fused to the C-terminus of the light chain of an IgG antibody against a checkpoint molecule, cancer cell or pathogen. The generated bispecific antibody is tested for its biological activity of activating immune cells in appropriate animal disease models (e.g., the above-mentioned cancer and infectious disease models) through multivalent cross-linking of co-stimulatory molecules.

[0205] Example 16: Activation of T cells by BS809

[0206] Approximately 100,000 JD / GITR cells were cultured in 96-well plates at 37°C in a 7.5% CO2 incubator 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), and the culture process was accompanied by: no antibody (a and f), 1 μg / ml HuPRO1-IgG1.AA ( Figure 8 Anti-PD-L1 in (b and g), 1 μg / ml HuGAB11-IgG1.AA ( Figure 8 1 μg / ml of NS0 cells were added to reactions a, b, c, d, and e. 100,000 NS0 / PD-L1 cells were added to reactions f, g, h, i, and j. After 1 day in culture, luciferase activity in the culture supernatants 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). The results are shown in Figure 8 shown.

[0207] The average relative luciferase units (RLU) in the culture supernatants of JD / GITR cells 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; (e) 422,955 for BS809 and NS0 cells. ; (f) NS0 / PD-L1 cells were 405,780; (g) HuPRO1-IgG1.AA and NS0 / PD-L1 cells were 443,588; (h) HuGAB11-IgG1.AA and NS0 / PD-L1 cells were 568,268; (i) HuPRO1-IgG1.AA, HuGAB11-Ig1.AA and NS0 / PD-L1 cells were 569,172; and (j) BS809 and NS0 / PD-L1 cells were 1,035,453. Luciferase activity in JD / GITR cells was significantly increased only in the presence of both a bispecific antibody that binds PD-L1 and GITR (BS809) and cells expressing PD-L1 (NS0 / PD-L1). The results showed that only BS809 could establish a bridge between JD / GITR and NS0 / PD-L1 cells, but HuPRO1-IgG1.AA, HuGAB11-IgG1.AA and the combination of these two monospecific antibodies did not lead to multimeric cross-linking of GITR on the surface of JD / GITR cells close to NS0 / PD-L1 cells, resulting in the activation of JD / GITR cells to increase the expression of luciferase.

[0208] Example 17: Activation of PHA-L-treated PBMCs with BS813, BS841 and BS839

[0209] The biological activity of BS813, BS841 and BS839 in enhancing immune responses was analyzed by measuring IL-2 expression in human T cells. The bispecific antibodies BS813, BS841 and BS839, each binding to PD-L1 and OX40, were purified from the culture supernatant of CHO-K1 cells stably transfected with pBS813, pBS841 and pBS839, respectively, using a protein A affinity column. As described above, human PBMCs were grown in RPMI-1640 medium containing 10% FBS and 10 μg / ml PHA-L for three days (3-day-old PBMCs). Approximately 100,000 3-day-old PBMC cells were cultured in 96-well plates pre-coated with 1 μg / ml mouse anti-human CD3 monoclonal antibody OKT3 at 200 μl / well for 1 day at 37°C in a 7.5% CO2 incubator with (a) no additional antibodies, (b) 1 μg / ml HuPRO1-IgG1.AA ( Fig. 9 1μg / ml of anti-PD-L1#1) and 1μg / ml of HuOHX14DS-IgG1.AA ( Fig. 9 OX40), (c) 1 μg / ml HuPRO2-IgG1.AA ( Fig. 9 1 μg / ml of anti-PD-L1 #2) and HuOHX14DS-IgG1.AA, (d) HuPRO5-IgG1.AA ( Fig. 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. The IL-2 concentration in the culture supernatant was measured using the human IL-2 ELISAMAX standard kit (BioLegend). The results are shown in Fig. 9 .

[0210] The mean IL-2 concentrations in human PBMC-derived T cells were (a) 439 pg / ml, without test antibody (control); (b) 324 pg / ml, combination of HuPRO1-IgG1.AA and HuOHX14DS-IgG1.A; (c) 281 pg / ml, combination of HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA; (d) 53 pg / ml, 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 the IL-2 level of the control or the IL-2 level of the combination of the two parental antibodies of BS813, BS841 and BS839, IL-2 expression was significantly increased only in the presence of one of the bispecific antibodies that bind PD-L1 and OX40 (BS813, BS841 and BS839). This is due to the bridging of PD-L1-expressing cells to OX40-expressing T cells through each of BS813, BS841 and BS839, resulting in multimeric cross-linking of OX40 at a location on the cell surface close to the PD-L1-expressing cells, and resulting in the activation of OX40-expressing T cells to upregulate IL-2 expression.

[0211] Example 18: Activation of SEB-treated PBMCs by BS813, BS841 and BS839

[0212] Superantigens such as SEB (Staphylococcus enterotoxin B) activate T cells by connecting MHC class II molecules on antigen presenting cells to the vβ element of the T cell receptor, resulting in the activation of T cells and the production of cytokines, including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor α (TNFα) and interferon gamma (IFNγ) (see, e.g., Krakauer et al., Toxins (Basel). August 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 in T cell-based cytokine secretion assays to monitor the activation of immune co-stimulatory molecules.

[0213] Approximately 400,000 human PBMCs were cultured in 96-well plates in 200 μl / well of RPMI-1640 medium containing 10% FBS and 1 μg / ml SEB, with (a) no antibody (control), (b) 1 μg / ml HuPRO1-IgG1.AA ( Fig.10 Anti-PD-L1 #1) and 1 μg / ml HuOHX14DS-IgG1.AA ( Fig.10 (c) 1 μg / ml HuPRO2-IgG1.AA ( Fig.10 Anti-PD-L1 #2) and 1 μg / ml HuOHX14DS-IgG1.AA, (d) HuPRO5-IgG1.AA ( Fig.10 (e) 1 μg / ml of BS813, (f) 1 μg / ml of BS841, and (g) 1 μg / ml of BS839. After one day of culture at 37°C in a 7.5% CO2 incubator, the IL-2 concentration in the culture supernatant was measured in triplicate using a human IL-2 ELISAMAX standard kit (BioLegend). The results are shown in Fig.10 .

[0214] The mean IL-2 concentrations were (a) 11.3 ng / ml without the test antibody; (b) 10.5 ng / ml, the combination of HuPRO1-IgG1.AA and HuOHX14DS-IgG1.A; (c) 10.0 ng / ml, the combination of HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA; (d) 10.0 ng / ml, the 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. IL-2 expression was significantly increased only in the presence of either of the bispecific antibodies (BS813, BS841, and BS839) that bind both PD-L1 and OX40 compared to the IL-2 levels of the control in which no antibody was added to SEB-treated PBMCs. The combination of the two parental antibodies of each of BS813, BS841 and BS839 did not increase IL-2 expression above the control level. This result suggests that each of BS813, BS841 and BS839 can establish a bridge between cells expressing PD-L1 and T cells expressing OX40, resulting in multimeric cross-linking of OX40 at a location on the cell surface close to cells expressing PD-L1, and leading to the activation of T cells expressing OX40 to upregulate IL-2 expression.

[0215] Example 19: Bispecific Antibodies with Disulfide-Linked Single-Chain Fvs

[0216] Brinkman et al. previously reported that the introduction of a disulfide bond stabilized the binding of VH and VL in an Fv format (dsFv) by converting an 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 numbering) (Proc. Natl. Acad. Sci. 90:7538-7542, 1993). It was also reported that a pair of cysteine ​​residues were introduced at different VH and VL positions to stabilize the Fv structure (Brinkman et al. supra; Young et al. FEBS Lett. 377:135-139, 1995; Schmiedl et al. Protein Eng. 13:724-730,2000).

[0217] The bispecific anti-PD-L1 / GITR antibody (BS809) of the present invention is modified to stabilize the single-chain Fv structure by introducing two cysteine ​​residues (underlined) in the anti-GITR variable region, one of which is located at position 44 in VH and the other at position 100 in VL, introduced by 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 regions of pBS809 and pBS853 are different. Vectors pBS809 and pBS853 carry the same light chain sequence (SEQ ID NO: 71). The amino acid sequence of the mature heavy chain encoded in pBS853 is: QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (SEQ ID NO: 106). Cysteine ​​substitutions are underlined.

[0218] 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 level of binding to PD-L1 and GITR as BS809 by ELISA.

[0219] Example 20: Bispecific Antibody Binding to PD-L1 and ICOS (BS859)

[0220] ICOS (inducible T cell co-stimulator), also known as CD278, is an immune co-stimulatory molecule belonging to the CD28 family. ICOS is required for multi-crosslinking on the surface of immune cells to initiate intracellular signal transduction, thereby enhancing immune response (Wikenheiser et al., Front. Immunol. 7: article 304, 2016).

[0221] Rat hybridomas producing IgG / κ monoclonal antibody TAM14 that specifically binds to human and cynomolgus monkey ICOS were isolated as described in Example 1, 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 human gamma-1 heavy chain was used as an immunogen. Sequencing and humanization of TAM14 VH and VL were performed as described by Tsurushita et al. (supra).

[0222] The amino acid sequence of humanized TAM14 (HuTAM14) VH is: MAVLVLLLCLVTFPSCALSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS (SEQ ID NO: 108). According to the definition of Kabat et al. (supra), 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 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.

[0223] The amino acid sequence of humanized TAM14 (HuTAM14) VL is MRTSIQLLGLLLFWLHDAQCDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIK (SEQ ID NO: 112). According to the definition of Kabat et al. (supra), 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 HuTAM14 VL was 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.

[0224] 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 SpeI and HindIII sites, (ii) the HuTAM14 VL exon is placed between NheI and EcoRI sites, and (iii) the two leucine residues at positions 234 and 235 (EU numbering of Kabat et al.) are replaced with alanine residues in CH2 (L234A and L235A, respectively) (SEQ ID NO: 20) to eliminate effector function (Hezareh et 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.

[0225] A mammalian expression vector designed for expressing a bispecific antibody that binds 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 into a single-chain Fv (scFv) format in the order of VL, polypeptide linker, and VH from N-terminus to C-terminus (HuTAM14.scFv; SEQ ID NO: 117). The N-terminus of HuTAM14.scFv was fused to the penultimate glycine residue in CH3 of pHuPRO1-IgG1.AA, and the polypeptide linker separated them (CH3-HuTAM14.scFv; SEQ ID NO: 118). In addition, in CH2, the two leucine residues at positions 234 and 235 (EU numbering) were replaced with alanine residues (L234A and L235A, respectively). The resulting vector, pBS859, expresses a bispecific IgG antibody, termed BS859, that binds human PD-L1 and ICOS.

[0226] The amino acid sequence of the mature heavy chain encoded in pBS859 is (SEQ ID NO: 119).

[0227] The amino acid sequence of the mature light chain encoded in pBS859 is identical to the mature light chain sequence encoded in pBS809 (SEQ ID NO: 71).

[0228] The schematic structure of the bispecific IgG antibody of the present invention (e.g., BS859) is as follows: Figure 2 shown.

[0229] Each of the three expression vectors, pHuPRO1-IgG1.AA, pHuTAM14-IgG1.AA and pBS859, was stably transfected into CHO-K1 cells as described above. HuPRO1-IgG1.AA, HuTAM14-IgG1.AA and BS859 were purified from the culture supernatant of 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 the three antibodies had only two major bands: about 50 kD heavy chain and 25 kD light chain (for HuPRO1-IgG1.AA and HuTAM14-IgG1.AA) or about 75 kD heavy chain and 25 kD light chain (for BS859).

[0230] The biological activity of BS859 in enhancing immune response was analyzed by measuring the expression levels of IL-2 and IL-10 in human T cells. Human peripheral blood mononuclear cells (PBMCs) from two donors (3486 and 4239) were obtained from iQ Biosciences (Berkeley, CA). Human PBMCs were grown in RPMI-1640 containing 10% FBS and 10 μg / ml phytohemagglutinin-L (PHA-L) at 37°C in a 7.5% CO2 incubator for three days to enrich T cells (3-day-old PBMCs).

[0231] Approximately 200,000 3-day-old PBMC cells were cultured in wells of 96-well plates pre-coated with 1 μg / ml mouse anti-human CD3 monoclonal antibody OKT3 and 1 μg / ml human PD-L1-Fc fusion protein (recombinant human B7-H1-Fc chimera, BioLegend, San Diego, CA) at 37°C in a 7.5% CO2 incubator in the presence of: (i) no additional antibody (control), (ii) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1), (iii) 1 μg / ml HuTAM14-IgG1.AA (anti-ICOS), (iv) 1 μg / ml HuPRO1-IgG1.AA and 1 μg / ml HuTAM14-IgG1.AA, and (v) 1 μg / ml BS859. The concentrations of IL-2 and IL-10 in the culture supernatant were measured using ELISAMAX Standard Set Human IL-2 and ELISAMAX Standard Set Human IL-10 (BioLegend, San Diego, CA), respectively. The results are shown in Fig.11 .

[0232] The average IL-2 concentrations in PBMC-derived T cells from donor 3486 were (i) 3.3 ng / ml, no 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.

[0233] The mean IL-10 concentration in PBMC-derived T cells from donor 3486 was (i) 604 pg / ml, no antibody (control); (ii) 676 pg / ml, HuPRO1-IgG1.AA; (iii) 527 pg / ml, HuTAM14-IgG1.AA; (iv) 699 pg / ml, HuPRO1-IgG1.AA and a mixture of HuTAM14-IgG1.AA; and (v) 1,201 pg / ml, BS859.

[0234] The mean IL-2 concentrations in PBMC-derived T cells from donor 4239 were (i) 0.01 ng / ml, no antibody (control); (ii) 0.15 ng / ml, HuPRO1-IgG1.AA; (iii) 0.01 ng / ml, HuTAM14-IgG1.AA; (iv) 0.06 ng / ml, HuPRO1-IgG1.AA and A mixture of HuTAM14-IgG1.AA; and (v) 13.6 ng / ml, BS859.

[0235] The mean IL-10 concentrations in PBMC-derived T cells from donor 4239 were (i) 238 pg / ml, no antibody (control), (ii) 252 pg / ml, HuPRO1-IgG1.AA; (iii) 217 ​​pg / ml, HuTAM14-IgG1.AA; (iv) 220 pg / ml, HuPRO1-IgG1.AA and A mixture of HuTAM14-IgG1.AA, and (v) 537pg / ml, BS859.

[0236] HuPRO1-IgG1.AA alone was able to increase IL-2 expression in PHA-L-treated PBMCs from two different donors compared to the respective control groups without antibody treatment. BS859 further significantly increased IL-2 expression in both donors.

[0237] The expression of IL-10 in PHA-L-treated PBMCs from two different donors was significantly increased only in the presence of the bispecific antibody of the invention (BS859) that binds both PD-L1 and ICOS.

[0238] Example 21: Bispecific Antibody Binding to PD-L1 and GITR (BS840)

[0239] The mammalian expression vector pBS840 designed for expressing a bispecific antibody that binds 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). The vector pBS840 expresses a bispecific IgG antibody called BS840 that binds to human PD-L1 and GITR.

[0240] The amino acid sequence of the mature heavy chain encoded in pBS840 is QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS(SEQ ID NO:120).

[0241] 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.

[0242] As mentioned above, expression vector pBS840 is stably transfected into CHO-K1 cells. BS840 is purified by protein A affinity column (as mentioned above) from the culture supernatant of the CHO-K1 stable transfectant obtained. By using the gel filtration of Superose 6 size exclusion 10 / 300 column (GE Healthcare Life Sciences, Pittsburgh, PA), the BS840 of A protein purification shows a single main peak of expected size (about 200kDa). The SDS-PAGE analysis under reducing conditions only shows two main bands of about 75kD heavy chain and 25kD light chain.

[0243] The biological activity of BS840 in enhancing immune response was studied using Jurkat Dual cells stably expressing human GITR (JD / GITR) as described above. Fig.12 The average relative luciferase units (RLU) in the culture supernatant of JD / GITR cells were: (a) 354,057, NS0 cells; (b) 341,277, HuPRO2-IgG1.AA ( Fig.12 Anti-PD-L1#2 in BS840; parental monospecific IgG antibody), HuGAB11-IgG1.AA ( Fig.12 ) and NS0 cells; (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 in JD / GITR cells was significantly increased only in the presence of both a bispecific antibody (BS840) that binds PD-L1 and GITR and cells expressing PD-L1 (NS0 / PD-L1).

[0244] Example 22: Bispecific antibody binding to PD-L1 and CD40 (BS846)

[0245] The mammalian expression vector pBS846 designed for expressing a bispecific antibody that binds 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). The vector pBS846 expresses a bispecific IgG antibody called BS846 that binds to human PD-L1 and CD40.

[0246] The amino acid sequence of the mature heavy chain encoded in pBS846 is (SEQ ID NO: 121).

[0247] The amino acid sequence of the mature light chain encoded in pBS846 is identical to the amino acid sequence of the light chain sequence encoded in pBS809 (SEQ ID NO: 71).

[0248] As mentioned above, expression vector pBS846 is stably transfected into CHO-K1 cells. BS846 is purified by protein A affinity column (as mentioned above) from the culture supernatant of their respective CHO-K1 stable transfectants. By using the gel filtration of Superose 6 size exclusion 10 / 300 columns (GE Healthcare Life Sciences, Pittsburgh, PA), the BS846 of A protein purification shows a single main peak of expected size (about 200kDa). The SDS-PAGE analysis under reducing conditions only shows two main bands of about 75kD heavy chain and 25kD light chain.

[0249] As described above, the biological activity of BS846 in enhancing CD40-mediated immune responses was studied using Ramos cells. In (a) no antibody and NS0 cells, (b) 1 μg / ml HuPRO1-IgG1.AA (anti-PD-L1) and NS0 cells, (c) 1 μg / ml HuACS2-IgG1.AA (anti-CD40) and NS0 cells, (d) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA and NS0 cells, (e) 1 μg / ml BS846 and NS0 cells, (f) no antibody and NS0 / PD-L1 cells, (g) 1 μg / ml HuPRO1-IgG1.AA and NS0 / PD-L1 cells, (h) 1 μg / ml HuACS2-IgG1.AA and NS0 / PD-L1 cells, (i) 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA and NS0 / PD-L1 cells, and (j) 1 μg / ml of BS846 and NS0 / PD-L1 cells, Ramos cells were cultured in DME medium containing 10% FBS for three days. The expression of CD95 in Ramos cells was analyzed by flow cytometry, and the flow cytometry used FITC-labeled mouse anti-human CD20 monoclonal antibody (Clone 2H7, BioLegend) to detect Ramos cells, and PE-labeled mouse anti-human CD95 antibody (Clone DX2, BioLegend) was used to monitor CD95 expression.

[0250] The percentages of CD95-positive populations in CD20-positive Ramos cells were: 0.3%, no antibody and NS0 cells (a); 0.3%, 1 μg / ml HuPRO1-IgG1.AA and NS0 cells (b); 0.3%, 1 μg / ml HuACS2-IgG1.AA and NS0 cells (c); 0.3%, 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA and NS0 cells (d); 2.5%, 1 μg / ml BS846 and NS0 cells (e); 0. 2%, no antibody and NS0 / PD-L1 cells (f); 0.3%, 1 μg / ml HuPRO1-IgG1.AA and NS0 / PD-L1 cells (g); 0.3%, 1 μg / ml HuACS2-IgG1.AA and NS0 / PD-L1 cells (h); 0.2%, 1 μg / ml HuPRO1-IgG1.AA, 1 μg / ml HuACS2-IgG1.AA and NS0 / PD-L1 cells (i); and 59.5%, 1 μg / ml BS846 and NS0 / PD-L1 cells (j) ( Fig.13 ). Only BS846, which can establish a bridge between Ramos and NS0 / PD-L1 cells, can significantly increase the expression of CD95 in Ramos cells, because BS846 can cross-link CD40 on the surface of Ramos cells at the junction with NS0 / PD-L1 cells and trigger CD40-mediated intracellular signal transduction to upregulate the expression of CD95 in Ramos cells.

[0251] Example 24: Disulfide-linked single-chain Fv

[0252] 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 replacing the amino acid residue at position 44 in VH with a cysteine ​​residue and replacing another amino acid residue at position 100 in VL with a cysteine ​​residue (Eu numbering) by site-directed mutagenesis to stabilize the structure (Brinkman et al., Proc. Natl. Acad. Sci. 90:7538-7542, 1993).

[0253] The amino acid sequence of the mature HuOHX14DS.scFv (HuOHX14DS.scFv.ds) with two cysteine ​​substitutions is DIQMTQSPSSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQ C LEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS (SEQ ID NO: 122).

[0254] The amino acid sequence of the mature HuACS2.scFv with two cysteine ​​substitutions (HuACS2.scFv.ds) is EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQ C LEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS (SEQ ID NO: 123).

[0255] The amino acid sequence of the mature HuTAM14.scFv with two cysteine ​​substitutions (HuTAM14.scFv.ds) is DIQMTQSPSSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFG C GTKVEIKGGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGK CLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS (SEQ ID NO: 124).

[0256] The generation of the amino acid sequence of the mature HuGAB11.scFv (HuGAB11.scFv.ds) with two cysteine ​​substitutions is described in Example 19 and is DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS (SEQ ID NO: 125).

[0257] In the amino acid sequence shown in this Example 24, the cysteine ​​substitutions are underlined.

[0258] Example 25: Construction of bispecific antibodies (BS883 and BS884) binding to PD-L1 and 4-1BB

[0259] 4-1BB, also known as CD137 and TNFRSF9, is an immune co-stimulatory molecule belonging to the TNF receptor superfamily. 4-1BB needs to be cross-linked on the cell surface of immune cells to initiate intracellular signal transduction, thereby enhancing immune response (Chester et al. 2016 Cancer Immunol. Immunother. 65: 1243-1248).

[0260] As described in Example 1, a mouse hybridoma producing IgG / κ monoclonal antibody FOB5 that specifically binds to human and cynomolgus monkey 4-1BB was isolated. The extracellular region of human 4-1BB fused to the Fc region of human γ-1 heavy chain (h4-1BB-Fc; SEQ ID NO: 126) was used as an immunogen. Sequencing and humanization of FOB5 VH and VL were performed as described by Tsurushita et al. (supra).

[0261] The amino acid sequence of humanized FOB5 (HuFOB5) VH is MERHWIFLFLFSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQGLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS (SEQ ID NO: 127). According to the definition of Kabat et al. (supra), 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 HuFOB5 VH was 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.

[0262] The amino acid sequence of humanized FOB5 (HuFOB5) VL is MDSQAQVLMLLLLWVSGTCGDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK (SEQ ID NO: 131). According to the definition of Kabat et al. (supra), 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 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.

[0263] The expression vector pHuFOB5-IgG1.AA has the same structure as pHuM195-IgG1 (Figure 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 numbering of Kabat et al.) are replaced with alanine residues in CH2 (L234A and L235A, respectively) (SEQ ID NO: 20) 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.

[0264] The mammalian expression vector designed to express a bispecific antibody that binds human PD-L1 (SEQ ID NO: 69) and human 4-1BB (SEQ ID NO: 135) was constructed by modifying pHuPRO1-IgG1.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 into single-chain Fv (scFv) forms according to the order of VL, polypeptide linker and VH from N-terminus to C-terminus. In addition, the glycine residue at position 44 of HuFOB5 VH was changed to a cysteine ​​residue, and the glycine residue at position 100 of HuFOB5 VL was also changed to 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 a polypeptide linker separates them (CH3-HuFOB5.scFv.LH.ds; SEQ ID NO: 137). The CH3 coding region in pHuPRO1-IgG1.AA is replaced with the coding region of CH3-FOB5.scFv.LH.ds. The resulting vector pBS883 expresses a bispecific IgG antibody called BS883 that binds to human PD-L1 and 4-1BB.

[0265] The amino acid sequence of the mature heavy chain encoded in pBS883 is QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFG C GTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQ C LEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS(SEQ ID NO:138). Cysteine substitutions are underlined.

[0266] 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.

[0267] Another mammalian expression vector designed to express a bispecific antibody that binds human PD-L1 (SEQ ID NO: 69) and human 4-1BB (SEQ ID NO: 135) was constructed by modifying pHuPRO1-IgG1.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 into scFv form in the order of VH, polypeptide linker and VL from N-terminus to C-terminus. In addition, the glycine residue at position 44 of HuFOB5 VH was changed to a cysteine ​​residue, and the glycine residue at position 100 of HuFOB5 VL was also changed to 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 a polypeptide 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 that binds to human PD-L1 and 4-1BB.

[0268] The amino acid sequence of the mature heavy chain encoded in pBS884 is QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQ C LEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFG C GTKVEIK (SEQ ID NO: 141). Cysteine ​​substitutions are underlined.

[0269] The amino acid sequence of the mature light chain encoded in pBS8834 is identical to the mature light chain sequence encoded in pBS809 (SEQ ID NO: 71).

[0270] The schematic structures of the bispecific IgG antibodies of the present invention (e.g., BS883 and BS884) are shown in Figure 2 .

[0271] Example 26: Blocking the interaction between PD-L1 and PD-1

[0272] By flow cytometry, NS0 / PD-L1 cells and PD-1-Fc fusion proteins were used to analyze the activity of HuPRO1-IgG1.AA, HuPRO2-IgG1.AA and HuPRO5-IgG1.AA in blocking the interaction of human PD-1 (SEQ ID NO: 142) with human PD-L1. In order to be used as a ligand bound to PD-L1, the extracellular region of human PD-1 was fused to the Fc region of human γ-1 heavy chain (hPD-1-Fc; SEQ ID NO: 143), expressed in NS0 cells, and purified by protein A affinity chromatography. In order to monitor the binding to NS0 / PD-L1 cells, hPD-1-Fc was labeled with fluorescein isothiocyanate (FITC) using standard binding methods. NS0 / PD-L1 cells were incubated with subsaturating concentrations of FITC-labeled hPD-1-Fc and different concentrations of test antibodies (HuPRO1-IgG1.AA, HuPRO2-IgG1.AA, or HuPRO5-IgG1.AA) in FACS buffer (PBS containing 0.5% BSA and 0.05% sodium azide) at 4°C for 60 min. After washing and resuspending in FACS buffer, cells were analyzed by flow cytometry. The half-maximal concentration (IC) of HuPRO1-IgG1.AA to block hPD-1-Fc binding to NS0 / PD-L1 cells 50 ) was 106ng / ml, while HuPRO2-IgG1.AA was 55ng / ml and HuPRO5-IgG1.AA was 87ng / ml. Sequence Listing

[0273] SEQ ID NO:1

[0274] Amino acid sequence of human CD33

[0275] MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSA APTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0276] SEQ ID NO:2

[0277] Amino acid sequence of HuM195 VH

[0278] MGWSWIFFFLLSGTASVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS

[0279] SEQ ID NO:3

[0280] Amino acid sequence of the CH1 region of the human gamma-1 heavy chain encoded in pHuM195-IgG1

[0281] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0282] SEQ ID NO:4

[0283] The amino acid sequence of the hinge region of the human γ-1 heavy chain encoded in pHuM195-IgG1

[0284] EPKSCDKTHTCPPCP

[0285] SEQ ID NO:5

[0286] Amino acid sequence of the CH2 region of the human gamma-1 heavy chain encoded in pHuM195-IgG1

[0287] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0288] SEQ ID NO:6

[0289] Amino acid sequence of the CH3 region of the human gamma-1 heavy chain encoded in pHuM195-IgG1

[0290] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0291] SEQ ID NO:7

[0292] Amino acid sequence of HuM195 VL

[0293] MEKDTLLLWVLLLWVPGSTGDIQMTQSPSSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK

[0294] SEQ ID NO:8

[0295] Amino acid sequence of the human kappa constant region encoded in pHuM195-IgG1

[0296] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0297] SEQ ID NO:9

[0298] Amino acid sequence of FLAG peptide

[0299] DYKDDDDK

[0300] SEQ ID NO:10

[0301] Amino acid sequence of the GPI anchor signal

[0302] PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0303] SEQ ID NO:11

[0304] Amino acid sequence of mature OX40-FLAG-GPI

[0305] LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNC TLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRATGGGDYKDDDDKGGGPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

[0306] SEQ ID NO:12

[0307] Amino acid sequence of HuOHX14DS VH

[0308] MGRLTSSFLLLIVPAYVLSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0309] SEQ ID NO:13

[0310] Amino acid sequence of CDR1 of HuOHX14DS VH

[0311] SYIMH

[0312] SEQ ID NO:14

[0313] Amino acid sequence of CDR2 of HuOHX14DS VH

[0314] YINPYNSGTKYNEKFKG

[0315] SEQ ID NO:15

[0316] Amino acid sequence of CDR3 of HuOHX14DS VH

[0317] YYGSTFTMDY

[0318] SEQ ID NO:16

[0319] Amino acid sequence of HuOHX14DS VL

[0320] MMSSAQFLGLLLLCFQGTRCDIQMTQSPSSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIK

[0321] SEQ ID NO:17

[0322] Amino acid sequence of CDR1 of HuOHX14DS VL

[0323] RASQDIRTYLN

[0324] SEQ ID NO:18

[0325] Amino acid sequence of CDR2 of HuOHX14DS VL

[0326] YTSRLHS

[0327] SEQ ID NO:19

[0328] Amino acid sequence of CDR3 of HuOHX14DS VL

[0329] QQGNTLPWT

[0330] SEQ ID NO:20

[0331] Amino acid sequence of CH2 region with L234A and L235 mutations

[0332] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0333] SEQ ID NO:21

[0334] Amino acid sequence of human OX40

[0335] MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0336] SEQ ID NO:22

[0337] Amino acid sequence of HuOHX14DS.scFv

[0338] DIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0339] SEQ ID NO:23

[0340] CH3 encoded in pBS824 and HuOHX14DS.scFv (CH3-HuOHX14DS.scFv) fused amino acid sequence

[0341] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0342] SEQ ID NO:24

[0343] Amino acid sequence of the mature heavy chain encoded in pBS824

[0344] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0345] SEQ ID NO:25

[0346] The amino acid sequence of the mature light chain encoded in pBS824

[0347] DIQMTQSPSSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0348] SEQ ID NO:26

[0349] Amino acid sequence of synthetic signal peptide

[0350] MGWSWIFFFLLSGTASVLS

[0351] SEQ ID NO:27

[0352] Amino acid sequence of the extracellular region of human CD33 encoded in pFCm267

[0353] MDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVH VTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVH

[0354] SEQ ID NO:28

[0355] Amino acid sequence of human epidermal growth factor receptor (EGFR)

[0356]

[0357] SEQ ID NO:29

[0358] Amino acid sequence of mouse 225 (Ch225) VH

[0359] MAVLALLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

[0360] SEQ ID NO:30

[0361] Amino acid sequence of mouse 225 (Ch225) VL

[0362] MRAPAQFLGFLLFWIPASRSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

[0363] SEQ ID NO:31

[0364] Amino acid sequence of hGITR-Fc

[0365] QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0366] SEQ ID NO:32

[0367] Amino acid sequence of HuGAB11 VH

[0368] MAVLGLLLCLVTFPSCVLSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0369] SEQ ID NO:33

[0370] Amino acid sequence of CDR1 of HuGAB11 VH

[0371] DYGV

[0372] SEQ ID NO:34

[0373] Amino acid sequence of CDR2 of HuGAB11 VH

[0374] VIWGGGGTYYNSALKS

[0375] SEQ ID NO:35

[0376] Amino acid sequence of CDR3 of HuGAB11 VH

[0377] HPYGHFGMDY

[0378] SEQ ID NO:36

[0379] Amino acid sequence of HuGAB11 VL

[0380] MRVLAELLGLLLFCFLGVRCDIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIK

[0381] SEQ ID NO:37

[0382] Amino acid sequence of CDR1 of HuGAB11 VL

[0383] HASQNINVWLS

[0384] SEQ ID NO:38

[0385] Amino acid sequence of CDR2 of HuGAB11 VL

[0386] KASNLHT

[0387] SEQ ID NO:39

[0388] Amino acid sequence of CDR3 of HuGAB11 VL

[0389] QQGQSYPLT

[0390] SEQ ID NO:40

[0391] Amino acid sequence of human GITR

[0392] MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTF SGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0393] SEQ ID NO:41

[0394] Amino acid sequence of HuGAB11.scFv

[0395] DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGG GSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0396] SEQ ID NO:42

[0397] Amino acid sequence of CH3 fused to HuGAB11.scFv (CH3-HuGAB11.scFv) encoded in pBS827

[0398] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0399] SEQ ID NO:43

[0400] Amino acid sequence of the mature heavy chain encoded in pBS827

[0401] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0402] SEQ ID NO:44

[0403] Amino acid sequence of the mature light chain encoded in pBS827

[0404] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0405] SEQ ID NO:45

[0406] Amino acid sequence of mature human GITR

[0407] QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTCTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTD CTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0408] SEQ ID NO:46

[0409] Amino acid sequence of the extracellular region of human EGFR encoded in pFCm507

[0410] LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0411] SEQ ID NO:47

[0412] Amino acid sequence of hCD40-Fc

[0413] EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0414] SEQ ID NO:48

[0415] Amino acid sequence of HuACS2 VH

[0416] MKLWLNWVFLLTLLHGIQCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0417] SEQ ID NO:49

[0418] Amino acid sequence of CDR1 of HuACS2 VH

[0419] SYWLH

[0420] SEQ ID NO:50

[0421] Amino acid sequence of CDR2 of HuACS2 VH

[0422] RIDPNSGDTKYNEKFKS

[0423] SEQ ID NO:51

[0424] Amino acid sequence of CDR3 of HuACS2 VH

[0425] YYYGRSYFDY

[0426] SEQ ID NO:52

[0427] Amino acid sequence of HuACS2 VL

[0428] MDFQVQIFSFLLISAVIISRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIK

[0429] SEQ ID NO:53

[0430] Amino acid sequence of CDR1 of HuACS2 VL

[0431] SASSSVSYMH

[0432] SEQ ID NO:54

[0433] Amino acid sequence of CDR2 of HuACS2 VL

[0434] DTSKLAS

[0435] SEQ ID NO:55

[0436] Amino acid sequence of CDR3 of HuACS2 VL

[0437] QQWSSNPLT

[0438] SEQ ID NO:56

[0439] Amino acid sequence of human CD40

[0440] MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGV SDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0441] SEQ ID NO:57

[0442] Amino acid sequence of HuACS2.scFv

[0443] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0444] SEQ ID NO:58

[0445] Amino acid sequence of the fusion of CH3 encoded in pBS828 and HuACS2.scFv (CH3-HuACS2.scFv)

[0446] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0447] SEQ ID NO:59

[0448] Amino acid sequence of the mature heavy chain encoded in pBS82

[0449] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0450] SEQ ID NO:60

[0451] Amino acid sequence of hPD-L1-Fc

[0452] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0453] SEQ ID NO:61

[0454] Amino acid sequence of HuPRO1 VH

[0455] MEWNWVVLFLLSLTAGVYAQVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSS

[0456] SEQ ID NO:62

[0457] Amino acid sequence of CDR1 of HuPRO1 VH

[0458] SSYIS

[0459] SEQ ID NO:63

[0460] Amino acid sequence of CDR2 of HuPRO1 VH

[0461] WIYAGTGGTSYNQKFTG

[0462] SEQ ID NO:64

[0463] Amino acid sequence of CDR3 of HuPRO1 VH

[0464] HEGVYWYFD

[0465] SEQ ID NO:65

[0466] Amino acid sequence of HuPRO1 VL

[0467] MDFQVQIFSFLLISAVIMSRGEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK

[0468] SEQ ID NO:66

[0469] Amino acid sequence of CDR1 of HuPRO1 VL

[0470] SASSSVSYMH

[0471] SEQ ID NO:67

[0472] Amino acid sequence of CDR2 of HuPRO1 VL

[0473] DTSNLAS

[0474] SEQ ID NO:68

[0475] Amino acid sequence of CDR3 of HuPRO1 VL

[0476] HQRSSYPWT

[0477] SEQ ID NO:69

[0478] Amino acid sequence of human PD-L1

[0479] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

[0480] SEQ ID NO:70

[0481] Amino acid sequence of the mature heavy chain encoded in pBS809

[0482] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0483] SEQ ID NO:71

[0484] The amino acid sequences of the mature light chains encoded in pBS809 and pBS813

[0485] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRPWIYDTSNLASGFPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0486] SEQ ID NO:72

[0487] Amino acid sequence of the mature heavy chain encoded in pBS813

[0488] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0489] SEQ ID NO:73

[0490] Amino acid sequence of the extracellular region of human PD-L1

[0491] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTH

[0492] SEQ ID NO:74

[0493] Amino acid sequence of HuPRO2 VH

[0494] MGWNWIFLFLSGTAGVHCQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTLTADKSSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSS

[0495] SEQ ID NO:75

[0496] Amino acid sequence of CDR1 of HuPRO2 VH

[0497] SYGIN

[0498] SEQ ID NO:76

[0499] Amino acid sequence of CDR2 of HuPRO2 VH

[0500] YIYPGSGGPVYNQKFKG

[0501] SEQ ID NO:77

[0502] Amino acid sequence of CDR3 of HuPRO2 VH

[0503] ENYRYWYFD

[0504] SEQ ID NO:78

[0505] Amino acid sequence of HuPRO2 VL

[0506] MHFQVQIFSFLLISASVIMSRGDIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK

[0507] SEQ ID NO:79

[0508] Amino acid sequence of CDR1 of HuPRO2 VL

[0509] SASSSVNYMH

[0510] SEQ ID NO:80

[0511] Amino acid sequence of CDR2 of HuPRO2 VL

[0512] STSNLAS

[0513] SEQ ID NO:81

[0514] Amino acid sequence of CDR3 of HuPRO2 VL

[0515] QQRSSYPLT

[0516] SEQ ID NO:82

[0517] Amino acid sequence of the mature heavy chain encoded in pBS841

[0518] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0519] SEQ ID NO:83

[0520] Amino acid sequence of the mature light chain encoded in pBS841

[0521] DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0522] SEQ ID NO:84

[0523] Amino acid sequence of HuPRO5 VH MMVLSLLYLLTALPGILSQVQLQESGPGLVKPSQTLSLTCTVSGDSISSGY WNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLS SVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSS

[0524] SEQ ID NO:85

[0525] Amino acid sequence of CDR1 of HuPRO5 VH

[0526] SGWY

[0527] SEQ ID NO:86

[0528] Amino acid sequence of CDR2 of HuPRO5 VH

[0529] YISYTGSTYSNPSLKS

[0530] SEQ ID NO:87

[0531] Amino acid sequence of CDR3 of HuPRO5 VH

[0532] SQNWERAWFAY

[0533] SEQ ID NO:88

[0534] Amino acid sequence of HuPRO5 VL

[0535] MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK

[0536] SEQ ID NO:89

[0537] Amino acid sequence of CDR1 of HuPRO5 VL

[0538] SASSSVSYMH

[0539] SEQ ID NO:90

[0540] Amino acid sequence of CDR2 of HuPRO5 VL

[0541] DTSKLAS

[0542] SEQ ID NO:91

[0543] Amino acid sequence of CDR3 of HuPRO5 VL

[0544] FQGYP

[0545] SEQ ID NO:92

[0546] Amino acid sequence of the mature heavy chain encoded in pBS839

[0547] QVQLQESGPGLVKPSQTLSLTCTVSGDSISSGYWNWIRQPPGKGLEYMGYISYTGSTYSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSQNWERAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQGLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0548] SEQ ID NO:93

[0549] Amino acid sequence of the mature light chain encoded in pBS839

[0550] DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSGYPFTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0551] SEQ ID NO:94

[0552] Amino acid sequence of CDR1 of HuM195 VH

[0553] DYNMH

[0554] SEQ ID NO:95

[0555] Amino acid sequence of CDR2 of HuM195 VH

[0556] YIYPYNGGTGYNQKFKS

[0557] SEQ ID NO:96

[0558] Amino acid sequence of CDR3 of HuM195 VH

[0559] GRPAMDY

[0560] SEQ ID NO:97

[0561] Amino acid sequence of CDR1 of HuM195 VL

[0562] RASESVDNYGISFMN

[0563] SEQ ID NO:98

[0564] Amino acid sequence of CDR2 of HuM195 VL

[0565] AASNQGS

[0566] SEQ ID NO:99

[0567] Amino acid sequence of CDR3 of HuM195 VL

[0568] QQSKEVPWT

[0569] SEQ ID NO:100

[0570] Amino acid sequence of CDR1 of Ch225 VH

[0571] NYGVH

[0572] SEQ ID NO:101

[0573] Amino acid sequence of CDR2 of Ch225 VH

[0574] VIWSGGNTDYNTPFTS

[0575] SEQ ID NO:102

[0576] Amino acid sequence of CDR3 of Ch225 VH

[0577] ALTYYDYEFAY

[0578] SEQ ID NO:103

[0579] Amino acid sequence of CDR1 of Ch225 VL

[0580] RASQSIGTNIH

[0581] SEQ ID NO:104

[0582] Amino acid sequence of CDR2 of Ch225 VL

[0583] YASESIS

[0584] SEQ ID NO:105

[0585] Amino acid sequence of CDR3 of Ch225 VL

[0586] QQNNNWPTT

[0587] SEQ ID NO:106

[0588] Amino acid sequence of the mature heavy chain encoded in pBS853

[0589] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFG C GTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGK C LEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0590] SEQ ID NO:107

[0591] Amino acid sequence of hICOS-Fc

[0592] GEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0593] SEQ ID NO:108

[0594] Amino acid sequence of HuTAM14 VH

[0595] MAVLVLLLCLVTFPSCALSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0596] SEQ ID NO:109

[0597] Amino acid sequence of CDR1 of HuTAM14 VH

[0598] SNSVS

[0599] SEQ ID NO:110

[0600] Amino acid sequence of CDR2 of HuTAM14 VH

[0601] AIWSGGSTDYNSALKS

[0602] SEQ ID NO:111

[0603] Amino acid sequence of CDR3 of HuTAM14 VH

[0604] WEQPYYFDY

[0605] SEQ ID NO:112

[0606] Amino acid sequence of HuTAM14 VL

[0607] MRTSIQLLGLLLFWLHDAQCDIQMTQSPSSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIK

[0608] SEQ ID NO:113

[0609] Amino acid sequence of CDR1 of HuTAM14 VL

[0610] QASQNIYKYIA

[0611] SEQ ID NO:114

[0612] Amino acid sequence of CDR2 of HuTAM14 VL

[0613] YTSTLES

[0614] SEQ ID NO:115

[0615] Amino acid sequence of CDR3 of HuTAM14 VL

[0616] LQYVNLY

[0617] SEQ ID NO:116

[0618] Amino acid sequence of human ICOS

[0619] MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

[0620] SEQ ID NO:117

[0621] Amino acid sequence of HuTAM14.scFv

[0622] DIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0623] SEQ ID NO:118

[0624] The amino acid sequence of CH3 encoded in pBS859 and fused with HuTAM14.scFv (CH3-HuTAM14.scFv)

[0625] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0626] SEQ ID NO:119

[0627] The amino acid sequence of the mature heavy chain encoded in pBS859

[0628] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKGLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0629] SEQ ID NO:120

[0630] Amino acid sequence of the mature heavy chain encoded in pBS840

[0631] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGINWVRQAPGQGLEWIGYIYPGSGGPVYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARENYRYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKALEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0632] SEQ ID NO:121

[0633] The amino acid sequence of the mature heavy chain encoded in pBS846

[0634] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0635] SEQ ID NO:122

[0636] Amino acid sequence of HuOHX14DS.scFv.ds

[0637] DIQMTQSPSSLSASVGDRVTITCRASQDIRTYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGNTLPWTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYIMHWVRQAPGQCLEWIGYINPYNSGTKYNEKFKGRVTITSDKSTSTAYMELSSLRSEDTAVYYCAHYYGSTFTMDYWGQGTTVTVSS

[0638] SEQ ID NO:123

[0639] Amino acid sequence of HuACS2.scFv.ds

[0640] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRRWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPLTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQCLEWIGRIDPNSGDTKYNEKFKSRATITVDKSTSTAYMELSSLRSEDTAVYYCARYYYGRSYFDYWGQGTTVTVSS

[0641] SEQ ID NO:124

[0642] Amino acid sequence of HuTAM14.scFv.ds

[0643] DIQMTQSPSSLSASVGDRVTITCQASQNIYKYIAWYQQKPGKAPKLLIRYTSTLESGTPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYVNLYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSISSNSVSWVRQPPGKCLEWMGAIWSGGSTDYNSALKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCTRWEQPYYFDYWGQGTMVTVSS

[0644] SEQ ID NO:125

[0645] Amino acid sequence of HuGAB11.scFv.ds

[0646] DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKVPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGQSYPLTFGCGTKVEIKGGGGSGGGGSGGGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTDYGVSWIRQPPGKCLEWLGVIWGGGGTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHPYGHFGMDYWGQGTTVTVSS

[0647] SEQ ID NO:126

[0648] Amino acid sequence of h4-1BB-Fc

[0649] FERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0650] SEQ ID NO:127

[0651] Amino acid sequence of HuFOB5 VH

[0652] MERHWIFLFLFSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQGLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0653] SEQ ID NO:128

[0654] Amino acid sequence of CDR1 of HuFOB5 VH

[0655] NYWMH

[0656] SEQ ID NO:129

[0657] Amino acid sequence of CDR2 of HuFOB5 VH

[0658] YINPSTGYTESNQKFKD

[0659] SEQ ID NO:130

[0660] Amino acid sequence of CDR3 of HuFOB5 VH

[0661] SYVGYYYAVDY

[0662] SEQ ID NO:131

[0663] Amino acid sequence of HuFOB5 VL

[0664] MDSQAQVLMLLLLWVSGTCGDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGGGTKVEIK

[0665] SEQ ID NO:132

[0666] Amino acid sequence of CDR1 of HuFOB5 VL

[0667] KSSQSLLYSNNEKNYLA

[0668] SEQ ID NO:133

[0669] Amino acid sequence of CDR2 of HuFOB5 VL

[0670] WASTRES

[0671] SEQ ID NO:134

[0672] Amino acid sequence of CDR3 of HuFOB5 VL

[0673] QQYYSYPYT

[0674] SEQ ID NO:135

[0675] Amino acid sequence of human 4-1BB

[0676] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFND QKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0677] SEQ ID NO:136

[0678] Amino acid sequence of HuFOB5.scFv.LH.ds

[0679] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGS GGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0680] SEQ ID NO:137

[0681] Amino acid sequence of CH3 fused to HuFOB5.scFv.LH.ds (CH3-HuFOB5.scFv.LH.ds) encoded in pBS883

[0682] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0683] SEQ ID NO:138

[0684] Amino acid sequence of the mature heavy chain encoded in pBS883

[0685] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSS

[0686] SEQ ID NO:139

[0687] Amino acid sequence of HuFOB5.scFv.HL.ds

[0688] QVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0689] SEQ ID NO:140

[0690] Amino acid sequence of CH3 encoded in pBS884 fused with HuFOB5.scFv.HL.ds (CH3-HuFOB5.scFv.HL.ds)

[0691] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0692] SEQ ID NO:141

[0693] Amino acid sequence of the mature heavy chain encoded in pBS884

[0694] QVQLVQSGAEVKKPGSSVKVSCKASGFTFSSSYISWVRQAPGQGLEWIAWIYAGTGGTSYNQKFTGRATITVDESTSTAYMELSSLRSEDTAVYYCARHEGVYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYIFINYWMHWVRAPGQCLEWIGYINPSTGYTESNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYVGYYYAVDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNEKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTIFTLTISSLQAEDVAVYYCQQYYSYPYTFGCGTKVEIK

[0695] SEQ ID NO:142

[0696] Amino acid sequence of human PD-1

[0697] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0698] SEQ ID NO:143

[0699] Amino acid sequence of hPD-1-Fc

[0700] LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

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, OX40, CD40, ICOS or 4-1BB. The bispecific antibody according to claim 1 , further comprising an Fc region.

3. The bispecific antibody of claim 1 or 2, wherein the first binding site antagonizes the binding of PD-L1 to PD1, and the second binding site agonizes GITR, OX40, CD40, ICOS or 4-1BB.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the first binding site comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 62-64, 75-77 or 85-87, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 66-68, 79-81 or 89-91, respectively; and the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 33-35, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 37-39, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 17-19, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 17-19, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 17-19, respectively, NO.49-51 of CDR H1, H2 and H3 and a mature light chain variable region including CDRL1, L2 and L3 of SEQ ID NO.53-55, respectively, or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.109-111, respectively and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.113-115, respectively, or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.128-130, respectively and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.132-134, respectively.

5. The bispecific antibody of claim 4, wherein the first binding site comprises a mature heavy chain variable region comprising 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 comprising 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 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, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 16, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 16, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 16, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 16 NO:48 and a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:48 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO:52, or the second binding region comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO:108 and a mature light chain variable region comprising residues 21-126 of SEQ ID NO:112, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:127 and a mature light chain variable region comprising residues 21-133 of SEQ ID NO:

131.

6. The bispecific antibody of claim 4, wherein the first binding site comprises a mature heavy chain variable region comprising 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 comprising 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 chain Fv fragment comprising SEQ ID NO: 22, 41, 57, 117, 122, 123, 124, 125, 136, or 139.

7. The bispecific antibody according to any one of the preceding claims, comprising a first pair of heavy chain and light chain variable regions forming a first binding site and a second pair of heavy chain and light chain variable regions forming a second binding site, wherein the C-terminus of the first pair of heavy chain and light chain variable regions are fused to the N-terminus of the heavy chain and light chain constant regions, and the second pair of heavy chain and light chain variable regions forms a scFv fused to the C-terminus of the heavy chain constant regions, and vice versa.

8. The bispecific antibody of claim 7, 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.

9. The bispecific antibody according to claim 8, wherein the light chain variable region of the scFv is fused to the C-terminus of the heavy chain constant region.

10. The bispecific antibody according to any one of the preceding claims, 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.

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

12. The bispecific antibody according to any one of the preceding claims, comprising two first binding sites and two second binding sites.

13. The bispecific antibody according to any one of the preceding claims, wherein the heavy chain constant region has at least one mutation that reduces FcRγ binding.

14. The bispecific antibody according to any one of claims 1 to 13, wherein the heavy chain constant region has at least one mutation that increases binding to FcRn.

15. A bispecific antibody comprising a first binding site that specifically binds to an antigen on a cancer cell or a pathogen-infected cell and a second binding site that specifically binds to a co-stimulatory molecule, wherein a first pair of heavy chain and light chain variable regions forms the first binding site and a second pair of heavy chain and light chain variable regions forms the second binding site, wherein the C-terminus of the first pair of heavy chain and light chain variable regions is fused to the N-terminus of the heavy chain and light chain constant regions, and the second pair of heavy chain and light chain variable regions forms an scFv fused to the C-terminus of the heavy chain constant region, and vice versa.

16. The bispecific antibody of claim 15, wherein the first binding site is linked to the N-termini of the heavy and light chain constant regions, and the second binding site is linked to the C-termini of the heavy chain constant region.

17. A monoclonal antibody that specifically binds to PD-L1, comprising a mature heavy chain variable region including CDRs H1, H2 and H3 of SEQ ID NO. 62-64, 75-77 or 85-87, respectively, and a mature light chain variable region including CDRL1, L2 and L3 of SEQ ID NO. 66-68, 79-81 or 89-91, respectively.

18. The monoclonal antibody of claim 17, comprising a mature heavy chain variable region comprising 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 comprising residues 23-127 of SEQ ID NO: 65, residues 23-128 of SEQ ID NO: 78, or residues 23-128 of SEQ ID NO: 88, respectively.

19. 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.

20. The bispecific antibody of claim 19, further comprising an IgG Fc region.

21. The bispecific antibody of claim 19 or 20, wherein the second binding site agonizes GITR, OX40, CD40, ICOS or 4-1BB.

22. The bispecific antibody according to any one of claims 19 to 21, wherein the first binding site comprises a mature heavy chain variable region comprising CDRH1, H2 and H3 of SEQ ID NOs. 94 to 96, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 97 to 99, respectively, and the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 33 to 35, respectively, and a mature light chain variable region comprising CDRL1, L2 and L3 of SEQ ID NOs. 37 to 39, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDRH1, H2 and H3 of SEQ ID NOs. 13 to 15, respectively, and a mature light chain variable region comprising CDRL1, L2 and L3 of SEQ ID NOs. 17 to 19, respectively; or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 49 to 51, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. NO.53-55 of CDRL1, L2 and L3; or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.109-111, respectively, and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.113-115, respectively; or the second binding region comprises a mature heavy chain variable region including CDRH1, H2 and H3 of SEQ ID NO.128-130, respectively, and a mature light chain variable region including CDRL1, L2 and L3 of SEQ ID NO.132-134, respectively.

23. The bispecific antibody of claim 22, wherein the first binding site comprises a mature heavy chain variable region comprising residues 20 to 135 of SEQ ID NO: 2 and a mature light chain variable region comprising residues 21-131 of SEQ ID NO: 7, 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, or the second binding site comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 16, or the second binding site comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 48 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO: 52, or the second binding site comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO: 108 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: NO:112, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:127 and a mature light chain variable region comprising residues 21-133 of SEQ ID NO:

131.

24. The bispecific antibody of claim 22, wherein the first binding site comprises a mature heavy chain variable region comprising residues 20 to 135 of SEQ ID NO: 2 and a mature light chain variable region comprising residues 20-131 of SEQ ID NO: 7, and the second binding region comprises a single chain Fv fragment comprising SEQ ID No: 22, 41, 57, 117, 122, 123, 124, 125, 136 or 139.

25. A bispecific antibody comprising 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. The bispecific antibody according to claim 25 , further comprising an Fc region.

27. The bispecific antibody of claim 25 or 26, wherein the first binding site antagonizes the binding of EGFR to EGF, and the second binding site antagonizes GITR, OX40, CD40, ICOS or 4-1BB.

28. The bispecific antibody of any one of claims 25-27, wherein the first binding site comprises a mature heavy chain variable region comprising CDRH1, H2 and H3 of SEQ ID NOs. 100-102, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 103-105, respectively, and the second binding region comprises a mature heavy chain variable region comprising CDRH1, H2 and H3 of SEQ ID NOs. 33-35, respectively, and a mature light chain variable region comprising CDRL1, L2 and L3 of SEQ ID NOs. 37-39, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 17-19, respectively, or the second binding region comprises a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 49-51, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. NO.53-55 of CDR L1, L2 and L3, or the second binding region comprises a mature heavy chain variable region including CDRH1, H2 and H3 of SEQ ID NO.109-111, respectively, and a mature light chain variable region including CDR L1, L2 and L3 of SEQ ID NO.113-115, respectively, or the second binding region comprises a mature heavy chain variable region including CDR H1, H2 and H3 of SEQ ID NO.128-130, respectively, and a mature light chain variable region including CDRL1, L2 and L3 of SEQ ID NO.132-134, respectively.

29. The bispecific antibody of claim 28, wherein the first binding site comprises a mature heavy chain variable region comprising residues 20 to 138 of SEQ ID NO: 29 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 30, 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, or the second binding site comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 16, or the second binding site comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO: 48 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO: 52, or the second binding site comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO: 108 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: NO:112, or the second binding region comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:127 and a mature light chain variable region comprising residues 21-133 of SEQ ID NO:

131.

30. The bispecific antibody of claim 28, wherein the first binding site comprises a mature heavy chain variable region comprising residues 100-138 of SEQ ID NO: 29 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO: 30, and the second binding region comprises a single chain Fv fragment comprising SEQ ID No: 22, 41, 57, 117, 122, 123, 124, 125, 136 or 139.

31. A monoclonal antibody comprising a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 13-15, respectively, and a mature light chain variable region comprising CDRL1, L2 and L3 of SEQ ID NOs. 17-19, respectively, or the monoclonal antibody comprising a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 49-51, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 53-55, respectively, or the monoclonal antibody comprising a mature heavy chain variable region comprising CDRH1, H2 and H3 of SEQ ID NOs. 109-111, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. 113-115, respectively, or the monoclonal antibody comprising a mature heavy chain variable region comprising CDR H1, H2 and H3 of SEQ ID NOs. 128-130, respectively, and a mature light chain variable region comprising CDR L1, L2 and L3 of SEQ ID NOs. The mature light chain variable region of CDRs L1, L2 and L3 of ID NOs. 132-134.

32. The monoclonal antibody of claim 31, comprising 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, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:12 and a mature light chain variable region comprising residues 21-127 of SEQ ID NO:16, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:48 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO:52, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-136 of SEQ ID NO:108 and a mature light chain variable region comprising residues 21-126 of SEQ ID NO:112, or the monoclonal antibody comprises a mature heavy chain variable region comprising residues 20-138 of SEQ ID NO:127 and a mature light chain variable region comprising residues 23-127 of SEQ ID NO:

52. The mature light chain variable region of residues 21-133 of NO:

131.

33. A pharmaceutical composition comprising the bispecific or monoclonal antibody of any one of the preceding claims and a pharmaceutically acceptable carrier.

34. A method of treating or achieving prevention of cancer comprising administering to a subject having cancer or at risk of cancer an effective regimen of a bispecific or monoclonal antibody as defined in any one of claims 1 to 32.

35. A method of treating an infection comprising administering to a subject having or at risk of infection an effective regimen of a bispecific antibody or monoclonal antibody as defined in any one of claims 1 to 32.

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