Modular tetravalent bispecific antibody platform

Abstract

To provide a better tetrameric bispecific antibody (tBsAb) that is easier to prepare, has better clinical stability and efficacy, and / or has reduced systemic toxicity.SOLUTION: In a tetravalent antibody molecule, a bispecific scFv fragment comprises a first binding site for a first antigen, and a second binding site for a second antigen, where the two binding sites are joined together via a linker domain. The linker domain comprises an immunoglobulin hinge region amino acid sequence, and IgG1 CH2 domain amino acid sequence. The linker domain is free from a CH3 domain amino acid sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority to USSN 62 / 408,271, filed October 14, 2016, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates generally to tetrameric bispecific antibody molecules, methods and systems for their production.

[0003] Government interests This invention was made with government support under [ ] awarded by [ ]. The government has certain rights in this invention. [Background technology]

[0004] Background of the Invention Bispecific antibodies (BsAbs) are antibodies or antibody-like molecules with two different binding specificities. BsAbs have wide applications in biomedicine, especially in immunotherapy for tumors. Currently, the focus of immunotherapy research is on how to harness the cell-mediated cytotoxicity of BsAbs to kill tumor cells. BsAbs can be designed to simultaneously target tumor cells and effector cells, triggering the destruction of tumor cells by the effector cells.

[0005] BsAbs can be prepared by methods such as chemical engineering, cell engineering, and genetic engineering. The advantage of genetic engineering is that antibodies can be easily modified, leading to the design and production of many different forms of bispecific antibody fragments, including diabodies, tandem ScFvs, and single-chain diabodies, as well as their derivatives. Because these BsAbs lack the IgG Fc domain, their small size enhances tumor penetration, but they have a significantly shorter half-life in vivo and lack the ADCC effect associated with the antibody constant region.

[0006] To improve stability and therapeutic potential, recombinant genetic engineering has been used to promote heterodimerization in heavy chains and produce higher yields of Fc-containing IgG-like bispecific antibodies. Several rational design strategies have been used to engineer antibody CH3 chains for heterodimerization, including disulfide bonds, salt bridges, and knobs-into-holes. The basis for creating knobs and holes in juxtaposed positions is that knob-hole interactions favor heterodimer formation, while knob-knob and hole-hole interactions prevent homodimer formation due to the lack of favorable interactions. While this knobs-into-hole approach solves the problem of heavy chain homodimerization, it does not address the issue of mispairing of light and heavy chains from two different antibodies. While it is possible to recognize the same light chain from two different antibodies, the possibilities for constructing BsAbs using the sequences of two antibodies that may share a common light chain are severely limited.

[0007] There is a need to provide better BsAbs that are easier to prepare, have better clinical stability and efficacy, and / or have reduced systemic toxicity. Summary of the Invention

[0008] The present invention provides superior tBsAbs that are easier to prepare, have better clinical stability and efficacy, and / or have reduced systemic toxicity.

[0009] One aspect of the present invention relates to a tetravalent antibody molecule. The tetravalent antibody may be a dimer of bispecific scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen. The two binding sites may be linked together via a linker domain. In embodiments, the scFv fragment is a tandem scFv, and the linker domain comprises the amino acid sequence of an immunoglobulin hinge region (e.g., the hinge region of IgG1, IgG2, IgG3, and IgG4). In embodiments, the amino acid sequence of the immunoglobulin hinge region includes a flexible linker amino acid sequence, such as the amino acid sequence TIFF2025142045000002.tif4128. In an embodiment, the linker domain comprises at least a portion of an immunoglobulin Fc domain, for example, an Fc domain of IgG1, IgG2, IgG3, and IgG4. At least a portion of the immunoglobulin Fc domain may be a CH2 domain. The Fc domain may be linked to the C-terminus of the amino acid sequence of an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, and IgG4 hinge region). The linker domain may be flanked at one or both ends by a flexible linker amino acid sequence It may also contain TIFF2025142045000003.tif5128.

[0010] In another aspect, the present invention relates to a nucleic acid construct. The construct may comprise a nucleic acid molecule encoding a light chain variable region and a heavy chain variable region of an antibody capable of specifically binding to a first antigen, a light chain variable region and a heavy chain variable region of an antibody capable of specifically binding to a second antigen, and a linker domain. In an embodiment, the linker domain is the amino acid sequence of an immunoglobulin hinge region (e.g., the hinge region of IgG1, IgG2, IgG3, and IgG4). In an embodiment, the linker domain is at least a portion of an immunoglobulin Fc domain, e.g., the Fc domain of IgG1, IgG2, IgG3, and IgG4. At least a portion of the immunoglobulin Fc domain may be a CH2 domain. The Fc domain may be linked to the C-terminus of the amino acid sequence of the immunoglobulin hinge region (e.g., the hinge region of IgG1, IgG2, IgG3, and IgG4). The linker domain may comprise a flexible linker amino acid sequence at one or both ends. It may also contain TIFF2025142045000004.tif4128.

[0011] Yet another aspect of the present invention is a vector comprising the nucleic acid construct of the above aspect.

[0012] Another aspect of the invention is a host cell (eg, T cells, B cells, follicular T cells, and NK cells) comprising a vector of the above-described aspects.

[0013] One aspect of the present invention is a chimeric antigen receptor (CAR). The CAR may comprise an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising any of the tetravalent antibody molecules of the above aspects or embodiments. In embodiments, the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain, and / or the transmembrane domain comprises CD28. In embodiments, the CAR further comprises one or more additional costimulatory molecules (e.g., CD28, 4-1BB, ICOS, and OX40) located between the transmembrane domain and the intracellular signaling domain, e.g., the CD3 zeta chain.

[0014] Yet another aspect of the present invention is a genetically modified cell. The genetically modified cell expresses and carries a chimeric antigen receptor of any of the above aspects or embodiments on its cell surface membrane. In embodiments, the cell is a T cell (e.g., CD4+ and / or CD8+) or an NK cell. The cell may comprise a mixed population of CD4+ and CD8+ cells.

[0015] An aspect of the present invention is a method of treating a disease or disorder. The method may comprise administering a tetravalent antibody molecule of any of the above aspects or embodiments. In embodiments, the disease or disorder is a CNS-related disease or disorder, e.g., a CNS cancer or a neurodegenerative disease. The CNS cancer may be glioblastoma (GBM). The neurodegenerative disease may be amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, or Huntington's disease. In embodiments, the tetravalent antibody molecule recognizes and / or is bound by a CNS transport receptor, e.g., transferrin receptor (TfR), VCAM-1, CD98hc, and insulin receptor. In this aspect, and any of the above aspects or embodiments, the tetravalent antibody molecule enhances transport across the blood-brain barrier.

[0016] Any of the above aspects or embodiments may be combined with any other aspect or embodiment.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0018] Other features and advantages of the present invention will be apparent from and encompassed by the following detailed description, and the claims. [The present invention 1001] A tetravalent antibody molecule that is a dimer of bispecific scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen, wherein the two binding sites are linked together via a linker domain. [The present invention 1002] The tetravalent antibody molecule of the present invention, wherein said scFv fragment is a tandem scFv. [The present invention 1003] 1001. The tetravalent antibody molecule of the present invention, wherein said linker domain comprises an immunoglobulin hinge region amino acid sequence. [The present invention 1004] The tetravalent antibody molecule of the present invention 1003, wherein said hinge region is an IgG1, IgG2, IgG3, or IgG4 hinge region. [The present invention 1005] The tetravalent antibody molecule of the present invention 1003 or 1004, wherein said immunoglobulin hinge region amino acid sequence is adjacent to a flexible linker amino acid sequence. [The present invention 1006] the flexible linker amino acid sequence is The tetravalent antibody molecule of the present invention 1005, including TIFF2025142045000005.tif4128. [The present invention 1007] The tetravalent antibody molecule of the present invention 1001 or 1002, wherein the linker domain comprises at least a portion of an immunoglobulin Fc domain. [The present invention 1008] The tetravalent antibody molecule of the present invention 1007, wherein said Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. [The present invention 1009] The tetravalent antibody molecule of the present invention 1007 or 1008, wherein at least a portion of the immunoglobulin Fc domain is a CH2 domain. [The present invention 1010] The tetravalent antibody molecule of any one of 1007 to 1009 of the present invention, wherein the Fc domain is linked to the C-terminus of an immunoglobulin hinge region amino acid sequence. [The present invention 1011] The tetravalent antibody molecule of the present invention, wherein the hinge region is an IgG1, IgG2, IgG3, or IgG4 hinge region. [The present invention 1012] The tetravalent antibody molecule of the invention 1009 or 1010, wherein said linker domain comprises a flexible linker amino acid sequence at one or both ends. [The present invention 1013] Each flexible linker amino acid sequence independently has the amino acid sequence The tetravalent antibody molecule of the present invention 1012, including TIFF2025142045000006.tif4128. [The present invention 1014] 1. A nucleic acid construct comprising a nucleic acid molecule encoding: a light chain variable region and a heavy chain variable region of an antibody capable of specifically binding to a first antigen; a light chain variable region and a heavy chain variable region of an antibody capable of specifically binding to a second antigen; and Linker domain. [The present invention 1015] 1014. The nucleic acid construct of the present invention, wherein the linker domain comprises an immunoglobulin hinge region amino acid sequence. [The present invention 1016] 1015. The nucleic acid construct of the present invention, wherein the hinge region is an IgG1, IgG2, IgG3, or IgG4 hinge region. [The present invention 1017] 1017. The nucleic acid construct of any one of claims 1014 to 1016, wherein the linker domain comprises at least a part of an immunoglobulin Fc domain. [The present invention 1018] 1017. The nucleic acid construct of the present invention, wherein the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. [The present invention 1019] The nucleic acid construct of the present invention 1017 or 1018, wherein at least a portion of the immunoglobulin Fc domain is a CH2 domain. [The present invention 1020] The nucleic acid construct of any one of claims 1017 to 1019, wherein the Fc domain is linked to the C-terminus of the hinge region. [The present invention 1021] 1020. The nucleic acid construct of any one of claims 1014 to 1020, wherein the linker domain comprises a flexible linker amino acid sequence at one or both ends. [The present invention 1022] Each flexible linker amino acid sequence independently has the amino acid sequence The nucleic acid construct of the present invention 1021, including TIFF2025142045000007.tif4128. [The present invention 1023] A vector comprising any one of the nucleic acid constructs of the present inventions 1014 to 1022. [The present invention 1024] A host cell comprising a vector of the present invention. [The present invention 1025] The host cell of the present invention 1024, which is a T cell, a B cell, a follicular T cell, or an NK cell. [The present invention 1026] A chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising a tetravalent antibody molecule of the present invention. [The present invention 1027] The CAR of the present invention 1026, wherein the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain. [The present invention 1028] 1026. The CAR of the present invention, wherein the transmembrane domain comprises CD28. [The present invention 1029] The CAR of the present invention 1026, further comprising one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain. [The present invention 1030] The CAR of the present invention, wherein the costimulatory molecule is CD28, 4-1BB, ICOS, or OX40. [The present invention 1031] The CAR of the present invention 1026, wherein the intracellular signaling domain comprises a CD3 zeta chain. [The present invention 1032] A genetically modified cell that expresses and carries any one of the chimeric antigen receptors of the present inventions 1026 to 1031 on its cell surface membrane. [The present invention 1033] The genetically modified cell of the present invention 1032, which is a T cell or an NK cell. [The present invention 1034] The genetically modified cell of the present invention 1033, wherein said T cells are CD4+ or CD8+. [This invention 1035] The genetically modified cells of the present invention 1034, comprising a mixed population of CD4+ and CD8+ cells. [The present invention 1036] A method for treating a disease or disorder, comprising administering any one of the tetravalent antibody molecules of the present inventions 1001 to 1012. [This invention 1037] The method of claim 1036, wherein said disease or disorder is a CNS-related disease or disorder. [The present invention 1038] The method of claim 1037, wherein said CNS-related disease or disorder is CNS cancer. [This invention 1039] The method of claim 1038, wherein said CNS cancer is glioblastoma (GBM). [The present invention 1040] 1037. The method of claim 1037, wherein said CNS-related disease or disorder is a neurodegenerative disease. [The present invention 1041] 1040. The method of claim 1040, wherein said neurodegenerative disease is amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, or Huntington's disease. [The present invention 1042] 1043. The method of any of claims 1037 to 1042, wherein said tetravalent antibody molecule recognizes and / or is bound by a CNS transport receptor. [This invention 1043] 1043. The method of claim 1042, wherein said CNS transport receptor is transferrin receptor (TfR), VCAM-1, CD98hc, or insulin receptor. [This invention 1044] 1043. The method of any of claims 1037 to 1042, wherein said tetravalent antibody molecule enhances transport across the blood-brain barrier. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the design and formation of a tetrameric bispecific antibody (tBsAb). [Figure 2] Schematic diagram of the pcDNA3.1\1 scFv-hinge-scFv expression vector. [Figure 3] Figure 3A is an SDS gel showing the synthetic tetramer linker digested with NotI and BsiWI and inserted into a tetramer expression vector, and Figure 3B is an SDS gel showing the purification of a tBsAb according to the invention. [Figure 4] Figure 4A shows a method for detecting the antibody binding affinity of tBsAb. Figures 4B-4C are graphs showing data when plates were coated with CCR4-Fc (B) or PD-L1-Fc (C) and then incubated with the tetravalent bispecific antibody (anti-CCR4 and anti-PD-L1) and a control antibody. The results showed that this tetravalent antibody was able to bind to both CCR4-Fc and PD-L1-Fc in a dose-dependent manner. [Figure 5] Figure 10 is a graph showing anti-CAIX-PD-L1 bispecific mAb binding to CAIX-Fc fusion protein. [Figure 6] Figure 10 is a graph showing the binding of anti-CAIX-PD-L1 bispecific mAb to PD-L1-Fc fusion protein. [Figure 7] Figure 7A shows how linker length can be varied to optimize binding of bispecific mAbs, and Figure 7B is a schematic representation of the tBsAb sequence. [Figure 8]Figure 8A shows the binding of αGITR-αPD-L1 tBsAb. The tBsAb binds to the GITR protein on T cells and PD-L1 on tumor cells. Figure 8B shows a schematic diagram of the tBsAb format, which is achieved via interchain disulfide bond formation between cysteine ​​residues in the hinge region. [Figure 9] Figure 9A shows the basic structure of two scFvs that combine to form a tBsAb. Figure 9B shows a bispecific dimeric taFv against GITR and PD-L1. Each VH and VL pair is linked by a 15-residue linker to form an scFv. The two scFvs are connected by a linker-hinge-linker (55 residues). The hinge region contains two cysteine ​​residues, allowing the two taFvs to pair via disulfide bridges under oxidizing conditions. [Figure 10] Figure 10A shows the basic structure of a tandem scFv. Figure 10B shows a trifunctional tBsAb against GITR and PD-L1, which has an additional CH2 domain. Each VH and VL pair is connected by a 15-residue linker. The two scFvs are connected by a linker-hinge-CH2-linker domain. The hinge region contains two cysteine ​​residues, allowing the two tandem scFvs to pair via disulfide bridges under oxidizing conditions. The N-terminus of the CH2 domain can bind to Fc-γ or C1q. The resulting format is a trifunctional tBsAb. [Figure 11] Mechanism of action of αGITR-αPD-L1 tBsAb. In Figure 11A, tumor cells overexpress PD-L1 protein. PD-1 / PD-L1 interaction inhibits effective T cell activation and promotes immunosuppression and adaptive immune tolerance. In Figure 11B, αGITR-αPD-L1 tBsAb can enhance immune responses. The αPD-L1 arm blocks the PD-1 / PD-L1 pathway, thereby inhibiting T cell exhaustion and abolishing Treg suppression. The αGITR arm acts as an agonist for the costimulatory GITR receptor, resulting in upregulation of GITR expression, which enhances T cell activation and proliferation. [Figure 12]Schematic diagram of the αGITR-αPD-L1 cloning process. The donor vector and pcDNA3.4 expression vector were digested with SfiI and NotI restriction enzymes. The VHGITR-VLGITR genes were isolated and then ligated together. The final plasmid yielded the αGITR-αPD-L1 clone. [Figure 13] Schematic diagram of the cloning process for the control plasmid (1). The pcDNA3.1 vector and the expression vector pcDNA3.4 were digested with SfiI and NotI restriction enzymes. The VHF10-VLF10 gene was isolated and then ligated into the pcDNA3.4 expression vector. The final plasmid yielded the αGITR-αPD-L1 clone. [Figure 14] Schematic diagram of the cloning process for control plasmids (2) and (3). The isolated F10 VH and VL DNA and the recipient vector pcDNA3.4 were digested with BsiWI and BamHI restriction enzymes and then ligated together. The final plasmids yielded the final αGITR1-αPD-L1 and αGITR10-αPD-L1 clones. [Figure 15] Figure 1 shows a schematic diagram of the cloning strategy for the αGITR-αPD-L1 construct with CH2. A HindIII restriction site was introduced into the pcDNA3.4 expression vector by site-directed mutagenesis. The isolated CH2 fragment and the expression vector were then digested and ligated together to yield the final αGITR-αPD-L1 construct with CH2. [Figure 16]Restriction enzyme analysis (REA) of the recipient pcDNA3.4 vector, six VHGITR-VLGITR inserts, and one VHF10-VLF10 insert. A 1% agarose gel of DNA electrophoresed in TAE buffer, stained with ethidium bromide, shows all plasmids digested with SfiI and NotI restriction enzymes. Lane 1: 7.5 kb of digested recipient pcDNA3.4 vector. The lower band between 500 and 1000 bp represents the previously used scFv insert (from Marasco Laboratory). Lanes 2-6: The lower band represents the 800 bp VHGITR-linker-VLGITR insert. The larger bands clustering at 8 kb represent the double-digested derivative vector. Lane 7: The 800 bp VHF10-linker-VLF10 insert is visualized in the lower band clustering at 500 to 1000 bp. The "bp" in the lanes represents a 1 kb DNA ladder (NEB). [Figure 17] REA of VHF10-linker-VLF10 cDNA and recipient pcDNA3.4 expression vectors (VHGITR1-VLGITR1 or VHGITR10-VLGITR10, respectively). A 1% agarose gel of DNA electrophoresed in TAE buffer, stained with ethidium bromide, is shown. The recipient expression vector and insert were digested with BsiWI and BamHI restriction enzymes. Lane 1: A single band converging at 800 bp represents the VHF10-linker-VLF10 (scFv) fragment isolated by PCR. Lanes 2 and 3: The top two bands visualize the pcDNA3.4 expression vectors containing VHGITR1-VLGITR1 (lane 2) and VHGITR10-VLGITR10 (lane 3). Both contain 7500 bp and are detectable by the correct level of ladder. The lower bands at 500-1000 bp in lanes 2 and 3 represent the VHPD-L1-VLPD-L1 fragments digested and separated from their vectors. The "bp" in the lanes corresponds to the 1 kb DNA ladder (NEB). [Figure 18]SDS-PAGE analysis of purified tBsAb. Coomassie blue-stained SDS gels of proteins electrophoresed in MES buffer are shown. 3-5 μg protein samples were loaded and separated on the gel under reducing (A) and non-reducing (B) conditions. Lanes 1-8: Under non-reducing conditions, SDS-PAGE revealed two major bands for each protein. The upper band had an apparent molecular weight of 80-115 kDa, and the lower band had a molecular weight of 70-80 kDa. In non-reducing SDS-gel analysis, several weak high-molecular-weight bands (>180 kDa) were observed. Under reducing conditions (10% DTT, 70°C for 10 min), SDS-gel analysis showed only a single band with an apparent molecular weight of 70-80 kDa. Lane 9: Under non-reducing conditions, a single band with an apparent molecular weight of just over 140 kDa was observed. Visualization of the two bands under reducing conditions shows the separated heavy and light chains (50 kDa and 25 kDa), highlighting the correct expression of αGITR IgG. The kDa in the lanes represent a benchmark prestained protein ladder (Invitrogen) under the corresponding conditions (4-12% gel concentration run in MES buffer). [Figure 19-1] Figure 1 shows the ELISA absorbance values ​​of αGITR-αPD-L1 tBsAb, F10-αPD-L1 tBsAb, and αPD-L1 mAb tested against passively immobilized PD-L1 antigen. A range of concentrations of αGITR-αPD-L1 (0.0001 mg / mL to 1 mg / mL, horizontal axis) were subjected to ELISA for PD-L1 antigen. Results show the mean and standard deviation of absorbance at 450 nm (vertical axis). Each sample was tested in triplicate at each concentration. Raw signal intensities were corrected for background signal by subtracting the mean signal from wells incubated without primary antibody from the mean signal from wells with primary antibody added. [Figure 19-2]Figure 1 shows the ELISA absorbance values ​​of αGITR-αPD-L1 tBsAb, F10-αPD-L1 tBsAb, and αPD-L1 mAb tested against passively immobilized PD-L1 antigen. A range of concentrations of αGITR-αPD-L1 (0.0001 mg / mL to 1 mg / mL, horizontal axis) were subjected to ELISA for PD-L1 antigen. Results show the mean and standard deviation of absorbance at 450 nm (vertical axis). Each sample was tested in triplicate at each concentration. Raw signal intensities were corrected for background signal by subtracting the mean signal from wells incubated without primary antibody from the mean signal from wells with primary antibody added. [Figure 20] Cell-based ELISA testing the binding of αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies to acetone-methanol-fixed GITR+-expressing CF2 cells. F10-αPD-L1 antibody represents a negative control. All antibodies were tested using 1:3 serial dilutions ranging from 3.3 mg / mL to 0.0046 mg / mL. All antibodies were tested on 1000 GITR+ CF2 cells per well. Each bar represents the mean obtained from triplicate samples (deviation indicated by the bar). Raw signal intensities were corrected for background signal by subtracting the mean signal from wells incubated without primary antibody from the mean signal from wells with primary antibody added. [Figure 21] Cell-based ELISA testing the binding of αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies to GITR+-expressing CF2 cells fixed with 8% paraformaldehyde. F10-αPD-L1 antibody represents a negative control. All antibodies were tested using 1:3 serial dilutions ranging from 3.3 mg / mL to 0.0046 mg / mL. All antibodies were tested on 1000 GITR+ CF2 cells per well. Each bar represents the mean obtained from triplicate samples (deviation indicated by the bar). Raw signal intensities were corrected for background signal by subtracting the mean signal from wells incubated without primary antibody from the mean signal from wells with primary antibody added. [Figure 22] This is a cell-based ELISA testing the binding of αGITR10-αPD-L1 and a commercially available αGITR10 mAb antibody to GITR+-expressing CF2 cells fixed with 8% paraformaldehyde. F10-αPD-L1 antibody represents a negative control. All antibodies were tested using 1:2 serial dilutions ranging from 5 mg / mL to 0.078 mg / mL. All antibodies were tested on 10,000 GITR+ CF2 cells per well. Each bar represents the average obtained from triplicate samples (deviation indicated by the bar). Raw signal intensities were corrected for background signal by subtracting the average signal from wells incubated without primary antibody from the average signal from wells with primary antibody added. [Figure 23A] Flow cytometry analysis of fluorescently activated αGITR10-αPD-L1 tBsAb (anti-His Alexa 488 (APC) conjugated) tested on GITR+ CF2 cells. [Figure 23B] Flow cytometry analysis of fluorescently activated αGITR10 IgG Ab (anti-human IgG Fc (conjugated to FITC)) tested on GITR+ CF2 cells. The horizontal line indicates the fluorescence intensity signal, and the vertical axis indicates cell number. Each individual image represents different concentrations of αGITR10-αPD-L1 with a constant cell number. [Figure 24A] Flow cytometry analysis of fluorescently activated αGITR1-αPD-L1 tBsAb (anti-His Alexa 488 (APC) conjugated) tested on GITR+ CF2 cells. Each individual image represents different concentrations of αGITR10-αPD-L1 with a constant cell number. The horizontal line indicates the fluorescence intensity signal, and the vertical axis indicates cell number. Each individual image represents different concentrations of αGITR10 IgG with a constant cell number. [Figure 24B]Flow cytometry analysis of fluorescently activated αGITR10 IgG Ab (anti-human IgG Fc (conjugated to FITC)) tested on GITR+ CF2 cells. The horizontal line indicates the fluorescence intensity signal, and the vertical axis indicates the cell number. Each individual image represents different concentrations of αGITR10 IgG with a constant cell number. [Figure 25] Restriction enzyme analysis (REA) of 16 clones. Shown is a 1% agarose gel of DNA electrophoresed in TAE buffer, stained with ethidium bromide. All plasmids were digested with HindIII and BamHI restriction enzymes. Two bands are shown in lane 10: the band clustered between 6 and 8 kb represents the digested recipient pcDNA3.4 vector (7.5 kb). The lower band between 500 and 1000 bp indicates that the fragments isolated with HindIII and BamHI restriction enzymes are close to the expected theoretical size of 800 bp. The "bp" in the lane represents the 1 kb DNA ladder. [Figure 26] REA of clone 10 (GITR10-PDL1 with HindIII) and GITR10-PDL1 (without the HindIII restriction site). Shown is a 1% agarose gel of DNA electrophoresed in TAE buffer, stained with ethidium bromide. Both plasmids were digested with HindIII alone (lane 1), NotI alone (lane 2), and HindIII and NotI simultaneously (lane 3). Digestion of clone 10 with a single enzyme (lanes 1 and 2) yielded a single band clustered around 8000 bp. Digestion of clone 10 with both enzymes (lane 3) yielded two fragments, the smaller of which clustered at less than 500 bp. Digestion of αGITR10-αPD-L1 with only the HindIII restriction site (lane 1) revealed supercoiled plasmid DNA. [Figure 27]Restriction enzyme digestion analysis of the vector GITR10-PDL1 (containing a HindIII restriction site) and the CH2 fragment. Shown is a 1% agarose gel of DNA electrophoresed in TAE buffer, stained with ethidium bromide. Lane 1: Single digestion of αGITR-αPD-L1 with HindIII. Lane 2: HindIII-digested CH2 fragment resulted in a band clustered below the 500 bp mark of the ladder. The "bp" in the lane corresponds to the 1 kb DNA ladder. [Figure 28] SDS-PAGE analysis of purified αGITR10-αPD-L1 BsAb with CH2. This figure shows a Coomassie Blue-stained SDS gel of proteins electrophoresed in MES buffer. 3-5 μg protein samples were loaded and separated on the gel under reducing (R) and non-reducing (NR) conditions. Under non-reducing conditions, SDS-PAGE revealed two major bands for each protein. The upper band had an apparent molecular mass of approximately 140 kDa, and the lower band had a molecular mass of 80 kDa, correlating with the theoretical sizes of dimeric (150 kDa) and monomeric (75 kDa) BsAb. SDS-gel analysis under reducing conditions (10% DTT, 70°C for 10 min) showed only a single band with an apparent molecular mass of approximately 80 kDa, confirming the correct expression of tBsAb, which can be reduced by its disulfide bridge in the hinge region. The "kDa" in the lanes represents a benchmark prestained protein ladder (Invitrogen) under the corresponding conditions (4-12% gel concentration run in MES buffer). [Figure 29]This is a cell-based ELISA testing the binding of αGITR10-αPD-L1 and αGITR10 IgG antibodies with CH2 to GITR+-expressing CF2 cells fixed with 8% paraformaldehyde. F10-αPD-L1 antibody represents a negative control. All antibodies were tested using 1:2 serial dilutions ranging from 5 mg / mL to 0.16 mg / mL. All antibodies were tested on 10,000 GITR+ CF2 cells per well. Each bar represents the average obtained from triplicate samples (deviation indicated by the bar). Raw signal intensities were corrected for background signal by subtracting the average signal from wells incubated without primary antibody from the average signal from wells with primary antibody added. [Figure 30] Figure 1 shows the ADCC activity of αGITR10-αPD-L1 antibody with CH2. The ADCC activity of αGITR10-αPD-L1 with CH2 was measured at various concentrations. All antibodies were serially diluted (1:2) from a highest concentration of 20 mg / mL down to 0.02 mg / mL and tested against 20,000 GITR+ CF2 cells per well. The effector cell (GITR+ CF2) to target cell (Wils-2) ratio was 5:1. αGITR IgG represents the positive control, and F10-αPD-L1 is the negative control. The vertical axis represents the raw luciferase activity in effector cells, quantified by luminescence readout. Each sample was tested in triplicate at each concentration, and the mean standard deviation is shown in parentheses. The background of GITR+ CF2 cells in RPMI medium was subtracted from the obtained values. [Figure 31]The αGITR10-αPD-L1 antibody with CH2 mediated mouse complement-mediated CDC activity. The percentage of lysis of GITR+ CF2 cells obtained by serial dilutions of the αGITR10-αPD-L1 tBsAb and control αGITR mAb (positive), αGITR10-αPD-L1 (negative) was determined by CDC assay. All antibodies were serially diluted (1:10) from a top concentration of 20 mg / mL down to 0.2 mg / mL and tested against 10,000 GITR+ CF2 cells per well. The vertical axis represents the percentage of lysis, calculated as the ratio of the signal intensity of the resulting sample to that of completely lysed GITR+ CF2 cells. Each sample was tested in triplicate at each concentration, and the mean standard deviation is shown in parentheses. The background concentration of GITR+ CF2 cells in RPMI medium was subtracted from the obtained values. Each bar represents a simple average obtained from triplicate samples (standard deviations are indicated by brackets). [Figure 32] This is a cell-based ELISA testing the binding of αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies to CF2 cells (without GITR expression) fixed with 8% paraformaldehyde. F10-αPD-L1 antibody represents a negative control. All antibodies were tested using 1:3 serial dilutions ranging from 3.3 mg / mL to 0.0046 mg / mL. All antibodies were tested on 1000 GITR-CF2 cells per well. Each bar represents the average obtained from triplicate samples (deviation indicated by the bar). Raw signal intensities were corrected for background signal by subtracting the average signal from wells incubated without primary antibody from the average signal from wells with primary antibody added. [Figure 33]This is a cell-based ELISA testing the binding of αGITR10-αPD-L1 and αGITR10 IgG antibodies to CF2 cells (without GITR expression) fixed with 8% paraformaldehyde. F10-αPD-L1 antibody represents a negative control. All antibodies were tested using 1:2 serial dilutions ranging from 5 mg / mL to 0.078 mg / mL. All antibodies were tested on 10,000 GITR-CF2 cells per well. Each bar represents the mean obtained from triplicate samples (deviation indicated by the bar). Raw signal intensities were corrected for background signal by subtracting the mean signal from wells incubated without primary antibody from the mean signal from wells with primary antibody added. [Figure 34] This is a cell-based ELISA testing the binding of αGITR10-αPD-L1 and αGITR10 IgG antibodies with CH2 to GITR- CF2 cells fixed with 8% paraformaldehyde. F10-αPD-L1 antibody represents a negative control. All antibodies were tested using 1:2 serial dilutions ranging from 5 mg / mL to 0.16 mg / mL. All antibodies were tested on 10,000 GITR+ CF2 cells per well. Each bar represents the mean obtained from triplicate samples (deviation indicated by the bar). Raw signal intensities were corrected for background signal by subtracting the mean signal from wells incubated without primary antibody from the mean signal from wells with primary antibody added. [Figure 35] Control setup for flow cytometry analysis of fluorescently activated αGITR1-αPD-L1 antibody. Lanes 1-6 show the control setup for GITR+ CF2 cells. Lanes 7-9 refer to the control setup for GITR- CF2 cells. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description of the Invention The present invention relates to bispecific antibodies containing two binding sites for each receptor (ie, tetravalent bispecific antibodies or "tBsAbs"), systems and methods for producing same.

[0021] The clinical development of bispecific antibodies (BsAbs) as therapeutic agents has been hindered by the difficulty of preparing material in sufficient quantity and quality using conventional methods. In recent years, various recombinant methods have been developed for the efficient production of BsAbs, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules most often have two antigen-binding capacities, each monovalent for its target antigen. The present invention provides an efficient approach for the production of novel tetravalent BsAbs (tBsAbs) with two antigen-binding sites for each of their target antigens by genetically engineering scFV bispecific antibodies and fusing them together.

[0022] Compared with bispecific / bivalent antibodies, tBsAbs bind more efficiently to both of their target antigens and are more effective at blocking ligand binding to receptors. Furthermore, expression of tBsAbs in mammalian cells resulted in higher production levels and better antibody activity. Importantly, tBsAbs exhibit greater stability and a longer half-life than monovalent bispecific antibodies. One drawback of monovalent bispecific antibodies is their small size and therefore short serum half-life, which necessitates continuous administration at low doses for several weeks. In contrast, the longer half-life of the tBsAbs of the present invention overcomes this challenge and is therefore more suitable for clinical applications. This design and expression of tBsAbs should be applicable to any antigen-specificity pair.

[0023] Preferably, the tBsAb is specific for BMCA, CAIX, CCR4, PD-L1, PD-L2, PD1, glucocorticoid-induced tumor necrosis factor receptor (GITR), severe acute respiratory syndrome (SARS), influenza, flavivirus, or Middle East respiratory syndrome (MERS).

[0024] Exemplary antibodies useful in constructing tBsAbs according to the present invention include, for example, the antibodies disclosed in WO / 2005 / 060520, WO / 2006 / 089141, WO / 2007 / 065027, WO / 2009 / 086514, WO / 2009 / 079259, WO / 2011 / 153380, WO / 2014 / 055 897, WO2015 / 143194, WO2015 / 164865, WO2013 / 166500, WO2014 / 144061, WO2016 / 057488, WO2016 / 054638, WO / 2016 / 164835, PCT / US2016 / 026232, PCT / US2017 / 050093, PCT / US2017 / 050327, and PCT / US2017 / 043504.

[0025] PDL1(68) An exemplary anti-PDL1 antibody has SEQ ID NO: 1485 VH The nucleotide sequence has SEQ ID NO: 1487 VLnucleotide sequences, a VH nucleotide sequence having SEQ ID NO: 1485 and a VL nucleotide sequence having SEQ ID NO: 1487, a VH nucleotide sequence having SEQ ID NO: 1489 and a VL nucleotide sequence having SEQ ID NO: 1491, a VH nucleotide sequence having SEQ ID NO: 1493 and a VL nucleotide sequence having SEQ ID NO: 1495, a VH nucleotide sequence having SEQ ID NO: 1497 and a VL nucleotide sequence having SEQ ID NO: 1499, a VH nucleotide sequence having SEQ ID NO: 1501 and a VL nucleotide sequence having SEQ ID NO: 1503, a VH nucleotide sequence having SEQ ID NO: 1505 and a VL nucleotide sequence having SEQ ID NO: 1507, a VH nucleotide sequence having SEQ ID NO: 1509 and a VL nucleotide sequence having SEQ ID NO: 1511, a VH nucleotide sequence having SEQ ID NO: 1513 and a VL nucleotide sequence having SEQ ID NO: 1515, The antibodies include antibodies having a VH nucleotide sequence having SEQ ID NO: 1517 and a VL nucleotide sequence having SEQ ID NO: 1519, a VH nucleotide sequence having SEQ ID NO: 1521 and a VL nucleotide sequence having SEQ ID NO: 1523, a VH nucleotide sequence having SEQ ID NO: 1525 and a VL nucleotide sequence having SEQ ID NO: 1527, a VH nucleotide sequence having SEQ ID NO: 1529 and a VL nucleotide sequence having SEQ ID NO: 1531, a VH nucleotide sequence having SEQ ID NO: 1533 and a VL nucleotide sequence having SEQ ID NO: 1535, and a VH nucleotide sequence having SEQ ID NO: 1537 and a VL nucleotide sequence having SEQ ID NO: 1539.

[0026] An exemplary anti-PDL1 antibody has SEQ ID NO:970 VH The amino acid sequence has SEQ ID NO:971 VLamino acid sequence, a VH amino acid sequence having SEQ ID NO: 1486 and a VL polypeptide sequence having SEQ ID NO: 1488, a VH amino acid sequence having SEQ ID NO: 1490 and a VL polypeptide sequence having SEQ ID NO: 1492, a VH amino acid sequence having SEQ ID NO: 1494 and a VL polypeptide sequence having SEQ ID NO: 1496, a VH amino acid sequence having SEQ ID NO: 1498 and a VL polypeptide sequence having SEQ ID NO: 1500, a VH amino acid sequence having SEQ ID NO: 1502 and a VL polypeptide sequence having SEQ ID NO: 1504, a VH amino acid sequence having SEQ ID NO: 1506 and a VL polypeptide sequence having SEQ ID NO: 1508, a VH amino acid sequence having SEQ ID NO: 1510 and a VL polypeptide sequence having SEQ ID NO: 1512, a VH amino acid sequence having SEQ ID NO: 1514 and a VL polypeptide sequence having SEQ ID NO: 1516, and a VL polypeptide sequence having SEQ ID NO: 1540.

[0027] In other embodiments, the anti-PDL1 antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1541, 1554, and 1569, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1584, 1599, and 1610, respectively; a heavy chain having three CDRs comprising the amino acid sequences of 1543, 1556, and 1571, and a light chain having three CDRs comprising the amino acid sequences of 1586, 1600, and 1612; or a heavy chain having three CDRs comprising the amino acid sequences of 1544, 1557, and 1572, and a light chain having three CDRs comprising the amino acid sequences of 1587, 1601, and 1613; or a heavy chain having three CDRs comprising the amino acid sequences of 1545, 1558, and 1573 and a light chain having three CDRs comprising the amino acid sequences of 1588, 1602, and 1614, or a heavy chain having three CDRs comprising the amino acid sequences of 1546, 1559, and 1574 and a light chain having three CDRs comprising the amino acid sequences of 1589, 1603, and 1615, or a heavy chain having three CDRs comprising the amino acid sequences of 1547, 1560, and 1575 and a light chain having three CDRs comprising the amino acid sequences of 1590, 1604, and 1616, or a heavy chain having three CDRs comprising the amino acid sequences of 1548, 1561, and 1576 and a light chain having three CDRs comprising the amino acid sequences of 1591, 1605, and 1617, or a heavy chain having three CDRs comprising the amino acid sequences of 1541, 1562, and 1577 and a light chain having three CDRs comprising the amino acid sequences of 1592, 1599, and 1618, or a heavy chain having three CDRs comprising the amino acid sequences of 1549, 1563, and 1578 and a light chain having three CDRs comprising the amino acid sequences of 1593, 1606, and 1619, or a heavy chain having three CDRs comprising the amino acid sequences of 1564, 1579 and a light chain having three CDRs comprising the amino acid sequences of 1594, 1607, 1620, or a heavy chain having three CDRs comprising the amino acid sequences of 1551, 1565, 1580 and a light chain having three CDRs comprising the amino acid sequences of 1595, 1599, 1621, or a heavy chain having three CDRs comprising the amino acid sequences of 1542, 1566, 1581 and a light chain having three CDRs comprising the amino acid sequences of 1596, 1599, 1622, orThe antibody has a heavy chain having three CDRs containing the amino acid sequence of 1582 and a light chain having three CDRs containing the amino acid sequences of 1597, 1608, and 1623, or a heavy chain having three CDRs containing the amino acid sequences of 1553, 1568, and 1583 and a light chain having three CDRs containing the amino acid sequences of 1598, 1609, and 1624.

[0028] SARS(26) An exemplary SARS neutralizing antibody has SEQ ID NO: 1626 VH The nucleotide sequence has SEQ ID NO: 1628 VL nucleotide sequence, a VH nucleotide sequence having SEQ ID NO: 1630 and a VL nucleotide sequence having SEQ ID NO: 1639, a VH nucleotide sequence having SEQ ID NO: 1634 and a VL nucleotide sequence having SEQ ID NO: 1640, a VH nucleotide sequence having SEQ ID NO: 1632 and a VL nucleotide sequence having SEQ ID NO: 1641, a VH nucleotide sequence having SEQ ID NO: 1633 and a VL nucleotide sequence having SEQ ID NO: 1642, a VH nucleotide sequence having SEQ ID NO: 1634 and a VL nucleotide sequence having SEQ ID NO: 1643, a VH nucleotide sequence having SEQ ID NO: 1635 and a VL nucleotide sequence having SEQ ID NO: 1644, a VH nucleotide sequence having SEQ ID NO: 1636 and a VL nucleotide sequence having SEQ ID NO: 1645, a VH nucleotide sequence having SEQ ID NO: 1637 and a VL nucleotide sequence having SEQ ID NO: 1646 The antibody includes an antibody having a VL nucleotide sequence having NO:1646.

[0029] CXCR4(33) Exemplary anti-CXCR4 antibodies include antibodies having a VH amino acid sequence having SEQ ID NO:771 and a VL amino acid sequence having SEQ ID NO:779, a VH amino acid sequence having SEQ ID NO:772 and a VL amino acid sequence having SEQ ID NO:780, a VH amino acid sequence having SEQ ID NO:773 and a VL amino acid sequence having SEQ ID NO:781, a VH amino acid sequence having SEQ ID NO:774 and a VL amino acid sequence having SEQ ID NO:782, a VH amino acid sequence having SEQ ID NO:775 and a VL amino acid sequence having SEQ ID NO:783, a VH amino acid sequence having SEQ ID NO:776 and a VL amino acid sequence having SEQ ID NO:784, a VH amino acid sequence having SEQ ID NO:777 and a VL amino acid sequence having SEQ ID NO:785, or a VH amino acid sequence having SEQ ID NO:778 and a VL amino acid sequence having SEQ ID NO:786.

[0030] In other embodiments, the anti-CXCR4 antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 803, 804, and 805, respectively, and a light chain having three CDRs comprising the amino acid sequences of 806, 807, and 808, respectively; a heavy chain having three CDRs comprising the amino acid sequences of 809, 810, and 811, respectively, and a light chain having three CDRs comprising the amino acid sequences of 812, 813, and 814, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of 815, 816, and 817, respectively, and a light chain having three CDRs comprising the amino acid sequences of 818, 819, and 820, respectively; a heavy chain having three CDRs each containing the amino acid sequences of 827, 828, and 829, respectively, and a light chain having three CDRs each containing the amino acid sequences of 830, 831, and 832, respectively; a heavy chain having three CDRs each containing the amino acid sequences of 833, 834, and 835, respectively, and a light chain having three CDRs each containing the amino acid sequences of 836, 837, and 838, respectively; or a heavy chain having three CDRs each containing the amino acid sequences of 839, 840, and 841, respectively, and a light chain having three CDRs each containing the amino acid sequences of 842, 843, and 844, respectively.

[0031] Carbonic anhydrase IX (40) An exemplary anti-CA IX antibody has SEQ ID NO:845 VH The amino acid sequence has SEQ ID NO:846 VLamino acid sequence, a VH amino acid sequence having SEQ ID NO:847 and a VL amino acid sequence having SEQ ID NO:868, a VH amino acid sequence having SEQ ID NO:848 and a VL amino acid sequence having SEQ ID NO:869, a VH amino acid sequence having SEQ ID NO:849 and a VL amino acid sequence having SEQ ID NO:870, a VH amino acid sequence having SEQ ID NO:850 and a VL amino acid sequence having SEQ ID NO:871, a VH amino acid sequence having SEQ ID NO:851 and a VL amino acid sequence having SEQ ID NO:872, a VH amino acid sequence having SEQ ID NO:852 and a VL amino acid sequence having SEQ ID NO:873, a VH amino acid sequence having SEQ ID NO:853 and a VL amino acid sequence having SEQ ID NO:874, a VH amino acid sequence having SEQ ID NO:854 and a VL amino acid sequence having SEQ ID NO:875, a VH amino acid sequence having SEQ ID NO:855 and a VL amino acid sequence having SEQ ID NO:876 a VH amino acid sequence having SEQ ID NO:861 and a VL amino acid sequence having SEQ ID NO:882; a VH amino acid sequence having SEQ ID NO:862 and a VL amino acid sequence having SEQ ID NO:883; a VH amino acid sequence having SEQ ID NO:863 and a VL amino acid sequence having SEQ ID NO:884; a VH amino acid sequence having SEQ ID NO:861 and a VL amino acid sequence having SEQ ID NO:885; a VH amino acid sequence having SEQ ID NO:862 and a VL amino acid sequence having SEQ ID NO:886; VH amino acid sequence having SEQ ID NO:864 and VL amino acid sequence having SEQ ID NO:885, VH amino acid sequence having SEQ ID NO:865 and VL amino acid sequence having SEQ ID NO:886The antibodies include an antibody having a VL amino acid sequence having SEQ ID NO:886, a VH amino acid sequence having SEQ ID NO:866 and a VL amino acid sequence having SEQ ID NO:887, a VH amino acid sequence having SEQ ID NO:867 and a VL amino acid sequence having SEQ ID NO:888.

[0032] In other embodiments, the anti-CA IX antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 803, 804, and 805, respectively, and a light chain having three CDRs comprising the amino acid sequences of 806, 807, and 808, respectively; a heavy chain having three CDRs comprising the amino acid sequences of 899, 915, and 909, and a light chain having three CDRs comprising the amino acid sequences of 905, 906, and 952; a heavy chain having three CDRs comprising the amino acid sequences of 899, 915, and 909, and a light chain having three CDRs comprising the amino acid sequences of 935, 943, and 953; or a heavy chain having three CDRs comprising the amino acid sequences of 899, 915, and 909, and a light chain having three CDRs comprising the amino acid sequences of 899, 915, and 909. a heavy chain having three CDRs comprising the amino acid sequence of 910, 916, 923 and a light chain having three CDRs comprising the amino acid sequence of 936, 944, 955; a heavy chain having three CDRs comprising the amino acid sequence of 999, 915, 909 and a light chain having three CDRs comprising the amino acid sequence of 936, 944, 956; or a heavy chain having three CDRs comprising the amino acid sequence of 911, 917, 924 and a light chain having three CDRs comprising the amino acid sequence of 911, 917, 924. a light chain having three CDRs comprising the amino acid sequences of 37, 945, and 957, or a heavy chain having three CDRs comprising the amino acid sequences of 899, 915, and 909 and a light chain having three CDRs comprising the amino acid sequences of 935, 946, and 958, or a heavy chain having three CDRs comprising the amino acid sequences of 899, 915, and 909 and a light chain having three CDRs comprising the amino acid sequences of 938, 946, and 959, or a heavy chain having three CDRs comprising the amino acid sequences of 899, 915, and 909 and a light chain having three CDRs comprising the amino acid sequences of 905, 946, and 960 or a heavy chain having three CDRs comprising the amino acid sequences of 899, 918, and 925 and a light chain having three CDRs comprising the amino acid sequences of 937, 947, and 955, or a heavy chain having three CDRs comprising the amino acid sequences of 899, 918, and 926 and a light chain having three CDRs comprising the amino acid sequences of 937, 945, and 957, or a heavy chain having three CDRs comprising the amino acid sequences of 912, 919, and 927 and a light chain having three CDRs comprising the amino acid sequences of 937, 943, and 961, ora heavy chain having three CDRs comprising the amino acid sequence of 928 and a light chain having three CDRs comprising the amino acid sequences of 937, 906, and 960, or a heavy chain having three CDRs comprising the amino acid sequences of 899, 918, and 928 and a light chain having three CDRs comprising the amino acid sequences of 937, 906, and 960, or a heavy chain having three CDRs comprising the amino acid sequences of 913, 920, and 929 and a light chain having three CDRs comprising the amino acid sequences of 939, 948, and 962, or a heavy chain having three CDRs comprising the amino acid sequences of 899, 918, and 930 and a light chain having three CDRs comprising the amino acid sequences of 935, 944, and 955, or a heavy chain having three CDRs comprising the amino acid sequences of 899, 921, and 931 and a light chain having three CDRs comprising the amino acid sequences of 935, 944, and 955 or a heavy chain having three CDRs comprising the amino acid sequences of 912, 919, 932 and a light chain having three CDRs comprising the amino acid sequences of 940, 949, 963; or a heavy chain having three CDRs comprising the amino acid sequences of 899, 915, 909 and a light chain having three CDRs comprising the amino acid sequences of 935, 943, 960; or a heavy chain having three CDRs comprising the amino acid sequences of 914, 922, 933 and a light chain having three CDRs comprising the amino acid sequences of 941, 950, 964; or a heavy chain having three CDRs comprising the amino acid sequences of 912, 918, 934 and a light chain having three CDRs comprising the amino acid sequences of 942, 951, 965.

[0033] CC-chemokine receptor 4 (CCR4) (048) An exemplary CC-chemokine receptor 4 (CCR4) antibody has SEQ ID NO:969 VH The nucleotide sequence has SEQ ID NO:971 VL The nucleotide sequence is SEQ ID NO:969 VH Nucleotide sequence and V having SEQ ID NO:972 L The present invention includes antibodies having the nucleotide sequences.

[0034] An exemplary CCR4 antibody has SEQ ID NO:970 VHThe amino acid sequence has SEQ ID NO:971 VL It includes an antibody having an amino acid sequence.

[0035] In other embodiments, the CCR4 antibody has a heavy chain with three CDRs comprising the amino acid sequences of SEQ ID NOs: 973, 974, and 975, respectively, and a light chain with three CDRs comprising the amino acid sequences of 976, 977, and 978, respectively.

[0036] Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (85) Exemplary anti-Middle East Respiratory Syndrome coronavirus (MERS-CoV) antibodies include antibodies having a VH nucleotide sequence having SEQ ID NO:677 and a VL nucleotide sequence having SEQ ID NO:679, a VH nucleotide sequence having SEQ ID NO:681 and a VL nucleotide sequence having SEQ ID NO:683, a VH nucleotide sequence having SEQ ID NO:685 and a VL nucleotide sequence having SEQ ID NO:687, a VH nucleotide sequence having SEQ ID NO:689 and a VL nucleotide sequence having SEQ ID NO:692, a VH nucleotide sequence having SEQ ID NO:693 and a VL nucleotide sequence having SEQ ID NO:695, a VH nucleotide sequence having SEQ ID NO:697 and a VL nucleotide sequence having SEQ ID NO:699, a VH nucleotide sequence having SEQ ID NO:701 and a VL nucleotide sequence having SEQ ID NO:703.

[0037] Exemplary anti-Middle East Respiratory Syndrome coronavirus (MERS-CoV) antibodies include antibodies having a VH amino acid sequence of SEQ ID NO:678 and a VL amino acid sequence having SEQ ID NO:680, a VH amino acid sequence of SEQ ID NO:682 and a VL amino acid sequence having SEQ ID NO:684, a VH amino acid sequence of SEQ ID NO:686 and a VL amino acid sequence having SEQ ID NO:688, a VH amino acid sequence of SEQ ID NO:690 and a VL amino acid sequence having SEQ ID NO:692, a VH amino acid sequence of SEQ ID NO:694 and a VL amino acid sequence having SEQ ID NO:696, a VH amino acid sequence of SEQ ID NO:698 and a VL amino acid sequence having SEQ ID NO:700, and a VH amino acid sequence of SEQ ID NO:702 and a VL amino acid sequence having SEQ ID NO:704.

[0038] In other embodiments, the anti-Middle East Respiratory Syndrome coronavirus (MERS-CoV) antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of 705, 706, and 707 and a light chain having three CDRs comprising the amino acid sequences of 722, 723, and 724; a heavy chain having three CDRs comprising the amino acid sequences of 708, 709, and 710 and a light chain having three CDRs comprising the amino acid sequences of 725, 726, and 727; a heavy chain having three CDRs comprising the amino acid sequences of 711, 712, and 713 and a light chain having three CDRs comprising the amino acid sequences of 728, 729, and 730; and a light chain having three CDRs comprising amino acid sequences of 731, 732, and 733; a heavy chain having three CDRs comprising amino acid sequences of 711, 735, and 716 and a light chain having three CDRs comprising amino acid sequences of 737, 738, and 739; a heavy chain having three CDRs comprising amino acid sequences of 717, 718, and 719 and a light chain having three CDRs comprising amino acid sequences of 736, 742, and 743; and a heavy chain having three CDRs comprising amino acid sequences of 714, 720, and 721 and a light chain having three CDRs comprising amino acid sequences of 740, 729, and 741.

[0039] GITR(93) Exemplary anti-human GITR antibodies include a VH nucleotide sequence having SEQ ID NO: 1361 and a VL nucleotide sequence having SEQ ID NO: 1363, a VH nucleotide sequence having SEQ ID NO: 1365 and a VL nucleotide sequence having SEQ ID NO: 1367, a VH nucleotide sequence having SEQ ID NO: 1369 and a VL nucleotide sequence having SEQ ID NO: 1371, a VH nucleotide sequence having SEQ ID NO: 1381 and a VL nucleotide sequence having SEQ ID NO: 1375, a VH nucleotide sequence having SEQ ID NO: 1377 and a VL nucleotide sequence having SEQ ID NO: 1379, a VH nucleotide sequence having SEQ ID NO: 1381 and a VL nucleotide sequence having SEQ ID NO: 1383, a VH nucleotide sequence having SEQ ID NO: 1385 and a VL nucleotide sequence having SEQ ID NO: 1387, a VH nucleotide sequence having SEQ ID NO: 1389 and a VL nucleotide sequence having SEQ ID NO: 1389 The antibodies include antibodies having a VL nucleotide sequence having SEQ ID NO:1391, a VH nucleotide sequence having SEQ ID NO:1393 and a VL nucleotide sequence having SEQ ID NO:1395, a VH nucleotide sequence having SEQ ID NO:1397 and a VL nucleotide sequence having SEQ ID NO:1398, or a VH nucleotide sequence having SEQ ID NO:1401 and a VL nucleotide sequence having SEQ ID NO:1403.

[0040] Exemplary anti-human GITR antibodies include a VH amino acid sequence having SEQ ID NO: 1362 and a VL amino acid sequence having SEQ ID NO: 1364, a VH amino acid sequence having SEQ ID NO: 1366 and a VL polypeptide sequence having SEQ ID NO: 1368, a VH amino acid sequence having SEQ ID NO: 1371 and a VL amino acid sequence having SEQ ID NO: 1372, a VH amino acid sequence having SEQ ID NO: 1382 and a VL amino acid sequence having SEQ ID NO: 1376, a VH nucleotide sequence having SEQ ID NO: 1378 and a VL nucleotide sequence having SEQ ID NO: 1380, a VH amino acid sequence having SEQ ID NO: 1382 and a VL polypeptide sequence having SEQ ID NO: 1384, a VH amino acid sequence having SEQ ID NO: 1386 and a VL amino acid sequence having SEQ ID NO: 1388, a VH amino acid sequence having SEQ ID NO: 1390 and a VL nucleotide sequence having SEQ ID NO: 1391, The antibodies include an antibody having a VL amino acid sequence having SEQ ID NO:1392, a VH amino acid sequence having SEQ ID NO:1394 and a VL polypeptide sequence having SEQ ID NO:1396, a VH amino acid sequence having SEQ ID NO:1399 and a VL amino acid sequence having SEQ ID NO:1400, or a VH amino acid sequence having SEQ ID NO:1402 and a VL amino acid sequence having SEQ ID NO:1404.

[0041] In other embodiments, the anti-human GITR antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of 1405, 1406, and 1407, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1408, 1409, and 1410; a heavy chain having three CDRs comprising the amino acid sequences of 1411, 1412, and 1413, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1414, 1415, and 1416; a heavy chain having three CDRs comprising the amino acid sequences of 1417, 1418, and 1419, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1420, 1421, and 1422; a light chain having three CDRs containing the amino acid sequences 1421, 1422, and 1423, respectively; a heavy chain having three CDRs containing the amino acid sequences 1423, 1424, and 1425, respectively, and a light chain having three CDRs containing the amino acid sequences 1426, 1427, and 1428, respectively; a heavy chain having three CDRs containing the amino acid sequences 1429, 1430, and 1431, respectively; and a light chain having three CDRs containing the amino acid sequences 1432, 1433, and 1434, respectively, and a light chain having three CDRs containing the amino acid sequences 1435, 1436, and 1437, respectively. a heavy chain having three CDRs each containing amino acid sequences 1438, 1439, and 1440; a heavy chain having three CDRs each containing amino acid sequences 1441, 1442, and 1443 and a light chain having three CDRs each containing amino acid sequences 1444, 1445, and 1446; a heavy chain having three CDRs each containing amino acid sequences 1447, 1448, and 1449 and a light chain having three CDRs each containing amino acid sequences 1450, 1451, and 1452; and a light chain having three CDRs each containing amino acid sequences 1453, 1454, and 1455. a heavy chain having three CDRs containing the amino acid sequences 1456, 1457, and 1458, respectively, and a light chain having three CDRs containing the amino acid sequences 1459, 1460, and 1461, respectively, and a light chain having three CDRs containing the amino acid sequences 1462, 1463, and 1464, respectively; or a heavy chain having three CDRs containing the amino acid sequences 1465, 1466, and 1467, respectively, and a light chain having three CDRs containing the amino acid sequences 1468, 1469, and 1470, respectively.

[0042] Flavivirus (73) Exemplary anti-West Nile virus envelope protein E (WNE) antibodies include antibodies having a VH nucleotide sequence having the VH amino acid sequence having SEQ ID NO:1224 and a VL amino acid sequence having SEQ ID NO:1226.

[0043] Exemplary anti-West Nile virus envelope protein E (WNE) antibodies include antibodies having a VH nucleotide sequence having SEQ ID NO:1225 and a VL nucleotide sequence having SEQ ID NO:1227.

[0044] In other embodiments, the anti-West Nile virus envelope protein E (WNE) antibody has a heavy chain with three CDRs comprising the amino acid sequences of 1244, 1245, and 1246, respectively, and a light chain with three CDRs comprising the amino acid sequences of 1247, 1248, and 1249.

[0045] CCR4(65) An exemplary anti-CC-chemokine receptor 4 (CCR4) antibody has SEQ ID NO: 1329 VH The nucleotide sequence has SEQ ID NO: 1331 VL The nucleotide sequence is SEQ ID NO: 1333 VH Nucleotide sequence and V having SEQ ID NO: 1335 L The nucleotide sequence is SEQ ID NO: 1337 VH Nucleotide sequence and V having SEQ ID NO: 1192 L The nucleotide sequence is SEQ ID NO: 1341 VH The nucleotide sequence has SEQ ID NO: 1343 VL having the nucleotide sequence, or SEQ ID NO: 1357 VH The nucleotide sequence has SEQ ID NO: 1359 VL The present invention includes antibodies having the nucleotide sequences.

[0046] An exemplary anti-CC-chemokine receptor 4 (CCR4) antibody is V having SEQ ID NO:1330. H V having the amino acid sequence SEQ ID NO: 1332 L V having the amino acid sequence SEQ ID NO:1334 H V having the amino acid sequence SEQ ID NO: 1336 L V having the amino acid sequence SEQ ID NO:1338 H V having the amino acid sequence SEQ ID NO: 1340 L V having the amino acid sequence SEQ ID NO:1342 H V having the amino acid sequence SEQ ID NO: 1344 L V having the amino acid sequence SEQ ID NO: 1358 H V having the amino acid sequence SEQ ID NO: 1360 L It includes an antibody having an amino acid sequence.

[0047] In other embodiments, the anti-CC-chemokine receptor 4 (CCR4) antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of 1203, 1208, and 1211, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1207, 1209, and 1216; or a heavy chain having three CDRs comprising the amino acid sequences of 1204, 1208, and 1212, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1207, 1209, and 1217; or a heavy chain having three CDRs comprising the amino acid sequences of 1204, 1208, and 1213, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1207, 1209, and 1217. a heavy chain having three CDRs comprising the amino acid sequences of 1205, 1208, and 1214, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1207, 1209, and 1218; a heavy chain having three CDRs comprising the amino acid sequences of 1206, 1208, and 1210, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1207, 1209, and 1220; or a heavy chain having three CDRs comprising the amino acid sequences of 1202, 1208, and 1210, respectively, and a light chain having three CDRs comprising the amino acid sequences of 1207, 1209, and 1219.

[0048] Human immunoglobulin heavy chain variable region germline gene VH1-69(57) Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibodies include antibodies having a VH nucleotide sequence having SEQ ID NO:1153 and a VL nucleotide sequence having SEQ ID NO:1155, or a VH nucleotide sequence having SEQ ID NO:1163 and a VL nucleotide sequence having SEQ ID NO:1155.

[0049] An exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody is a VH1-69 antibody having SEQ ID NO:1154. H V having the amino acid sequence SEQ ID NO: 1156 L V having the amino acid sequence SEQ ID NO: 1164H V having the amino acid sequence SEQ ID NO: 1156 L It includes an antibody having an amino acid sequence.

[0050] In other embodiments, the anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody has a heavy chain with three CDRs comprising the amino acid sequences of 1157, 1158, and 1159, respectively, and a light chain with three CDRs comprising the amino acid sequences of 1160, 1161, and 1162.

[0051] Influenza (49) Exemplary anti-influenza antibodies include a VH nucleotide sequence having SEQ ID NO:981 and a VL nucleotide sequence having SEQ ID NO:983, a VH nucleotide sequence having SEQ ID NO:985 and a VL nucleotide sequence having SEQ ID NO:989, a VH nucleotide sequence having SEQ ID NO:987 and a VL nucleotide sequence having SEQ ID NO:991, a VH nucleotide sequence having SEQ ID NO:993 and a VL nucleotide sequence having SEQ ID NO:997, a VH nucleotide sequence having SEQ ID NO:995 and a VL nucleotide sequence having SEQ ID NO:999, a VH nucleotide sequence having SEQ ID NO:1001 and a VL nucleotide sequence having SEQ ID NO:1005, a VH nucleotide sequence having SEQ ID NO:1003 and a VL nucleotide sequence having SEQ ID NO:1007, a VH nucleotide sequence having SEQ ID NO:1009 and a VL nucleotide sequence having SEQ ID NO:1011, The antibodies include an antibody having a VH nucleotide sequence having SEQ ID NO:1013 and a VL nucleotide sequence having SEQ ID NO:1015, as well as an antibody having a VH nucleotide sequence having SEQ ID NO:1017 and a VK nucleotide sequence having SEQ ID NO:1019, a VH nucleotide sequence having SEQ ID NO:1020 and a VL nucleotide sequence having SEQ ID NO:1022.

[0052] Exemplary anti-influenza antibodies include a VH amino acid sequence having SEQ ID NO:982 and a VL amino acid sequence having SEQ ID NO:984, a VH amino acid sequence having SEQ ID NO:986 and a VL amino acid sequence having SEQ ID NO:988, a VH amino acid sequence having SEQ ID NO:986 and a VL amino acid sequence having SEQ ID NO:990, a VH amino acid sequence having SEQ ID NO:992 and a VL amino acid sequence having SEQ ID NO:994, a VH amino acid sequence having SEQ ID NO:992 and a VL amino acid sequence having SEQ ID NO:996, a VH amino acid sequence having SEQ ID NO:998 and a VL amino acid sequence having SEQ ID NO:1000, a VH amino acid sequence having SEQ ID NO:998 and a VL amino acid sequence having SEQ ID NO:1002, a VH amino acid sequence having SEQ ID NO:1004 and a VL amino acid sequence having SEQ ID NO:1006, These antibodies include an antibody having a VH amino acid sequence having SEQ ID NO:1008 and a VL amino acid sequence having SEQ ID NO:1010, a VH amino acid sequence having SEQ ID NO:1012 and a VK amino acid sequence having SEQ ID NO:1014, and a VH amino acid sequence having SEQ ID NO:1016 and a VL amino acid sequence having SEQ ID NO:1018.

[0053] In other embodiments, the anti-influenza antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of 1023, 1031, and 1039 and a light chain having three CDRs comprising the amino acid sequences of 1047, 1059, and 1071; a heavy chain having three CDRs comprising the amino acid sequences of 1023, 1032, and 1040 and a light chain having three CDRs comprising the amino acid sequences of 1048, 1060, and 1072; a heavy chain having three CDRs comprising the amino acid sequences of 1025, 1032, and 1040 and a light chain having three CDRs comprising the amino acid sequences of 1057, 1059, and 1071; a light chain having three CDRs containing amino acid sequences of 1069 and 1081; a heavy chain having three CDRs containing amino acid sequences of 1026, 1033, and 1041, and a light chain having three CDRs containing amino acid sequences of 1049, 1061, and 1073; a heavy chain having three CDRs containing amino acid sequences of 1026, 1033, and 1041, and a light chain having three CDRs containing amino acid sequences of 1054, 1066, and 1078; a heavy chain having three CDRs comprising amino acid sequences of 1027, 1034, and 1042 and a light chain having three CDRs comprising amino acid sequences of 1056, 1068, and 1080; a heavy chain having three CDRs comprising amino acid sequences of 1028, 1035, and 1043 and a light chain having three CDRs comprising amino acid sequences of 1051, 1063, and 1065; a heavy chain having three CDRs comprising the amino acid sequences 1029, 1037, and 1045 and a light chain having three CDRs comprising the amino acid sequences 1053, 1065, and 1077; or a heavy chain having three CDRs comprising the amino acid sequences 1030, 1038, and 1046 and a light chain having three CDRs comprising the amino acid sequences 1058, 1070, and 1082.

[0054] Influenza (78) An exemplary anti-influenza antibody has SEQ ID NO:397 VH The nucleotide sequence has SEQ ID NO: 398 VL The nucleotide sequence is SEQ ID NO:399 VH Nucleotide sequence and V having SEQ ID NO: 400 L The nucleotide sequence is SEQ ID NO:401 VH Nucleotide sequence and V having SEQ ID NO: 402 L The nucleotide sequence is SEQ ID NO:403 VH The nucleotide sequence has SEQ ID NO: 404 VL having the nucleotide sequence SEQ ID NO:405 VH The nucleotide sequence has SEQ ID NO: 406 VL having the nucleotide sequence SEQ ID NO:407 VH The nucleotide sequence has SEQ ID NO: 408 VL having the nucleotide sequence SEQ ID NO:409 VH The nucleotide sequence has SEQ ID NO: 410 VL having the nucleotide sequence SEQ ID NO:411 VH The nucleotide sequence has SEQ ID NO: 412 VL having the nucleotide sequence SEQ ID NO:413 VH The nucleotide sequence has SEQ ID NO: 414 VL having the nucleotide sequence SEQ ID NO:415 VH The nucleotide sequence has SEQ ID NO: 416 VL having the nucleotide sequence SEQ ID NO:417 VH The nucleotide sequence has SEQ ID NO: 418 VL having the nucleotide sequence SEQ ID NO:419 VH The nucleotide sequence has SEQ ID NO: 420 VLhaving the nucleotide sequence SEQ ID NO:421 VH The nucleotide sequence has SEQ ID NO: 422 VL having the nucleotide sequence SEQ ID NO:423 VH The nucleotide sequence has SEQ ID NO: 424 VL having the nucleotide sequence SEQ ID NO:425 VH The nucleotide sequence has SEQ ID NO: 426 VL having the nucleotide sequence SEQ ID NO:427 VH The nucleotide sequence has SEQ ID NO: 428 VL having the nucleotide sequence SEQ ID NO:429 VH The nucleotide sequence has SEQ ID NO: 430 VL having the nucleotide sequence SEQ ID NO:431 VH The nucleotide sequence has SEQ ID NO: 432 VL having the nucleotide sequence SEQ ID NO:433 VH The nucleotide sequence has SEQ ID NO: 434 VL having the nucleotide sequence SEQ ID NO:435 VH The nucleotide sequence has SEQ ID NO: 436 VL having the nucleotide sequence SEQ ID NO:437 VH The nucleotide sequence has SEQ ID NO: 438 VL having the nucleotide sequence SEQ ID NO:439 VH The nucleotide sequence has SEQ ID NO: 440 VL having the nucleotide sequence SEQ ID NO:441 VH The nucleotide sequence has SEQ ID NO: 442 VLnucleotide sequence, or a VH nucleotide sequence having SEQ ID NO: 541 and a VL nucleotide sequence having SEQ ID NO: 542, or a VH nucleotide sequence having SEQ ID NO: 543 and a VL nucleotide sequence having SEQ ID NO: 544, or a VH nucleotide sequence having SEQ ID NO: 545 and a VL nucleotide sequence having SEQ ID NO: 546, or a VH nucleotide sequence having SEQ ID NO: 547 and a VL nucleotide sequence having SEQ ID NO: 548, or a VH nucleotide sequence having SEQ ID NO: 549 and a VL nucleotide sequence having SEQ ID NO: 550, or a VH nucleotide sequence having SEQ ID NO: 551 and a VL nucleotide sequence having SEQ ID NO: 552, or a VH nucleotide sequence having SEQ ID NO: 553 and a VL nucleotide sequence having SEQ ID NO: 554, or a VH nucleotide sequence having SEQ ID NO: 555 and a VL nucleotide sequence having SEQ ID NO: 556 or a VH nucleotide sequence having SEQ ID NO: 557 and a VL nucleotide sequence having SEQ ID NO: 558, or a VH nucleotide sequence having SEQ ID NO: 559 and a VL nucleotide sequence having SEQ ID NO: 560, or a VH nucleotide sequence having SEQ ID NO: 561 and a VL nucleotide sequence having SEQ ID NO: 562, or a VH nucleotide sequence having SEQ ID NO: 563 and a VL nucleotide sequence having SEQ ID NO: 564, or a VH nucleotide sequence having SEQ ID NO: 565 and a VL nucleotide sequence having SEQ ID NO: 566, or a VH nucleotide sequence having SEQ ID NO: 567 and a VL nucleotide sequence having SEQ ID NO: 568, or a VH nucleotide sequence having SEQ ID NO: 569 and a VL nucleotide sequence having SEQ ID NO: 570, or a VH nucleotide sequence having SEQ ID NO: 571 and a VL nucleotide sequence having SEQ ID NO: 572. VL nucleotide sequence having SEQ ID NO:572a VH nucleotide sequence having SEQ ID NO:573 and a VL nucleotide sequence having SEQ ID NO:574, or a VH nucleotide sequence having SEQ ID NO:575 and a VL nucleotide sequence having SEQ ID NO:576, or a VH nucleotide sequence having SEQ ID NO:577 and a VL nucleotide sequence having SEQ ID NO:578, or a VH nucleotide sequence having SEQ ID NO:579 and a VL nucleotide sequence having SEQ ID NO:580, or a VH nucleotide sequence having SEQ ID NO:581 and a VL nucleotide sequence having SEQ ID NO:582, or a VH nucleotide sequence having SEQ ID NO:583 and a VL nucleotide sequence having SEQ ID NO:584, or a VH nucleotide sequence having SEQ ID NO:585 and a VL nucleotide sequence having SEQ ID NO:586, or a VH nucleotide sequence having SEQ ID NO:587 and a VL nucleotide sequence having SEQ ID NO:588, or or a VH nucleotide sequence having SEQ ID NO:591 and a VL nucleotide sequence having SEQ ID NO:592; or a VH nucleotide sequence having SEQ ID NO:593 and a VL nucleotide sequence having SEQ ID NO:594; or a VH nucleotide sequence having SEQ ID NO:595 and a VL nucleotide sequence having SEQ ID NO:596; or a VH nucleotide sequence having SEQ ID NO:597 and a VL nucleotide sequence having SEQ ID NO:598; or a VH nucleotide sequence having SEQ ID NO:599 and a VL nucleotide sequence having SEQ ID NO:600.

[0055] An exemplary anti-influenza antibody has SEQ ID NO:469 VH The amino acid sequence has SEQ ID NO: 470 VL having the amino acid sequence SEQ ID NO:471VH V having the amino acid sequence SEQ ID NO:472 L Polypeptide sequence, having SEQ ID NO:473 VH V having the amino acid sequence SEQ ID NO:474 L The amino acid sequence is SEQ ID NO:475 VH The amino acid sequence has SEQ ID NO: 476 VL having the amino acid sequence, or SEQ ID NO:477 VH The nucleotide sequence has SEQ ID NO: 478 VL The nucleotide sequence is SEQ ID NO:479 VH The amino acid sequence has SEQ ID NO: 480 VL having the amino acid sequence SEQ ID NO:481 VH The amino acid sequence has SEQ ID NO: 482 VL having the amino acid sequence SEQ ID NO:483 VH The amino acid sequence has SEQ ID NO: 484 VL The amino acid sequence is SEQ ID NO:485 VH The amino acid sequence has SEQ ID NO: 486 VL having the amino acid sequence SEQ ID NO:487 VH The amino acid sequence has SEQ ID NO: 488 VL having the amino acid sequence SEQ ID NO:489 VH The amino acid sequence has SEQ ID NO: 490 VL having the amino acid sequence SEQ ID NO:491 VH The amino acid sequence has SEQ ID NO: 492 VL having the amino acid sequence SEQ ID NO:493 VH The amino acid sequence has SEQ ID NO: 494 VL having the amino acid sequence SEQ ID NO:495 VH The amino acid sequence has SEQ ID NO: 496 VL having the amino acid sequence SEQ ID NO:497 VHThe amino acid sequence has SEQ ID NO: 498 VL having the amino acid sequence SEQ ID NO:499 VH The amino acid sequence has SEQ ID NO: 500 VL The amino acid sequence is SEQ ID NO:501 VH The amino acid sequence has SEQ ID NO: 502 VL The amino acid sequence is SEQ ID NO:503 VH The amino acid sequence has SEQ ID NO: 504 VL The amino acid sequence is SEQ ID NO:505 VH The amino acid sequence has SEQ ID NO:506 VL having the amino acid sequence SEQ ID NO:507 VH The amino acid sequence has SEQ ID NO: 508 VL having the amino acid sequence SEQ ID NO:509 VH The amino acid sequence has SEQ ID NO:510 VL The amino acid sequence is SEQ ID NO:511 VH The amino acid sequence has SEQ ID NO: 512 VL The amino acid sequence is SEQ ID NO:513 VH The amino acid sequence has SEQ ID NO: 514 VL The amino acid sequence is SEQ ID NO:515 VH The amino acid sequence has SEQ ID NO: 516 VL The amino acid sequence is SEQ ID NO:517 VH The amino acid sequence has SEQ ID NO: 518 VL having the amino acid sequence SEQ ID NO:519 VH The amino acid sequence has SEQ ID NO: 520 VL having the amino acid sequence SEQ ID NO:521 VH The amino acid sequence has SEQ ID NO: 522 VL having the amino acid sequence SEQ ID NO:523 VH The amino acid sequence has SEQ ID NO: 524VL The amino acid sequence is SEQ ID NO:525 VH The amino acid sequence has SEQ ID NO: 526 VL having the amino acid sequence SEQ ID NO:527 VH The amino acid sequence has SEQ ID NO: 528 VL having the amino acid sequence SEQ ID NO:529 VH The amino acid sequence has SEQ ID NO: 530 VL having the amino acid sequence SEQ ID NO:531 VH The amino acid sequence has SEQ ID NO: 532 VL having the amino acid sequence SEQ ID NO:533 VH The amino acid sequence has SEQ ID NO: 534 VL having the amino acid sequence SEQ ID NO:535 VH The amino acid sequence has SEQ ID NO: 536 VL having the amino acid sequence SEQ ID NO:537 VH The amino acid sequence has SEQ ID NO: 538 VL having the amino acid sequence SEQ ID NO:539 VH The amino acid sequence has SEQ ID NO: 540 VLamino acid sequences, a VH amino acid sequence having SEQ ID NO:601 and a VL amino acid sequence having SEQ ID NO:602, a VH amino acid sequence having SEQ ID NO:603 and a VL amino acid sequence having SEQ ID NO:604, a VH amino acid sequence having SEQ ID NO:605 and a VL amino acid sequence having SEQ ID NO:606, a VH amino acid sequence having SEQ ID NO:607 and a VL amino acid sequence having SEQ ID NO:608, a VH amino acid sequence having SEQ ID NO:609 and a VL amino acid sequence having SEQ ID NO:610, a VH amino acid sequence having SEQ ID NO:611 and a VL amino acid sequence having SEQ ID NO:612, a VH amino acid sequence having SEQ ID NO:613 and a VL amino acid sequence having SEQ ID NO:614, a VH amino acid sequence having SEQ ID NO:615 and a VL amino acid sequence having SEQ ID NO:616, a VH amino acid sequence having SEQ ID NO:617 and a VL amino acid sequence having SEQ ID NO:617 a VH amino acid sequence having SEQ ID NO: 627 and a VL amino acid sequence having SEQ ID NO: 628; a VH amino acid sequence having SEQ ID NO: 629 and a VL amino acid sequence having SEQ ID NO: 630; a VH amino acid sequence having SEQ ID NO: 631 and a VL amino acid sequence having SEQ ID NO: 632; a VH amino acid sequence having SEQ ID NO: 633 and a VL amino acid sequence having SEQ ID NO: 634; a VH amino acid sequence having SEQ ID NO: 635 and a VL amino acid sequence having SEQ ID NO: 636; a VH amino acid sequence having SEQ ID NO: 637 and a VL amino acid sequence having SEQ ID NO: 638; a VH amino acid sequence having SEQ ID NO: 639 and a VL amino acid sequence having SEQ ID NO: 640; a VH amino acid sequence having SEQ ID NO: 641 and a VL amino acid sequence having SEQ ID NO: 642; a VH amino acid sequence having SEQ ID NO: 643 and a VL amino acid sequence having SEQ ID NO: 644; a VH amino acid sequence having SEQ ID NO:635 and a VL amino acid sequence having SEQ ID NO:636; a VH amino acid sequence having SEQ ID NO:637 and a VL amino acid sequence having SEQ ID NO:638;a VH amino acid sequence having SEQ ID NO: 647 and a VL amino acid sequence having SEQ ID NO: 648; a VH amino acid sequence having SEQ ID NO: 649 and a VL amino acid sequence having SEQ ID NO: 650; a VH amino acid sequence having SEQ ID NO: 651 and a VL amino acid sequence having SEQ ID NO: 652; a VH amino acid sequence having SEQ ID NO: 653 and a VL amino acid sequence having SEQ ID NO: 654; a VH amino acid sequence having SEQ ID NO: 655 and a VL amino acid sequence having SEQ ID NO: 656; a VH amino acid sequence having SEQ ID NO: 657 and a VL amino acid sequence having SEQ ID NO: 658; a VH amino acid sequence having SEQ ID NO: 659 and a VL amino acid sequence having SEQ ID NO: 660; a VH amino acid sequence having SEQ ID NO: 661 and a VL amino acid sequence having SEQ ID NO: 662; a VH amino acid sequence having SEQ ID NO: 663 and a VL amino acid sequence having SEQ ID NO: 663; These antibodies include an antibody having a VH amino acid sequence having SEQ ID NO:655 and a VL amino acid sequence having SEQ ID NO:656, a VH amino acid sequence having SEQ ID NO:657 and a VL amino acid sequence having SEQ ID NO:658, and a VH amino acid sequence having SEQ ID NO:659 and a VL amino acid sequence having SEQ ID NO:660.

[0056] In other embodiments, the anti-influenza antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1, 37, and 73, respectively, and a light chain having three CDRs comprising the amino acid sequences of 109, 145, and 181, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of 2, 38, and 74, respectively, and a light chain having three CDRs comprising the amino acid sequences of 110, 146, and 182, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of 3, 39, and 75, respectively, and a light chain having three CDRs comprising the amino acid sequences of 111, 147, and 183, respectively; or a heavy chain having three CDRs containing amino acid sequences of 4, 40, and 76 and a light chain having three CDRs containing amino acid sequences of 112, 148, and 184, respectively; a heavy chain having three CDRs containing amino acid sequences of 5, 41, and 77, respectively, and a light chain having three CDRs containing amino acid sequences of 113, 149, and 185, respectively; a heavy chain having three CDRs containing amino acid sequences of 6, 42, and 78, respectively, and a light chain having three CDRs containing amino acid sequences of 114, 150, and 186, respectively; or a heavy chain having three CDRs containing amino acid sequences of 7, 4, and 76, respectively. a heavy chain having three CDRs containing amino acid sequences of 3, 79 and a light chain having three CDRs containing amino acid sequences of 115, 151 and 187, respectively; a heavy chain having three CDRs containing amino acid sequences of 8, 44 and 80, respectively and a light chain having three CDRs containing amino acid sequences of 116, 152 and 188, respectively; a heavy chain having three CDRs containing amino acid sequences of 9, 45 and 81, respectively and a light chain having three CDRs containing amino acid sequences of 117, 153 and 189, respectively; a heavy chain having three CDRs containing an amino acid sequence of 11, 47, and 82 and a light chain having three CDRs containing amino acid sequences of 118, 154, and 190, respectively; a heavy chain having three CDRs containing amino acid sequences of 11, 47, and 83, respectively, and a light chain having three CDRs containing amino acid sequences of 119, 155, and 191, respectively; a heavy chain having three CDRs containing amino acid sequences of 12, 48, and 84, respectively, and a light chain having three CDRs containing amino acid sequences of 120, 156, and 192, respectively; or a heavy chain having three CDRs containing amino acid sequences of 13, 49, and 83, respectively, and a light chain having three CDRs containing amino acid sequences of 119, 155, and 191, respectively.a heavy chain having three CDRs containing an amino acid sequence of 14, 50, and 86, respectively, and a light chain having three CDRs containing amino acid sequences of 122, 158, and 194, respectively; a heavy chain having three CDRs containing amino acid sequences of 15, 51, and 87, respectively, and a light chain having three CDRs containing amino acid sequences of 123, 159, and 195, respectively; or a heavy chain having three CDRs containing amino acid sequences of 16, 52, and 88, respectively; a heavy chain having three CDRs each containing 124, 160, and 196 amino acid sequences, and a light chain having three CDRs each containing 125, 161, and 197 amino acid sequences, or a heavy chain having three CDRs each containing 17, 53, and 89 amino acid sequences, and a light chain having three CDRs each containing 125, 161, and 197 amino acid sequences, or a heavy chain having three CDRs each containing 18, 54, and 90 amino acid sequences, and a light chain having three CDRs each containing 126, 162, and 198 amino acid sequences, or a heavy chain having three CDRs each containing 19, 55, and 91 amino acid sequences, and a light chain having three CDRs each containing 12 a light chain having three CDRs each containing an amino acid sequence of 7, 163, or 199; or a heavy chain having three CDRs each containing an amino acid sequence of 20, 56, or 92, respectively, and a light chain having three CDRs each containing an amino acid sequence of 128, 164, or 200, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 21, 57, or 93, respectively, and a light chain having three CDRs each containing an amino acid sequence of 129, 165, or 201, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 22, 58, or 94, respectively, and a light chain having three CDRs each containing an amino acid sequence of 130, 166, or 202, respectively. a heavy chain having three CDRs each containing an amino acid sequence of 23, 59, and 95, respectively, and a light chain having three CDRs each containing an amino acid sequence of 131, 167, and 203, respectively; a heavy chain having three CDRs each containing an amino acid sequence of 24, 60, and 96, respectively, and a light chain having three CDRs each containing an amino acid sequence of 132, 168, and 204, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 25, 61, and 95, respectively, and a light chain having three CDRs each containing an amino acid sequence of 133, 169, and 205, respectively;Alternatively, a heavy chain having three CDRs each containing an amino acid sequence of 26, 62, or 96, and a light chain having three CDRs each containing an amino acid sequence of 134, 170, or 206, or a heavy chain having three CDRs each containing an amino acid sequence of 27, 63, or 97, and a light chain having three CDRs each containing an amino acid sequence of 135, 171, or 207, or a heavy chain having three CDRs each containing an amino acid sequence of 28, 64, or 98, and a light chain having three CDRs each containing an amino acid sequence of 136, 172, or 208, or a heavy chain having three CDRs each containing an amino acid sequence of 29, 65, or 99, and a light chain having three CDRs each containing an amino acid sequence of 137, 173, and 209, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 30, 66, and 100, respectively, and a light chain having three CDRs each containing an amino acid sequence of 138, 174, and 210, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 31, 67, and 101, respectively, and a light chain having three CDRs each containing an amino acid sequence of 139, 175, and 211, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 32, 68, and 102, respectively. a heavy chain having three CDRs and a light chain having three CDRs each containing an amino acid sequence of 140, 176, or 212; a heavy chain having three CDRs each containing an amino acid sequence of 33, 69, or 103, respectively, and a light chain having three CDRs each containing an amino acid sequence of 141, 177, or 213, respectively; a heavy chain having three CDRs each containing an amino acid sequence of 34, 70, or 104, respectively, and a light chain having three CDRs each containing an amino acid sequence of 142, 178, or 214, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 35, 71, or 105, respectively, and a light chain having the heavy chain having three CDRs each containing an amino acid sequence of 35, 71, or 105, respectively; a light chain having three CDRs each containing an amino acid sequence of 143, 179, or 215, or a heavy chain having three CDRs each containing an amino acid sequence of 36, 72, or 106, and a light chain having three CDRs each containing an amino acid sequence of 144, 180, or 216, or a heavy chain having three CDRs each containing an amino acid sequence of 217, 247, or 277, and a light chain having three CDRs each containing an amino acid sequence of 307, 337, or 367, or a heavy chain having three CDRs each containing an amino acid sequence of 218, 248, or 278, and a light chain having three CDRs each containing an amino acid sequence of 308, 338, ora light chain having three CDRs containing an amino acid sequence of 368; a heavy chain having three CDRs containing amino acid sequences of 219, 249, and 279, respectively, and a light chain having three CDRs containing amino acid sequences of 309, 339, and 369, respectively; a heavy chain having three CDRs containing amino acid sequences of 220, 250, and 280, respectively, and a light chain having three CDRs containing amino acid sequences of 310, 340, and 370, respectively; or a heavy chain having three CDRs containing amino acid sequences of 221, 251, and 281, respectively, and a light chain having three CDRs containing amino acid sequences of 311, 341, and 371, respectively. a light chain having three CDRs each containing an amino acid sequence of 222, 252, and 282, respectively, and a light chain having three CDRs each containing an amino acid sequence of 312, 342, and 372, respectively; a heavy chain having three CDRs each containing an amino acid sequence of 223, 253, and 283, respectively, and a light chain having three CDRs each containing an amino acid sequence of 313, 343, and 373, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 224, 254, and 284, respectively, and a light chain having three CDRs each containing an amino acid sequence of 314, 344, and 374, respectively. a light chain having three CDRs, or a heavy chain having three CDRs each containing an amino acid sequence of 225, 255, and 285, and a light chain having three CDRs each containing an amino acid sequence of 315, 345, and 375, or a heavy chain having three CDRs each containing an amino acid sequence of 226, 256, and 286, and a light chain having three CDRs each containing an amino acid sequence of 316, 346, and 376, or a heavy chain having three CDRs each containing an amino acid sequence of 227, 257, and 287, and a light chain having three CDRs each containing an amino acid sequence of 317, 347, and 377, or a heavy chain having three CDRs each containing an amino acid sequence of 228, 258, and 288, and a light chain having three CDRs each containing an amino acid sequence of 318, 348, and 378, or a heavy chain having three CDRs each containing an amino acid sequence of 229, 259, and 289, and a light chain having three CDRs each containing an amino acid sequence of 319, 349, and 379, or a heavy chain having three CDRs each containing an amino acid sequence of 230, 260, and 290, and a light chain having three CDRs each containing an amino acid sequence of 320, 350, and 380,or a heavy chain having three CDRs each containing the amino acid sequences of 231, 261, and 291, and a light chain having three CDRs each containing the amino acid sequences of 321, 351, and 381, or a heavy chain having three CDRs each containing the amino acid sequences of 232, 262, and 292, and a light chain having three CDRs each containing the amino acid sequences of 322, 352, and 382, ​​or a heavy chain having three CDRs each containing the amino acid sequences of 233, 263, and 293, and a light chain having three CDRs each containing the amino acid sequences of 323, 353, and 383, or a heavy chain having three CDRs each containing an amino acid sequence of 234, 273, and 294, and a light chain having three CDRs each containing an amino acid sequence of 324, 354, and 384, or a heavy chain having three CDRs each containing an amino acid sequence of 235, 274, and 295, and a light chain having three CDRs each containing an amino acid sequence of 325, 355, and 385, or a heavy chain having three CDRs each containing an amino acid sequence of 236, 275, and 296, and a light chain having three CDRs each containing an amino acid sequence of 326, 356, and 386, or a heavy chain having three CDRs containing amino acid sequences of 237, 276, and 297 and a light chain having three CDRs containing amino acid sequences of 327, 357, and 387, respectively; a heavy chain having three CDRs containing amino acid sequences of 237, 277, and 298, respectively, and a light chain having three CDRs containing amino acid sequences of 328, 358, and 388, respectively; a heavy chain having three CDRs containing amino acid sequences of 238, 278, and 299, respectively, and a light chain having three CDRs containing amino acid sequences of 329, 359, and 389, respectively; a heavy chain having three CDRs containing amino acid sequences of 279 and 300 and a light chain having three CDRs containing amino acid sequences of 330, 360 and 390, respectively; a heavy chain having three CDRs containing amino acid sequences of 240, 280 and 301, respectively, and a light chain having three CDRs containing amino acid sequences of 331, 361 and 391, respectively; a heavy chain having three CDRs containing amino acid sequences of 241, 281 and 302, respectively, and a light chain having three CDRs containing amino acid sequences of 332, 362 and 392, respectively; or a heavy chain having three CDRs containing amino acid sequences of 242, 282 and 303, respectively, and a light chain having three CDRs containing amino acid sequences of 332, 362 and 392, respectively.a heavy chain having three CDRs containing the amino acid sequence of 303 and a light chain having three CDRs containing the amino acid sequences of 333, 363, and 393, respectively; or a heavy chain having three CDRs containing the amino acid sequences of 243, 283, and 304, respectively, and a light chain having three CDRs containing the amino acid sequences of 334, 364, and 394, respectively; or a heavy chain having three CDRs containing the amino acid sequences of 244, 284, 305, and a light chain having three CDRs each containing an amino acid sequence of 335, 365, and 395, respectively; or a heavy chain having three CDRs each containing an amino acid sequence of 245, 285, and 306, respectively, and a light chain having three CDRs each containing an amino acid sequence of 336, 366, and 396, respectively.

[0057] Other anti-influenza antibodies include those having the amino acid or nucleic acid sequences shown in Table 1 below.

[0058] Table 1A: Antibody 3I14 variable region nucleic acid sequences TIFF2025142045000008.tif64152

[0059] Table 1B: Antibody 3I14 variable region amino acid sequence TIFF2025142045000009.tif39152

[0060] (Table 1C) Antibody 3I14V L D94N variable region nucleic acid sequence TIFF2025142045000010.tif26152

[0061] (Table 1C) Antibody 3I14V L D94N variable region amino acid sequence TIFF2025142045000011.tif15152

[0062] 3I14 and 3I14V L The amino acid sequences of the complementarity determining regions of the heavy and light chains of the D94N influenza neutralizing antibody are shown in Table 2 below.

[0063] (Table 2) TIFF2025142045000012.tif39150

[0064] CC-chemokine receptor 4 CCR4(94) Exemplary CCR4 antibodies include antibodies having a VH nucleotide sequence having SEQ ID NO:1678 and a VL nucleotide sequence having SEQ ID NO:1679, or a VH nucleotide sequence having SEQ ID NO:1680 and a VL nucleotide sequence having SEQ ID NO:1681, or a VH nucleotide sequence having SEQ ID NO:1682 and a VL nucleotide sequence having SEQ ID NO:1683, or a VH nucleotide sequence having SEQ ID NO:1684 and a VL nucleotide sequence having SEQ ID NO:1685, or a VH nucleotide sequence having SEQ ID NO:1686 and a VL nucleotide sequence having SEQ ID NO:1687, or a VH nucleotide sequence having SEQ ID NO:1688 and a VL nucleotide sequence having SEQ ID NO:1689.

[0065] Exemplary anti-CCR4 antibodies include antibodies having a VH amino acid sequence having SEQ ID NO:1690 and a VL amino acid sequence having SEQ ID NO:1691, or a VH amino acid sequence having SEQ ID NO:1692 and a VL amino acid sequence having SEQ ID NO:1693, or a VH amino acid sequence having SEQ ID NO:1694 and a VL amino acid sequence having SEQ ID NO:1695, or a VH amino acid sequence having SEQ ID NO:1696 and a VL amino acid sequence having SEQ ID NO:1697, or a VH amino acid sequence having SEQ ID NO:1698 and a VL amino acid sequence having SEQ ID NO:1699, or a VH amino acid sequence having SEQ ID NO:1700 and a VL amino acid sequence having SEQ ID NO:1701.

[0066] In other embodiments, the anti-influenza antibody is a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1702, 1703, 1704, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1705, 1706, 1707, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1708, 1709, 1710, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1711, 1712, 1713, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1714, 1715, 1716, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1717, 1718, 1719, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1720, 1721, 1722, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753 or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1726, 1727, and 1728, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1729, 1730, and 1731, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1732, 1733, and 1734, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1735, 1736, and 1737, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1738, 1739, and 1740, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1741, 1742, and 1743, respectively.

[0067] Human immunoglobulin heavy chain variable region germline gene (VH1-69) (133) Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibodies comprise a VH nucleotide sequence having SEQ ID NO:1744 and a VL nucleotide sequence having SEQ ID NO:1745, or a VH nucleotide sequence having SEQ ID NO:1748 and a VL nucleotide sequence having SEQ ID NO:1749, or a VH nucleotide sequence having SEQ ID NO:1752 and a VL nucleotide sequence having SEQ ID NO:1753.

[0068] Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibodies include a VH amino acid sequence having SEQ ID NO:1746 and a VL amino acid sequence having SEQ ID NO:1747, or a VH amino acid sequence having SEQ ID NO:1750 and a VL amino acid sequence having SEQ ID NO:1751, or a VH amino acid sequence having SEQ ID NO:1754 and a VL amino acid sequence having SEQ ID NO:1755.

[0069] In other embodiments, the anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody has a heavy chain with three CDRs comprising the amino acid sequences of SEQ ID NOs: 1756, 1757, and 1758, respectively, and a light chain with three CDRs comprising the amino acid sequences of SEQ ID NOs: 1759, 1760, and 1761, respectively.

[0070] Zika virus antibody (140) Exemplary antibodies that target and neutralize Zika virus include antibodies having a VH nucleotide sequence having SEQ ID NO:1762 and a VL nucleotide sequence having SEQ ID NO:1763.

[0071] Exemplary antibodies that target and neutralize Zika virus include antibodies having a VH amino acid sequence having SEQ ID NO:1764 and a VL amino acid sequence having SEQ ID NO:1765.

[0072] In other embodiments, an exemplary antibody that targets and neutralizes Zika virus has a heavy chain with three CDRs comprising the amino acid sequences of SEQ ID NOs: 1766, 1767, and 1768, respectively, and a light chain with three CDRs comprising the amino acid sequences of SEQ ID NOs: 1769, 1770, and 1771, respectively.

[0073] Glucocorticoid-induced tumor necrosis factor receptor (GITR) (141) An exemplary anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody has a VH nucleotide sequence having SEQ ID NO:1772 and a VL nucleotide sequence having SEQ ID NO:1773, or a VH nucleotide sequence having SEQ ID NO:1774 and a VL nucleotide sequence having SEQ ID NO:1775, or a VH nucleotide sequence having SEQ ID NO:1776 and a VL nucleotide sequence having SEQ ID NO:1777, or a VH nucleotide sequence having SEQ ID NO:1778 and a VL nucleotide sequence having SEQ ID NO:1779, or a VH nucleotide sequence having SEQ ID NO:1780 and a VL nucleotide sequence having SEQ ID NO:1781, or a VH nucleotide sequence having SEQ ID NO:1782 and a VL nucleotide sequence having SEQ ID NO:1783, or a VH nucleotide sequence having SEQ ID NO:1784 and a VL nucleotide sequence having SEQ ID NO:1785, or or a VH nucleotide sequence having SEQ ID NO: 1786 and a VL nucleotide sequence having SEQ ID NO: 1787, or a VH nucleotide sequence having SEQ ID NO: 1788 and a VL nucleotide sequence having SEQ ID NO: 1789, or a VH nucleotide sequence having SEQ ID NO: 1790 and a VL nucleotide sequence having SEQ ID NO: 1791, or a VH nucleotide sequence having SEQ ID NO: 1792 and a VL nucleotide sequence having SEQ ID NO: 1793, or a VH nucleotide sequence having SEQ ID NO: 1794 and a VL nucleotide sequence having SEQ ID NO: 1795, or a VH nucleotide sequence having SEQ ID NO: 1796 and a VL nucleotide sequence having SEQ ID NO: 1797.

[0074] Exemplary anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) antibodies include a VH amino acid sequence having SEQ ID NO:1798 and a VL amino acid sequence having SEQ ID NO:1799, or a VH amino acid sequence having SEQ ID NO:1800 and a VL amino acid sequence having SEQ ID NO:1801, or a VH amino acid sequence having SEQ ID NO:1802 and a VL amino acid sequence having SEQ ID NO:1803, or a VH amino acid sequence having SEQ ID NO:1804 and a VL amino acid sequence having SEQ ID NO:1805, or a VH amino acid sequence having SEQ ID NO:1806 and a VL amino acid sequence having SEQ ID NO:1807, or a VH amino acid sequence having SEQ ID NO:1808 and a VL amino acid sequence having SEQ ID NO:1809, or a VH amino acid sequence having SEQ ID NO:1810 and a VL amino acid sequence having SEQ ID NO:1811, or or a VH amino acid sequence having SEQ ID NO: 1812 and a VL amino acid sequence having SEQ ID NO: 1813, or a VH amino acid sequence having SEQ ID NO: 1814 and a VL amino acid sequence having SEQ ID NO: 1815, or a VH amino acid sequence having SEQ ID NO: 1816 and a VL amino acid sequence having SEQ ID NO: 1817, or a VH amino acid sequence having SEQ ID NO: 1818 and a VL amino acid sequence having SEQ ID NO: 1819, or a VH amino acid sequence having SEQ ID NO: 1820 and a VL amino acid sequence having SEQ ID NO: 1821, or a VH amino acid sequence having SEQ ID NO: 1822 and a VL amino acid sequence having SEQ ID NO: 1823.

[0075] In other embodiments, the anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody comprises a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1824, 1825, and 1826, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1827, 1828, and 1829, respectively; a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1830, 1831, and 1832, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1833, 1834, and 1835, respectively; a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1836, 1837, and 1838, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1839, 1840, and 1841, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1839, 1840, and 1841, respectively. a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1842, 1843, 1844 and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1845, 1846, 1847, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1848, 1849, 1850, respectively and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1851, 1852, 1853, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1854, 1855, 1856, respectively and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1857, 1858, 1859, respectively; or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1860, 1861, 1862, respectively and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1900, 1901, 1902, 1903, 1904, 1905, 1906, a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1863, 1864, 1865, or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1866, 1867, 1868, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1869, 1870, 1871, respectively, or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1872, 1873, 1874, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1873, 1874, respectively, and a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923,a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1875, 1876, 1877, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1878, 1879, 1880, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1881, 1882, 1883, respectively, or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1884, 1885, 1886, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1887, 1888, 1889, respectively, or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1890, 1891, 1892, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1893, 1894, 1895, respectively, or a heavy chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1894, 1895, 1896, respectively, and a light chain having three CDRs comprising the amino acid sequences of SEQ ID NOs: 1897, 1898, 1899, respectively, It has a heavy chain with three CDRs comprising the amino acid sequences of SEQ ID NOs:1896, 1897, and 1898, and a light chain with three CDRs comprising the amino acid sequences of SEQ ID NOs:1899, 1900, and 1901, respectively.

[0076] A tetravalent antibody is a dimer of bispecific scFv fragments having a first binding site for a first antigen and a second binding site for a second antigen. The scFv is preferably a tandem scFv. The variable domains of the two binding sites are linked together via a linker domain. In a preferred embodiment, the linker domain comprises an immunoglobulin hinge region amino acid sequence. The hinge region is that of IgG1, IgG2, IgG3, or IgG4. Exemplary hinge region amino acid sequences are: Includes TIFF2025142045000013.tif11144.

[0077] In some embodiments, the linker domain further comprises at least a portion of an immunoglobulin Fc domain. At least a portion of the immunoglobulin Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. At least a portion of the immunoglobulin Fc domain is linked to the C-terminus of the hinge region. By at least a portion of an immunoglobulin Fc domain is meant, for example, an immunoglobulin CH2 domain amino acid sequence, a CH3 domain amino acid sequence, a CH4 domain amino acid sequence, or any combination thereof.

[0078] Incorporating at least a portion of an immunoglobulin Fc domain (e.g., a CH2 domain) provides a third functional binding site (i.e., an Fc effector function), resulting in a trifunctional bispecific antibody. Therefore, it may be preferable to modify at least a portion of the effector function, e.g., to enhance the efficacy of the tBsAb. For example, amino acid substitutions, insertions, or deletions can be introduced into at least a portion of the immunoglobulin Fc domain to generate a tBsAb with improved uptake capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Alternatively, at least a portion of the immunoglobulin Fc domain can be glycosylated to improve the stability and solubility of the tBsAb. For example, at least a portion of the immunoglobulin Fc domain is glycosylated at the amino acid corresponding to the asparagine at amino acid position 297. While glycosylation is important for stability, defucosylation of the CH2 carbohydrate can also increase binding affinity to FcγR and further enhance ADCC.

[0079] In certain embodiments, the tBsAbs of the present invention may comprise Fc variants containing amino acid substitutions that alter the antigen-independent effector functions of the antibody, particularly its circulating half-life. Such antibodies exhibit increased or decreased binding to FcRn and therefore have increased or decreased serum half-lives, respectively, compared to antibodies lacking these substitutions. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in mammalian therapeutic methods where a long half-life of the administered antibody is desirable, e.g., for treating chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to mammals where a short circulation time may be advantageous, e.g., for in vivo diagnostic imaging, or in situations where the starting antibody has toxic side effects when present in the circulation for an extended period of time. In one embodiment, the Fc domain contains one or more amino acid substitutions within the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop consists of amino acid residues 280 to 299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in International PCT Application Publication No. WO 05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, an antibody or fragment thereof of the present invention comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0080] Preferably, at least a portion of the Fc domain is a CH2 domain amino acid sequence. An exemplary CH2 domain amino acid sequence is: Includes TIFF2025142045000014.tif4128.

[0081] In other embodiments, the amino acid sequence of the immunoglobulin hinge region or the amino acid sequence of the immunoglobulin hinge region Fc domain is flanked by a flexible linker amino acid sequence. The flexible linker amino acid sequence can be, for example, Includes TIFF2025142045000015.tif4128.

[0082] Enlarging the linker by adding multiple repeats (e.g., four or more) will result in predominantly monomeric scFvs, which can therefore increase epitope accessibility. Linker length and composition can be selected to optimize stability and functional activity, taking into account the topography of the epitope on the target protein.

[0083] Also included in the present invention is a nucleic acid construct comprising nucleic acid molecules encoding: a light chain and a heavy chain variable region of an antibody that specifically binds to a first antigen, a light chain and a heavy chain variable region of an antibody that specifically binds to a second antigen, and a linker domain.

[0084] In yet a further aspect, the present invention provides genetically modified cells that express and carry the tBsAb of the present invention on their cell surface membrane. The cells are T cells, B cells, follicular T cells, or NK cells. The T cells are CD4+ or CD8+. The cells are a mixed population of CD4+ and CD8+ cells. The cells are further modified to express and secrete the tBsAb.

[0085] The vector comprises a nucleic acid construct according to the invention and the host cell, eg, a mammalian cell, expresses the vector of the invention.

[0086] Chimeric Antigen Receptor The tBsAbs of the invention can be used to generate chimeric antigen receptors (CARs), which generally comprise at least one transmembrane polypeptide comprising at least one extracellular ligand-binding domain comprising a tBsAb of the invention, and a transmembrane polypeptide comprising at least one intracellular signaling domain.

[0087] In a preferred embodiment, the transmembrane domain further comprises a stalk region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term "stalk region" refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region is used to provide more flexibility and accessibility to the extracellular ligand-binding domain. The stalk region may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The stalk region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the stalk region may be a synthetic sequence corresponding to a naturally occurring stalk sequence, or may be a completely synthetic stalk sequence. In a preferred embodiment, the stalk region is a portion of the human CD8 α chain.

[0088] The signaling domain or intracellular signaling domain of the CAR of the present invention is responsible for intracellular signal transduction following binding of the extracellular ligand-binding domain to a target, resulting in immune cell activation and an immune response. In other words, the signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "signaling domain" refers to the portion of a protein that transmits an effector signal function signal and instructs the cell to perform a specific function.

[0089] Signaling domains include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as ITAMs (immunoreceptor tyrosine-based activation motifs). ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that serve as binding sites for the syk / zap70 class of tyrosine kinases. Examples of ITAMs used in the present invention can include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR can include the CD3 zeta signaling domain or the cytoplasmic domain of the Fc epsilon RI beta or gamma chain. In another preferred embodiment, signaling is mediated by CD3 zeta with co-stimulation provided by CD28 and / or tumor necrosis factor receptor (TNFr), such as, for example, 4-1BB or OX40.

[0090] In certain embodiments, the intracellular signaling domain of the CAR of the present invention comprises a costimulatory signal molecule. In some embodiments, the intracellular signaling domain comprises two, three, four, or more costimulatory molecules in tandem. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.

[0091] A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal in addition to the primary signal provided by, for example, engagement of the TCR / D3 complex with a peptide-loaded MHC molecule, and mediating T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAMs, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to toll ligand receptors, and antibodies that specifically bind to B7-H3. Costimulatory ligands also include, among others, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0092] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. In another specific embodiment, the signaling domain is a TNFR-associated factor 2 (TRAF2) binding motif, i.e., the intracytoplasmic tail of a costimulatory TNFR member family. The cytoplasmic tails of costimulatory TNFR family members contain a TRAF2-binding motif consisting of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD), where X is any amino acid. TRAF proteins are recruited to the intracellular tails of many TNFRs in response to receptor trimerization.

[0093] Distinguishing features of suitable transmembrane polypeptides include their ability to be expressed on the surface of immune cells, particularly lymphocytes or natural killer (NK) cells, and to interact with each other to direct the immune cell's cellular response to a specific target cell. Different transmembrane polypeptides of the CAR of the present invention, including extracellular ligand-binding domains and / or signaling domains, interact with each other to participate in signal transduction after binding to a target ligand and induce an immune response. The transmembrane domains can be derived from either natural or synthetic sources. Although the transmembrane domains can be derived from any membrane-associated or transmembrane protein, specific transmembrane domains that best fit the chimera are preferred, such as those that promote self-aggregation or increase basal CAR cell activation in the absence of target binding, which can lead to premature depletion.

[0094] The term "portion" as used herein refers to any subset of a molecule, such as a shorter peptide. Alternatively, functional variants of a polypeptide's amino acid sequence can be prepared by mutations in the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can also be made to arrive at the final construct, provided that the final construct exhibits the desired activity, particularly specific anti-target cell immune activity. The functionality of the CAR of the present invention in host cells can be detected in assays suitable for demonstrating the signal transduction ability of the CAR upon binding to a specific target. Such assays are available to those skilled in the art. For example, these assays allow for the detection of signal transduction pathways resulting from target binding, such as assays involving measurement of increased calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover, or the resulting affected production of interleukin (IL) 2, interferon gamma, GM-CSF, IL-3, and IL-4.

[0095] How to use The tBsAb, tBsAb-expressing cell, or CAR according to the present invention can be used to treat cancer, viral infection, or autoimmune disease in patients in need thereof. In another embodiment, the tBsAb, tBsAb-expressing cell, or CAR according to the present invention can be used to produce a medicament for the treatment of cancer, viral infection, or autoimmune disease in patients in need thereof.

[0096] The treatment can be ameliorative, curative, or preventative. It can be part of autologous immunotherapy or allogeneic immunotherapy. Autologous means that the cells, cell lines, or cell populations used to produce tBsAb or tBsAb-expressing cells are derived from the patient or a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells or cell populations used to produce tBsAb or tBsAb-expressing cells are derived from a donor rather than from the patient.

[0097] The treatment can be used to treat patients diagnosed with cancer, viral infection, autoimmune disease, or graft-versus-host disease (GvHD). Cancers that can be treated include tumors that are not vascularized or not yet substantially vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or solid tumors. Cancer types to be treated with the CARs of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0098] The treatment may be in combination with one or more therapies for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0099] In a further embodiment, the compositions of the invention are administered to a patient in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell depletion therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH.

[0100] definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., a "bispecific antibody" is understood to refer to one or more bispecific antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0101] As used herein, the term "polypeptide" encompasses the singular "polypeptide" and the plural "polypeptides" and is intended to refer to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids is included in the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-natural amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0102] As used herein in reference to cells, nucleic acids such as DNA or RNA, the term "isolated" refers to a molecule separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. As used herein, the term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a cell or polypeptide that has been isolated from other cellular proteins or tissues. Isolated polypeptide is meant to encompass both purified and recombinant polypeptides.

[0103] As used herein, the term "recombinant" with respect to a polypeptide or polynucleotide intends a form of polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which is one that can be created by combining polynucleotides or polypeptides that do not normally occur together.

[0104] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, preferably less than 25% identity, with one of the sequences of the present disclosure.

[0105] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage of "sequence identity" to another sequence (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), meaning that, when aligned, that percentage of bases (or amino acids) are identical in comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for the alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: Genetic Code=Standard, Filter=None, Strand=Both, Cutoff=60, Predict=10, Matrix=BLOSUM62, Description=50 sequences, Sort=By High Score, Database=Non-Redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+SwissProtein+SPupdate+PIR. Details of these programs can be found on the World Wide Web (www)ncbi.nlm.nih.gov / blast / Blast.cgi, last accessed May 21, 2008. Biologically equivalent polynucleotides are those that have the specified percentages of homology above and encode polypeptides having the same or similar biological activity.

[0106] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity with the nucleotide sequence of a nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence with a degree of homology to its complement or its complement. In one embodiment, a nucleic acid homolog can hybridize to a nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0107] Hybridization reactions can be performed under conditions of different "stringency." Generally, low stringency hybridization reactions are performed at about 40°C in a solution of about 10xSSC or equivalent ionic strength / temperature. Medium stringency hybridizations are typically performed at about 50°C in about 6xSSC, and high stringency hybridization reactions are generally performed at about 60°C in about 1xSSC. Hybridization reactions can also be performed under "physiological conditions," as known to those skilled in the art. Non-limiting examples of physiological conditions include the temperature, ionic strength, pH, and Mg normally found in cells. 2+ is the concentration.

[0108] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), with uracil (U) substituted for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" refers to the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term "polymorphism" refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene that exists in at least two different forms, i.e., two different nucleotide sequences, is called a "polymorphic region of a gene." A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.

[0109] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present disclosure that is a polynucleotide encompasses the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to make up the double-stranded form.

[0110] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide if, in its natural state, or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragment thereof. The antisense strand is the complement of the nucleic acid from which the coding sequence can be deduced.

[0111] As used herein, the term "detectable label" refers to a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to a polynucleotide or protein, such as an antibody, to produce a composition to be detected, e.g., a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that will provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition that is detectable. Labels may be suitable for small-scale detection or more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected or quantified. A simply detected response generally involves a response whose presence is simply confirmed, whereas a quantified response generally involves a response that has a quantifiable value (e.g., reportable as a numerical value), such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable response can occur directly, using a luminophore or fluorophore associated with the component of the assay that actually participates in the binding, or indirectly, using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0112] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody or any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples include, but are not limited to, the complementarity-determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0113] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0114] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L In some embodiments, the variable regions are joined with a short linker peptide of 10 to about 25 amino acids. This linker may be glycine-rich for flexibility, or serine- or threonine-rich for solubility, and may be a fusion protein of the variable regions of V. H N-terminus of V L The ScFv molecule can be linked to the C-terminus of the ScFv or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0115] A "tandem scFv" consists of two scFvs linked via a short linker, which allows free rotation of the two separate antigen-binding units and thus provides a flexible structure.

[0116] The term antibody encompasses a wide variety of biochemically distinguishable polypeptides. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, with several subclasses within these (e.g., gamma 1-gamma 4). It is the nature of this chain that determines the "class" of an antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in light of this disclosure and are therefore within the scope of this disclosure. While all immunoglobulin classes are expressly within the scope of this disclosure, the following discussion generally relates to immunoglobulin molecules of the IgG class. Regarding IgG, a typical immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 daltons. The four chains are typically joined by disulfide bonds in a "Y" configuration, where the light chains surround the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

[0117] The antibodies, antigen-binding polypeptides, variants or derivatives thereof of the disclosure include polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, such as Fab, Fab', and F(ab)2, Fd, Fv, single chain Fv (scFv), disulfide-linked Fv (sdFv), V, VF, VH ... K or V HImmunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0118] Light chains are classified as either kappa or lambda. Each heavy chain class can combine with either kappa or lambda light chains. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0119] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, light (V K ) Chain and Heavy (V H It will be understood that the variable domains of both the light chain (CK) and heavy chain (CH1, CH2, or CH3) portions determine antigen recognition and specificity. Conversely, the constant domain of the light chain (CK) and the constant domain of the heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region: the CH3 and CK domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.

[0120] As mentioned above, the variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. K Domain and VH A subset of domains, or complementarity determining regions (CDRs), combine to form the variable regions that define the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is represented by the V H Chain and V K Each chain is defined by three CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some instances, for example, in certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule may consist only of heavy chains, without including light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0121] In natural antibodies, each antigen-binding domain contains six "complementarity-determining regions" (CDRs), short, noncontiguous sequences of amino acids specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domain, called "framework" regions, show less inter-molecular variation. The framework regions adopt a predominantly β-sheet structure, while the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act as a scaffold, ensuring that the CDRs are properly oriented by inter-chain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. The amino acids that comprise the CDR and framework regions, respectively, can be readily identified by one of skill in the art for any given heavy or light chain variable region because they have been precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983), and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), the entire contents of which are incorporated herein by reference).

[0122] Where there is more than one definition of a term used and / or accepted in the art, the definition of the term used herein is intended to encompass all such meanings unless expressly stated otherwise. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., US Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), the entire contents of which are incorporated herein by reference. The Kabat and Chothia definitions of CDRs include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to an antibody CDR or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above-cited references are set forth in the table below for comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which residues comprise a particular CDR, given the amino acid sequence of the variable region of an antibody.

[0123] also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0124] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue from the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about the 33rd amino acid residue after the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG, where X is any amino acid. CDR-L1 begins at about residue 24 (i.e., following the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins about the 16th residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins about residue 33 after the end of CDR-L2 (i.e., after the cysteine ​​residue), includes about 7-11 residues, and ends with a sequence related to WGXG, where X is any amino acid.

[0125] The antibodies disclosed herein can be of any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In another embodiment, the variable region can be chondricthoid in origin (e.g., from sharks).

[0126] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise a polypeptide chain comprising a CH1 domain, a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain, a polypeptide chain comprising a CH1 domain and a CH3 domain, a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of the CH2 domain). As discussed above, those skilled in the art will appreciate that heavy chain constant regions can be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0127] The heavy chain constant regions of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide can comprise a CH1 domain derived from an IgG molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region can comprise a hinge region derived, in part, from an IgG molecule and in part from an IgG3 molecule. In another example, the heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG molecule and in part from an IgG4 molecule.

[0128] As used herein, the term "light chain constant region" includes amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0129] A "light chain-heavy chain pair" refers to an assembly of light and heavy chains that can form dimers via disulfide bonds between the CL domains of the light chains and the CH1 domains of the heavy chains.

[0130] As discussed above, the subunit structures and three-dimensional configuration of the constant regions of the various immunoglobulin classes are well known. H The term "CH1 domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. H It is adjacent to the domain and amino-terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0131] As used herein, the term "CH2 domain" includes, for example, the portion of a heavy chain molecule extending from about residue 244 to residue 360 ​​of an antibody using standard numbering schemes (residues 244 to 360 in the Kabat numbering system and residues 231 to 340 in the EU numbering system; see Kabat et al., US Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact, native IgG molecules. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of an IgG molecule and comprises approximately 108 residues.

[0132] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to function independently. The hinge region can be divided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0133] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229 in the EU numbering system), and the two heavy chains are linked by two disulfide bonds.

[0134] As used herein, the term "chimeric antibody" refers to any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial, or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site is of non-human origin (e.g., murine or primate) and the constant region is human.

[0135] As used herein, "percent humanization" is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain, then subtracting that number from the total number of amino acids, dividing it by the total number of amino acids, and multiplying by 100.

[0136] "Specifically binds" or "having specificity" generally means that an antibody binds to an epitope via its antigen-binding domain and that the binding involves a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with higher specificity than to the related epitope "D."

[0137] As used herein, the terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of disease symptoms, reduction in extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, remission or improvement of the disease state, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0138] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, farm animals, livestock, zoo animals, sport animals, or pet animals (dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bovines, etc.).

[0139] As used herein, phrases such as "for a patient in need of treatment" or "subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of an antibody or composition of the present disclosure used, for example, for detection, for diagnostic procedures, and / or for treatment.

[0140] This disclosure describes the development of bispecific antibodies applicable to cancer therapy. To this end, two tetrameric bispecific antibodies (tBsAbs) with dual specificities for GITR and PD-L1 proteins were created. The advantage of these constructs is that they enhance anti-tumor responses by activating T cells and overriding the suppression of regulatory T cells. An anti-CCR4-anti-PDL1 tBsAb (Figures 3 and 4) and an anti-CAIX-anti-PDL1 (Figures 5 and 6) are also described.

[0141] Two different formats of tandem scFv fragment dimerization units in the pcDNA3.4 mammalian expression vector are described herein. In the first construct, the tandem scFv contains two scFvs derived from different parent antibodies. The αGITR scFv and αPD-L1 scFv are linked in tandem by an IgG1 hinge region between two flexible linkers. The first construct has the structure VH GITR10-linker-VL GITR10-linker-hinge-linker-VH PD-L1-linker-VL PD-L1, and its sequence was confirmed by DNA sequencing. The second format is identical to the first format. In addition, it contains an additional domain, such as a CH2 domain, introduced between the hinge and one of the linker regions. The second construct has the structure VH GITR-linker-VL GITR-linker-hinge-CH2-linker-VH PD-L1-linker VL PD-L1. In contrast to the first construct, the second construct contains an Fc domain, potentially resulting in a trifunctional tBsAb.

[0142] Both tBsAbs were successfully expressed by transient transfection in HEK cells and purified by affinity chromatography using Ni-NTA agarose.

[0143] The purified proteins were evaluated by SDS-PAGE, and the results shown showed that under reducing conditions, the protein profiles of all tBsAbs showed one single band, which was in exact agreement with the theoretical values ​​of tandem scFv (taFv): 65 kDa for αGITR-αPD-L1 taFv and 75 kDa for αGITR-αPD-L1 taFv with CH2. Under non-reducing conditions, the predicted molecular weights of αGITR-αPD-L1 (130 kDa) and αGITR-αPD-L1 (150 kDa) tBsAbs were consistent with the apparent molecular weights (see Figure 18).

[0144] ELISA and flow cytometry demonstrated the biological activity of the newly designed tBsAb. In ELISA, the retained biological activity of the αPD-L1 arm of the generated BsAb was preserved in vitro, showing similar binding activity to αPD-L1 mAb. Nonspecific binding of the tBsAb was ruled out, as CCR4, which is not bound by the αGITR-αPD-L1 antibody, showed no signal. Furthermore, similar binding activity of the αGITR arm to GITR protein was observed for both generated BsAbs (αGITR-αPD-L1 and αGITR-αPD-L1 with CH2). Nonspecific binding of this arm was also ruled out, as the αGITR-αPD-L1 antibody showed no binding specificity to GITR-CF2 cells. When αGITR IgG was compared with each BsAb, αGITR IgG showed higher binding in ELISA experiments, suggesting a lower affinity of the tBsAb. Nevertheless, depending on the spatial arrangement of the antigen binding site and the bivalency of the antigen surface distribution of the tBsAb, avidity can be increased, which can compensate for weaker binding.

[0145] Flow cytometry analysis of αGITR-αPD-L1 tested with GITR+ CF2 cells suggests that the novel tBsAb recognizes the GITR protein in its native conformation when expressed on cells. Similar binding affinity of αGITR-αPD-L1 tBsAb compared to GITR mAb was observed. Thus, ELISA and flow cytometry analyses demonstrated the ability of αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2 to specifically recognize their corresponding antigens when expressed on cells, as in vivo. These characterization studies demonstrated similar binding behavior of αGITR-αPD-L1 and αGITR-αPD-L1 with CH2.

[0146] An important aspect of αGITR10-αPD-L1 bearing CH2 is its ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), which further contribute to the beneficial effects of tBsAb in targeting tumor cells for destruction.

[0147] In ADCC assays using GITR+ CF2 as target cells and WIL2-S as effector cells (E / T=5:1), αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2 showed surprising results. For αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2 antibodies, the raw signals of luciferase activity decreased at high antibody concentrations and were substantially lower than the signals for target cells and effector cells alone (see Figure 30).

[0148] The method described herein allows for the generation of tBsAbs involving only one cloning step. tBsAbs are small molecules of only approximately 150 kDa, yet possess dual affinities for the GITR protein and the PD-L1 antigen. Such tBsAbs may be crucial for effective cancer therapy. [Example]

[0149] Example 1: Cloning of αGITR-αPD-L1 tetrameric bispecific antibody (tBsAb) Cloning strategy The goal was to clone a plasmid containing two recombinant single-chain variable fragments (scFv) derived from different parent antibodies linked by a flexible linker. One scFv was directed against the GITR protein, and the other against PD-L1. Such a plasmid would produce two scFvs covalently linked by a linker-hinge-linker domain, resulting in a tetrameric bispecific antibody (αGITR-αPD-L1 tBsAb).

[0150] The mammalian expression vector pcDNA3.4 plasmid contains the construct (V H GITR-Linker-V L GITR-Linker-Hinge-Linker-V H PD-L1-Linker-V L The basic structure of the pcDNA3.4 expression vector was a V fused to an N-terminal 6xHis tag. H X -Linker-V L X -Linker-Hinge-Linker-V H PD-L1-Linker-V L The PD-L1 gene was included (Figure 12).

[0151] Restriction enzyme digestion and ligation The six αGITR scFv gene sequences were individually cloned into the pcDNA3.4 expression vector. H GITR-Linker-V L The GITR gene sequence is V H GITRL1-V L GITRL1, V H GITRL10-V L GITRL10, V H GITRL11-V L GITRL11, V H GITRL14-V L GITRL14, V H GITRL15-VL GITRL15 and V H GITRL17-V L The fragments were labeled GITRL17. All six αGITR gene sequences were flanked by SfiI and NotI restriction sites and isolated by digestion from the corresponding donor plasmids (Table 1). Similarly, the pcDNA3.4 expression vector was also digested with SfiI and NotI restriction enzymes. The digested vectors and fragments were analyzed on a 1% agarose gel and purified using a QIAquick Gel Extraction Kit. The overhanging inserts from the SfiI and NotI digestions were ligated into the corresponding vector pcDNA3.4 at a 5-fold molar ratio using a T4 Ligation Kit. Fifty nanograms of recipient vector was used per ligation reaction. The ligation products were V H GITR-Linker-V L GITR-Linker-Hinge-Linker-V H PD-L1-Linker-V L This resulted in the final structure of PD-L1 (Figure 12).

[0152] Three additional clones were constructed, each producing a control antibody. H F10-V L The F10 gene was selected as the "control arm" because the F10 binding domain has no binding affinity to either the GITR protein or the PD-L1 protein. Therefore, this domain was defined as a negative control. F10 is an antibody evaluated against influenza HA protein. To maintain the same antibody format, its gene sequence is V, ... H GITR1-linker-V L GITR1 or V H PD-L1-Linker-V L The three control plasmids replace only one of the PD-L1 genes. The sequences of the three control plasmids are as follows: (1)V H F10-Linker-V L F10-Linker-Hinge-Linker-V H PD-L1-Linker-V LPD-L1 (F10-αPD-L1) (2)V H GITR1-linker-V L GITR1-linker-hinge-linker-V H F10-Linker-V L F10 (αGITR1-F10) (3) V H GITR10-Linker-V L GITR10-linker-hinge-linker-V H F10-Linker-V L F10 (αGITR10-F10).

[0153] Construction of plasmid (1) V H F10-Linker-V L The F10 gene was isolated from the pcDNA3.1 vector via digestion with SfiI and NotI REs. For the expression vector, the same pcDNA3.4 vector was used (Figure 13). It contains SfiI and NotI restriction sites at the desired insertion site and was therefore digested with the corresponding restriction enzymes. The final plasmid was obtained by ligating both digestion products together. Ligation was performed overnight at 16°C using a T4 Ligation Kit.

[0154] Construction procedures for plasmids (2) and (3) To replace the αPD-L1 scFv in the above construct, BsiWI and BamHI restriction sites and V H F10-Linker-V L Forward and reverse primers were designed and synthesized to introduce the 5' and 3' ends of the F10 fragment, respectively. After PCR amplification, V H F10-Linker-V L The PCR product containing F10 and the pcDNA3.4 expression vector were digested with BsiWI and BamHI and transformed into the previously constructed expression plasmid, V H GITR 1 -Linker-V L GITR 1-Linker-Hinge-Linker-V H PD-L1-Linker-V L PD-L1 and V H GITR 10 -Linker-V L GITR 10 -Linker-Hinge-Linker-V H PD-L1-Linker-V L PD-L1 V H PD-L1-Linker-V L This was used to replace the DNA fragment encoding PD-L1. The digested expression vector and insert were gel purified (1% agarose) using a QIAquick gel extraction kit and then ligated together by quick ligation (5 minutes at room temperature). This procedure resulted in plasmids (2) and (3) (Figure 14).

[0155] Primer design for construction of control plasmid constructs As described above, for the control plasmids (2) and (3), V H F10-Linker-V L Two primers were designed to isolate F10. The forward primer (5'-3') was designed to bind to the 3' end of the complementary strand of DNA, and the reverse primer (3'-5') was designed to bind to the 3' end of the main strand of DNA, and they were reverse complementary. The primers were approximately 20 bp long, with optimal melting temperatures of 62-65°C, not deviating by more than ±1°C. The forward primer (containing a BsiWI restriction site (number 1)) and the reverse primer (containing a BamHI restriction site (number 2)) were synthesized by Genewiz. For the PCR reaction, 100 ng of DNA template (pcDNA3.1) was used in a thermal cycle. The PCR products were purified using a QIAquick PCR Purification Kit according to the manufacturer's protocol and analyzed on a 1% agarose gel.

[0156] Example 2: Cloning of αGITR10-αPD-L1 tetrameric bispecific antibody (tBsAb) containing the CH2 domain Cloning strategy The purpose of this example was to introduce the CH2 domain from IgG1 into the previously constructed plasmid and to generate V H GITR-Linker-V L GITR-linker-hinge-CH2-linker-V H PD-L1-Linker-V L The objective of this study was to provide the basic structure of PD-L1. The addition of CH2 confers effector functions, resulting in a trifunctional tBsAb.

[0157] The pcDNA3.4 expression vector containing αGITR10-αPDL1 served as a template used for constructing a new plasmid. A novel restriction site, HindIII, was introduced by site-directed mutagenesis between the IgG1 hinge region and the linker (GGGGS)6. This newly constructed restriction site served as a cloning site for the IgG1 constant CH2 domain (see Figure 15). The HindIII restriction site was selected for several reasons. It is unique in the plasmid, and its genomic sequence is dissimilar to that of its adjacent coding region. Nevertheless, HindIII is characterized by several disadvantages, such as its relatively long length (6 nucleotides), which likely reduces mutagenesis efficiency.

[0158] The IgG1 plasmid was used as a template to isolate the CH2 domain. The CH2 sequence was amplified by PCR using primers containing the restriction site HindIII. The pcDNA3.4 expression vector (V H GITR 10 -Linker-V L GITR 10 -Linker-Hinge-HindIII * -V H PD-L1-Linker-V LThe PD-L1 and amplified CH2 fragment were digested with the corresponding restriction enzymes. The digested vector and fragment were gel purified (1% agarose) using a QIAquick gel extraction Kit. The overhanging insert from the HindIII digestion was ligated into a vector (pcDNA3.4) at 20x insert using a Quick Ligation Kit to generate a new plasmid, V H GITR 10 -Linker-V L GITR 10 -Linker-Hinge-CH2-Linker-V H PD-L1-Linker-V L PD-L1 was constructed.

[0159] Site-directed mutagenesis Mutagenesis of the GITR10-PDL1 vector was achieved using the QuikChange Lightning Site-Directed Mutagenesis Kit (Aligent Technologies®) according to the manufacturer's protocol. Two oligonucleotide primers, each complementary to opposite strands of the vector, were synthesized. Both primers contained a HindIII site as the desired mutation.

[0160] Primers were designed to contain a HindIII mutation in the center of the primer flanked by 7–10 bases. The oligonucleotide primers were used for extension with PfuUltra HF DNA Polymerase during thermal cycling. This approach allowed for the generation of mutant plasmids containing staggered nicks. During subsequent thermal cycling, the products were treated with DpnI to digest the parent DNA template, including methylated and hemimethylated DNA. As a control, the 4.5 kb pWhitescript plasmid was used to test the mutant plasmids. The pWhitescript plasmid encodes a stop codon (TAA) at the position where the glutamine codon appears in the β-galactosidase gene of pBluescript II, which normally abolishes the blue color of colonies on LB-ampicillin agar plates containing IPTG and X-gal. However, when the oligonucleotide control primers created a point mutation in the 4.5 kb pWhitescript control plasmid, they reverted the T residue of the stop codon to a C, thereby resulting in a blue phenotype on medium containing IPTG and X-gal. After cycling, 2 μL of DpnI restriction enzyme was added (37°C, 5 minutes) to digest the parent dsDNA. The mutagenized plasmids were then transformed into XL10Gold® Ultracompetent cells and spread onto LB-ampicillin agar plates containing 80 μg / ml X-gal and 20 mM IPTG (37°C, >16 hours). The following day, 16 clones were picked from the LB-ampicillin plates, purified using a QIAprep spin Miniprep Kit, and digested with HindIII and NotI restriction enzymes to identify successfully mutagenized clones. Positive clone #10 (GITR10-PDL1 with HindIII) was subjected to another digestion and compared with the original plasmid GITR10-PDL1 (without HindIII). Each sample was digested individually with HindIII or BamHI, as well as simultaneously with HindIII and BamHI-HF, resulting in a total of six digests (see Table 1 below).

[0161] Table 1. Parameters and volumes for all six restriction enzyme digests. TIFF2025142045000016.tif54146Six samples were incubated at 37°C for 2 hours and analyzed on a 1% agarose gel.

[0162] Bacteria containing the positive mutant clone number 10 were amplified overnight in 120 mL of YT medium at 37°C, and the plasmid DNA was subsequently purified using a QIAGEN Plasmid Maxi Kit. The correct construct, containing a HindIII restriction site, was confirmed by sequencing (Genewiz®, using predesigned primers). Glycerol stocks were prepared and stored at -80°C. The recipient plasmid and CH2 fragment containing the HindIII domain were digested with HindIII and then ligated together. The ligation product was transformed into XL10-Gold® Ultracompetent cells by heat pulse according to the protocol described herein. The correct plasmid was verified by sequencing (Genewiz®).

[0163] transformation The ligation products were transformed into XL10-Gold® Ultracompetent cells by heat pulsing. The cells were slowly thawed on ice. For each transformation, 45 μL of cells were mixed with 2 μL of β-mercaptoethanol and 1.5 μL of the DNA of interest. The transformation reactions were incubated for 30 minutes and then heat pulsed for 40 seconds in a 42°C water bath. 0.5 mL of SOCMedium (Life Technologies®) was added to each tube and incubated at 37°C for 1 hour. The transformation reactions were grown overnight on LB-ampicillin plates at 37°C.

[0164] Several colonies per ligation sample were individually picked and grown in 1.5 mL of 2-YT medium for 8 hours. Plasmids from selected colonies were purified using the QIAprep Spin Miniprep Kit as specified by the manufacturer. Correct plasmids were verified by sequencing (Genewiz®). Bacteria from positive clones were grown overnight in 120 mL of YT medium (37°C, 240 rpm), and plasmid DNA was purified using the QIAGEN Plasmid Maxi Kit (according to the manufacturer's protocol). Glycerol stocks were prepared by adding 400 μL of glycerol and 600 μL of culture to a cryotube vial and then stored at -80°C.

[0165] Cell culture and transfection For protein expression, the 293F human cell line was obtained from Life Technologies®, and the 293T adherent cell line was obtained from the ATCC Cell Bank. For cell-based ELISA assays, the CF2-GITR cell line was generated in the Marasco Laboratory to express GITR on the cell surface.

[0166] 293F cells in suspension for protein expression Suspension cultures of 293F cells (derived from human embryonic kidney, HEK, cells) were maintained in Erlenmeyer flasks (Corning®) and 293 Freestyle medium (Life Technologies®) at 37° C. and 5% CO. Cells were passaged in logarithmic growth phase and diluted to an optimal density (200,000 cells / mL) with fresh medium for continued growth.

[0167] 293T and CF2-GITR adherent cells Adherent 293T or CF2-GITR cells were maintained in 75 cm flasks (Cellstar) in DMEM medium (Life Technologies®) supplemented with 10% FBS (fetal bovine serum) (Life Technologies®) and 1% SP (sodium pyruvate) (Life Technologies®) at 37°C and 5% CO2. For continued growth, cells were passaged at 80-100% confluence and seeded at an optimal density (2 × 10) in fresh medium. 6 cells).

[0168] Transfection For the production of tetrameric bispecific antibodies (tBsAb) (αGITR1-αPD-1L1, αGITR10-αPD-L1, αGITR11-αPD-L1, αGITR14-αPD-L1, αGITR15-αPD-L1, αGITR17-αPDL1, and αGITR10-αPD-L1 (with CH2)) and control antibodies (αGITR1-F10, αGITR10-F10, F10-αPD-L1, αGITR IgG), 293F or 293T cells were transfected with the corresponding plasmids.

[0169] Polyethylenimine (PEI)-mediated transient transfection in 293F HEK cells One day before transfection, cells were plated at 6 × 10 in a total volume of 300 mL. 5 On the day of transfection, the cells were passaged to a final concentration of 1.0 × 10 cells / mL. 6 ~1.4×10 6The corresponding plasmid was prepared for transfection. The overall charge of the transfection complex was determined by the ratio of transfection reagent to DNA. The negative charge contributed by the phosphates in the DNA backbone was offset by the positive charge of the transfection reagent. This allowed for good complex formation and neutralization of the electrostatic repulsion exerted on the DNA by the negatively charged cell membrane. A 1:1 ratio of plasmid:PEI allowed complete binding of the polymer to the DNA and complete condensation to protect the cargo, but excess PEI was important to overcome the inhibitory effect of the anionic cell surface. For every million cells, 1 μg of plasmid and 3 μg of PEI were used for transfection, each diluted separately in 15 mL of Opti-MEM (reduced serum medium) (Life Technologies®). The diluted PEI was added to the plasmid and incubated at room temperature for 20 minutes. Neutralization efficiency increases with exposure time to the PEI-DNA complex, but excessively long exposure to the lipid reagent can be toxic. PEI / plasmid complexes were incubated with 293F suspension cells (1 × 10 6 The cells were then poured into a flask (300 mL per flask, 300 mL cells / mL) and incubated at 37°C at 140 rpm for 6 days.

[0170] Polyethylenimine (PEI)-mediated transient transfection in 293T HEK cells Transfection of 293T HEK cells with PEI cells followed the same protocol as described above for 293F suspension HEK cells, with some modifications. Transfection was performed on 293T cells growing at 80% confluence in tissue culture dishes (200 mm) in diluted DMEM medium supplemented with 10% FBS. For 40 μg of DNA, 200 μg of PEI was used (1:5 ratio), and each was separately diluted in 1 mL of Opti-MEM (reduced serum medium) (Life Technologies®). The diluted PEI was added to the plasmid and stored at room temperature for 20 minutes. The DNA / PEI complex was gently added dropwise to the dish to prevent cell dissociation and death. The cells were then incubated at 37°C for 2 days.

[0171] Example 3: Protein purification Ni-NTA purification of bispecific antibodies The 293 HEK cell suspension was harvested and centrifuged at 5000 rpm for 35 minutes at 4°C. To purify the bispecific antibody via the N-terminal 6xHis tag, the filtered supernatant (0.22 μm PEV, Costar®) was incubated with 1 mL of Ni-NTA agarose (Qiagen) for 2 hours (240 rpm, room temperature). The supernatant was passed twice through a 15 mL Ni-NTA Sepharose gravity-flow column. After washing, the column containing the beads was washed with four column volumes of Ni-NTA wash buffer (0.02 M imidazole, 0.3 M NaCl, 1 M Tris HCl, pH = 7.0), and the protein was slowly eluted with 13 mL of Ni-NTA elution buffer (0.5 M imidazole, 0.3 NaCl, 0.02 Tris HCl, pH = 7.0). The eluted protein was buffer exchanged into PBS buffer using a centrifugal filter unit 100,000 MW (Amicon®). The yield of tBsAb was measured using a NanoDrop ND-1000.

[0172] Protein A purification of αGITR IgG antibody αGITR IgG antibody was collected from a suspension of 293 HEK cells and centrifuged at 5000 rpm for 35 minutes at 4°C. To purify αGITR IgG antibody via the Fc domain, the filtered supernatant was incubated with 1 mL of Protein A (GE Lifesciences) for 2 hours (room temperature, shaking) and then passed twice through a 15 mL gravity-flow column (Biorad), followed by washing with 10 mL of PBS. αGITR IgG was eluted with 2 mL of TEA (100 nM), and 200 μL of Tris-HCl (1 M, pH = 7) was added to the eluate to neutralize the TEA. An additional 2 mL of PBS was added to the column and collected in a tube along with the eluted protein.

[0173] Example 4: Protein characterization SDS-PAGE analysis Protein purity was verified using SDS-PAGE analysis according to the NuPAGE® Technical Guide (Invitrogen). NuPAGE Bis-Tris Gels (4–12%) (Novex) were used in MES-SDS running buffer, with total protein amounts ranging from 3 μg to 5 μg. Protein samples were mixed with 4x LDS sample buffer (Novex) containing dodecyl sulfate to denature the proteins. Protein samples were further boiled at 100°C for 10 min under reducing conditions. Samples were then loaded onto Novex Bis-Tris Gels in MES-SDS running buffer. Gels were run in an Xcell SureLock Mini-Cell at 200 V for 35 min, followed by Coomassie G-250 staining using simplyBlue™ Safe Stain (Novex).

[0174] Direct ELISA of αGITR-αPD-L1 against passively adsorbed soluble PD-L1 antigen Maxisorb 96-well plates (Costar®) were coated overnight at room temperature with 100μL of 5μg / mL PD-L1 rabbit Fc antigen and CCR4 protein (negative control) in PBS. The following day, the plates were washed three times with PBS and blocked with 200μL of blocking solution (2% BSA in PBS) at room temperature for 2 hours. The plates were washed three times with PBS. Primary antibodies, αGITR1-αPD-L1, αGITR10-αPD-L1, αGITR11-αPD-L1, αGITR14-αPD-L1, αGITR15-αPD-L1, αGITR17-αPD-L1, F10-αPD-L1 BsAB, and a commercially available anti-mouse PD-L1 mAb (Biolegend), prepared at various concentrations in 1x PBS, were added to the wells (100μL) and incubated at room temperature for 2 hours. The highest antibody concentration tested was 1 μg / mL, followed by 1 × 10 5 The samples were serially diluted 10-fold to 1 μg / mL dilution. Each sample was run in triplicate at all concentrations. Several controls were set up and are listed in the table below (Table 2). A 96-well plate (Costar) was washed three times with 1x PBS buffer. Solutions of secondary antibody (6x His-HRP (Thermoscientific) and goat anti-mouse IgG Fc, HRP conjugate (Thermoscientific) were diluted in 1x PBS (1:2000 and 1:5000). Secondary antibody (100 μL) was added to each well and incubated for 2 hours at room temperature. Finally, each well was washed four times with PBS. The 96-well plate was developed with 100 μL of TBM substrate solution (Thermoscientific). After development, 100 μL of phosphoric acid stop solution (Thermoscientific) was added. End-point OD data were recorded at 450 nm using a Bio-Rad Benchmark Plus and analyzed using Microplate Manager 5.2.1 software.

[0175] Table 2. Experimental overview of test samples and controls for the direct ELISA of αGITR-αPD-L1 to passively adsorbed PD-1 antigen. TIFF2025142045000017.tif126147

[0176] GITR + Cell-based ELISA of αGITR-αPD-L1 BsAb against CF2 For cell-based ELISA, αGITR1-αPD-L1, αGITR10-αPD-L1, and αGITR10-αPD-L1 antibodies with CH2 were tested for their retention of binding ability to GITR+ CF2 cells. A total of four ELISA experiments were set up.

[0177] In the first cell-based ELISA, αGITR1-F10 and αGITR10-F10 tetrameric bispecific antibodies (tBsAb) were analyzed. For seeding of GITR+ CF2 cells and GITR- CF2 cells (negative control), 1,000 cells per well were added to 200 μL of 1% DMEM medium and incubated overnight to allow adhesion. The following day, cells were fixed with 100 μL of acetone-methanol solution (1:1 ratio) and incubated at room temperature for 20 minutes. The acetone-methanol solution was aspirated from the plate, and the cells were washed three times with 1x PBS. The overall assay procedure and color development were performed according to the ELISA protocol described in Section 2.6.2. Primary antibodies, αGITR1-αPD-L1 and αGITR10-αPD-L1, were tested at various concentrations. The tBsAb was serially diluted in 1× incubation buffer in thirds, with a highest concentration of 3.33 mg / mL and a lowest concentration of 0.0411 mg / mL. Several controls were set up and are listed in the table below (Table 3).

[0178] Table 3: Experimental overview of test samples and controls for cell-based ELISA of αGITR1-αPD-L1 and αGITR10-αPD-L1 on GITR+ CF2 cells. TIFF2025142045000018.tif126148

[0179] After evaluating the results of the cell-based ELISA (Figure 20), a second cell-based ELISA experiment was repeated using the same procedure as described above, except that the cells were fixed with 8% paraformaldehyde.

[0180] A third cell-based ELISA was performed to compare the αGITR10-αPD-L1 tBsAb with the commercially available human αGITR mAb. + CF2 cells and GITR - For seeding of CF2 cells (negative control), 10,000 cells per well were added to 200 μL of 1% DMEM medium and incubated overnight to allow adhesion. The following day, cells were fixed with 100 μL of 8% paraformaldehyde and incubated at room temperature for 20 minutes. The paraformaldehyde solution was aspirated from the plate, and the cells were washed three times with 1x PBS. General assay procedures and color development were performed according to the ELISA protocol described herein. Primary antibodies, αGITR10-αPD-L1, and αGITR10 mAb were tested at various concentrations. Antibodies were serially diluted (1:2) in 1x incubation buffer, with a maximum concentration of 5 mg / mL and a minimum concentration of 0.078 mg / mL. Several controls were set up and are listed in the table below (Table 4).

[0181] Table 4. Experimental overview of test samples and controls for cell-based ELISA of αGITR10-αPD-L1 and αGITR mAb on GITR+ CF2 cells. TIFF2025142045000019.tif206145

[0182] A fourth ELISA was performed to compare the αGITR10-αPD-L1 tBsAb with CH2 with the commercially available αGITR mAb. The assay procedure was identical to the third ELISA (described above).

[0183] Flow cytometry analysis of αGITR1-αPD-L1 and αGITR10-αPD-L1 The biological activity of αGITR on GITR+ CF2 cells was analyzed by fluorescence-activated cell sorting (FACS). GITR+ CF2 cells and GITR- CF2 cells were cultured at 75 cm 2 Cells were grown in flasks (Cellstar) until they reached approximately 80% confluence. They were dissociated and resuspended by adding 1:10 diluted trypsin containing 0.25% trypsin-EDTA (Life Technologies) in PBS, and then added to a 96-well round-bottom plate in FACS buffer (PBS, 1% FBS, 2mM EDTA). In the next step, αGITR1-αPD-L1 and αGITR10-αPD-L1 were added at various concentrations for 1 hour at 4°C. The highest concentration of antibody tested was 100μg / mL, followed by two-fold serial dilutions down to a dilution of 0.05μg / mL. Primary antibodies were detected with His-tag Alexa Fluor 488 conjugates (Biotech). Secondary antibodies were diluted in PBS (Life Technologies) and added to each well for 30 minutes. Cells were then washed three times with PBS buffer and resuspended in FACS buffer. A total of 10,000 events were analyzed by FACSCalibur. Results were analyzed by FlowJo 10.1 software. Several controls were performed and are listed in the table below. (Table 5)

[0184] Table 5. Experimental overview of control samples for FACS analysis of αGITR1-αPD-L1α and GITR10-αPD-L1 on GITR+ CF2 cells and GITR- CF2 cells. TIFF2025142045000020.tif199152

[0185] Example 5: Functional testing ADCC assay of αGITR-αPDL1 with CH2 against GITR+ CF2 cells The antibody-dependent cellular cytotoxicity of αGITR-αPD-L1 bearing CH2 against GITR+ CF2 cells was analyzed using the ADCC Reporter Bioassay Complete Kit (WIL2-S) (Promega) and performed according to the manufacturer's protocol. The aim was to test αGITR10-αPD-L1 bearing CH2 for ADCC. The assay was performed using ADCC reporter cells (WIL2-S) harboring an Fcγ receptor and a response element-driven luciferase gene.

[0186] GITR + CF2 cells and GITR - CF2 cells 75cm 2 Cells were grown in flasks (Cellstar) until they reached approximately 80% confluence. They were dissociated by adding a 1:10 dilution of 0.25% trypsin-EDTA (Life Technologies) in PBS and tested for viability. + CF2 cells were used as target cells, with 2 × 10 cells per well diluted in RPMI 1640 medium (Life Technologies® serum-free). 4 Cells were seeded into 96-well flat-bottom microplates (PerkinElmer) at a density of 10 × 10 cells per well. αGITR10-αPD-L1 (with CH2) and controls (αGITR10-IgG (positive control) and GITR10-PD-L1 and F10-PDL1 (negative controls)) were serially diluted in ADCC assay medium. The four antibodies were added concentration-dependently, starting at 20 mg / mL (highest concentration), followed by 2 mg / mL, 0.2 mg / mL, and 0.02 mg / mL (1:10 serial dilutions), and incubated at room temperature for 5 minutes. Following incubation, effector cells WIL2-S were suspended in ADCC assay medium and plated at 10 × 10 cells per well. 6Cells were added to the target cell / antibody mixture at 1000 kJ / well. The effector cell to target cell ratio was set at 5:1 (E / T). After approximately 6 hours of incubation at 37°C (5% CO2), an equal volume of Bio-Gio luciferase assay reagent (Promega) was added to the wells and incubated (room temperature, 10 minutes). Cell luminescence was measured using a Polarstar Omega. Assays were performed in triplicate. All data were plotted using Excel.

[0187] GITR + CDC assay of αGITR-αPDL1 with CH2 on CF2 cells To test the complement-dependent cytotoxicity (CDC) of the αGITR10-α-PDL1 tBsAb bearing CH2, baby rabbit complement (Cedarlane Laboratories) was used in the CellTox™ Green Cytotoxicity assay (Promega), which uses CellTox Green Dye (Promega), which binds to DNA in living cells. The fluorescent signal generated by the dye binding to dead cell DNA is proportional to cytotoxicity. The assay was performed according to the manufacturer's protocol. The experimental procedure and setup for testing complement-dependent cytotoxicity were similar to those for the CDC test described above, except that assay development and analysis were performed using the CellTox™ Green Cytotoxicity assay (Promega). The antibodies tested for complement-dependent cytotoxicity were αGITR10-αPDL1 and the αGITR10-α-PDL1 tetrameric bispecific antibody (tBsAb) bearing CH2. αGITR mAb was used as a positive control, and F10-αPD-L1 was used as a negative control.

[0188] After approximately 4 hours of incubation at 37°C (5% CO), an equal volume of CellTox Green Dye assay reagent (Promega) was added to the wells and incubated (room temperature, 10 minutes). Fluorescence was measured using a Polarstar Omega. Assays were performed in triplicate. All data were plotted using Excel.

[0189] Example 6: Isolation and characterization of αGITR-αPD-L1 BsAb Generation of expression vectors A total of six vectors (αGITR-αPD-L1) were constructed to produce the desired tBsAb, and an additional three vectors were for the production of control Abs (αGITR1-F10, αGITR10-F10, and F10-αPD-L1). Expression vectors were generated according to the cloning strategy described above.

[0190] The recipient expression vector pcDNA 3.4 and all donor vectors (six V H GITR-Linker V L GITR and insert and one V H F10-V L The fragments (F10 insert) were digested with SfiI and NotI restriction enzymes, and the fragments were separated on a 1% agarose gel and stained with ethidium bromide. The SfiI and NotI digestion patterns of the seven digests were consistent with the theoretical calculations. The digested recipient vector pcDNA3.4 vector contained 7500 bp and was detectable by the ladder at the correct level (lane 1, 8000 bp). The smaller fragments in lane 1, representing 500-1000 bp, corresponded to the V fragments of the previously used scFvs. H X -Linker V L X The GITR insert (lanes 2-6) and F10 insert (lane 7) clustered at 500-1000 bp. The larger bands seen at the 8000 bp level (lanes 2-7) represent the corresponding derivative vectors.

[0191] Two additional control plasmids (2) and (3) were constructed. The recipient expression vector pcDNA3.4 encoding the αGITR1-αPDL1 and αGITR10-αPDL1 scFvs was digested with BsiWI and BamHI Re to produce the V H PD-L1-Linker-V L PD-L1 fragment V H F10-Linker-V LThe F10 fragment was replaced with the V from the pcDNA3.1 donor vector. H F10-Linker-V L To isolate the F10 fragment, forward and reverse primers (number 1 and number 2) containing BsiWI and BamHI restriction sites were designed. cDNA was isolated using PCR, followed by digestion with BsiWI and BamHI REs. Gel analysis of all three digests was consistent with the theoretical values. As expected, PCR of the F10 fragment showed only one band at the correct position relative to the ladder. Two digested recipient vectors (V H GITR1-V L GITR1 or V H GITR10-V L The fragment containing GITR10 is approximately 8000 bp in size, which is consistent with the theoretical size of the vector (7500 bp).

[0192] All digested fragments were extracted and purified from agarose gel, and the respective ligation reactions were carried out. The resulting plasmids were successfully constructed and confirmed by sequencing (Genewiz).

[0193] Expression of GITR-PDL1 bispecific antibody and αGITR-IgG αGITR-αPD-L1 protein was expressed in 293F HEK cells and isolated by Ni-NTA purification. αGITR IgG protein was expressed in HEK293F cells and isolated by Protein A purification. Yields were measured by NanoDrop spectrophotometer and are listed in Table 6.

[0194] Table 6. Antibody yield of 293F HEK expression TIFF2025142045000021.tif50143

[0195] SDS-PAGE analysis The purity of tBsAbs αGITR1-αPDL1, αGITR10-αPDL1, αGITR11-αPDL1, αGITR14-αPDL1, αGITR15-αPDL1, αGITR17-αPDL1, and F10-αPD-L1 was analyzed by SDS-PAGE. Protein samples of 3 μg to 5 μg were loaded onto the gel, separated by electrophoresis, and stained with Coomassie blue.

[0196] Notably, under non-reducing conditions, two bands are particularly noteworthy. The upper band is in the 115 kDa and 140 kDa range. The quantitative dominance of this band in each protein profile and its apparent molecular size, similar to that of the αGITR-αPD-L1 tetrameric bispecific antibody (tBsAb) (130 kDa), indicate successful antibody production. The lower band is between 70 and 80 kDa and may therefore consist of a significant amount of monomeric tandem scFv (65 kDa). Separately, several weak bands above 140 kDa are observable, suggesting the formation of aggregates.

[0197] Under reducing conditions, only one band between 70 and 80 kDa was observed, suggesting the reduction of disulfide bonds in the tBsAb to a tandem scFv (65 kDa). The discrepancy in molecular weight between the apparent and theoretical values ​​may be due to post-translational modifications (such as glycosylation and phosphorylation) and the conformation of the protein as it runs on SDS-PAGE. The differential loading on the gel may explain the difference in band intensity between the αGITR-αPD-L1 tBsAbs.

[0198] Furthermore, the purity of αGITR-IgG was analyzed by SDS-PAGE. Under nonreducing conditions, analysis revealed one band with an apparent molecular weight of 140 kDa, which is approximately equal to the theoretical calculated molecular weight of αGITR IgG (150 kDa). Reducing SDS analysis revealed two bands, suggesting successful reduction of the disulfide bonds of αGITR IgG to yield a heavy chain (50 kDa) and a light chain (25 kDa).

[0199] Direct ELISA of αGITR-αPD-L1 BsAb against passively adsorbed PD-L1 antigen Direct ELISA of the αGITR-αPD-L1 BsAbs was performed to characterize their reactivity to the PD-L1 antigen. As shown in Figure 19, reactivity to the PD-L1 antigen could be observed for all αGITR-αPD-L1 tBsAbs, but nonspecific adhesion to CCR4 was not observed (not shown). The readout signals were very similar for all αGITR-αPD-L1 tBsAbs at all concentrations. The highest ELISA signal was measured at the highest concentration. Furthermore, the absorbance values ​​of αGITR-αPD-L1 tBsAb binding were similar to those for the commercially available αPD-L1 mAb, with the intensity signal decreasing at lower concentrations. The ELISA showed no saturation at high concentrations, with very weak signals at concentrations below 0.01 mg / mL.

[0200] Cell-based ELISA of αGITR-αPD-L1 tBsAb against GITR+ CF2 Previous studies of αGITR IgG demonstrated that αGITR1 IgG and αGITR10 IgG had the best characteristics; therefore, in this project, the following experiments were narrowed down to αGITR10-αPD-L1 and αGITR1-αPD-L1 tBsAb. A cell-based enzyme-linked immunosorbent assay (ELISA) was used to test various concentrations of αGITR1-αPD-L1 and αGITR10-αPD-L1 against GITR+ CF2 cells and analyze their reactivity. As shown in Figure 20, reactivity against GITR+ CF2 cells could be observed for the αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies. The OD values ​​of αGITR1-αPD-L1 and αGITR10-αPD-L1 depended on their respective concentrations. Consistent with expectations, stronger signals were measured at higher concentrations, which then gradually decreased as the concentration decreased.

[0201] The signal intensity of αGITR10-αPD-L1 was superior to that of αGITR1-αPD-L1 at all concentrations. Surprisingly, the negative control F10-αPD-L1 antibody not only exhibited absorbance but also appeared to exhibit concentration-dependent behavior. No readout signal was detected below the 0.1235 mg / mL threshold for αGITR1-αPD-L1 or F10-αPD-L1. Overall, the mean standard deviation was very high.

[0202] Due to the surprising results of the previous ELISA (see Figure 20), the experiment was repeated. The setup remained identical, except that GITR+ CF2 cells were fixed with 8% paraformaldehyde instead of acetone-methanol solution. The results of this second approach revealed similar signal readout observations for the αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies, but with slightly higher absorbance values ​​(see Figure 21). However, the F10-αPD-L1 antibody continued to show signal activity, and its absorbance was still dependent on the concentration used. The tBsAb showed no binding whatsoever when incubated with GITR- CF2 cells. See Figure 32.

[0203] A third cell-based ELISA was performed to compare the αGITR10-αPD-L1 antibody with commercially available αGITR IgG. Reactivity of both antibodies was observed with GITR+ CF2 cells (Figure 22), but not with GITR- CF2 cells (see Figure 33). Again, the results with αGITR10-αPD-L1 were consistent with previously recorded data. The signal intensity of the αGITR mAb was superior to that of αGITR10-αPD-L1 at all concentrations. Surprisingly, no saturation of the signal readout was observed at higher concentrations. The control antibody F10-αPD-L1 (negative control) showed concentration-dependent signal activity against GITR+ CF2 cells, but not against CF2 cells (without GITR+ expression). See Figure 33.

[0204] Flow cytometry analysis of αGITR-αPD-L1 BsAb against GITR+ cells Flow cytometry analysis evaluated the binding of the αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies to GITR+ CF2 cells (Figures 23 and 24). The results show that both antibodies (co-stained with APC-labeled His-tag Alexa Fluor 488) can specifically bind to GITR+ CF2. Furthermore, the tBsAb showed no reactivity to GITR- CF2 (Figure 34). Note that some nonspecific binding was caused by the secondary antibody, as shown in the control (Figure 34). Comparing the two antibodies with each other indicates that they exhibit similar binding under identical conditions. Therefore, only the αGITR10-αPD-L1 tBsAb was selected for further characterization. Standard assays of αGITR1 IgG and αGITR10 IgG revealed similar binding properties when compared to the tBsAb.

[0205] Example 7: Isolation and characterization of αGITR-αPD-L1 bsAb with CH2 Generation of bacterial expression vectors In previous studies, the αGITR10 mAb was shown to be the best characterized, and therefore αGITR10-αPD-L1 was chosen as the expression vector for engineering a new construct containing the CH2 domain. The vector was generated according to the cloning strategy described above and contained the following genes in the following order: V H GITR-Linker-V L GITR-linker-hinge-CH2-linker-V H PD-L1-Linker-V L PD-L1 was introduced.

[0206] Site-directed mutagenesis allowed the introduction of a HindIII restriction site into the recipient pcDNA3.4 vector between the IgG1 hinge region and the linker (GGGGS)6. After transformation into E. coli strain XL10-Gold® Ultracompetent cells, 16 clones were selected and then DNA purified. Restriction enzyme digestion analysis using HindIII and BamHI restriction enzymes displayed on a 1% agarose gel was performed to test for correct introduction of the HindIII restriction site (see Figure 25). Of the 16 clones, only clone #10 showed two bands. The size of the smaller band, clustered between 500 and 1000 bp, corresponds to the theoretical size (800 bp) of the HindIII and BamHI digestion. Because HindIII and BamHI represent the only restriction sites in the plasmid, this result indicated successful introduction of HindIII into the DNA of clone #10 cells.

[0207] Further gel analysis of clone #10 was performed to compare it with the original GITR10-PDL1 (which does not contain a HindIII restriction site). See Figure 26. Clone #10 (GITR10-PDL1 with HindIII) and GITR10-PDL1 (without HindIII) were each subjected to three separate digests. The first digestion was performed with HindIII restriction enzyme alone, the second digestion was performed with NotI restriction enzyme alone, and the third digestion was performed with both HindIII and NotI restriction enzymes. Digestion of clone #10 with a single enzyme resulted in an open circular structure clustered around 8000 bp. In contrast, double digestion of clone #10 with HindIII and NotI restriction enzymes yielded two bands. The lower band clustered at less than 500 bp, corresponding to the theoretically calculated value for the HindIII / NotI digestion fragment (117 bp). The αGITR10-αPD-L1 plasmid does not contain a HindIII restriction site, and therefore, gel analysis of a single HindIII digest revealed supercoiled plasmid DNA, as expected. These results strongly suggest the correct introduction of a HindIII restriction site.

[0208] Sequencing of clone 10 (Genewiz) confirmed the correct introduction of a HindIII site between the hinge and linker domains. However, deletion of five linker repeats out of a total of six (GGGGS) repeats occurred during site-directed mutagenesis. As a result, the new construct displayed only one linker repeat instead of six. Nevertheless, it was decided to continue with the plasmid construction using this newly created plasmid containing the hinge region followed by one single linker repeat (GGGGS).

[0209] Two primers (forward and reverse) were designed to isolate the CH2 domain from the IgG1 plasmid. Each primer contained a HindIII restriction site. The recipient vector GITR10-PDL1 (containing the HindIII site) and the CH2 fragment were single-digested with HindIII restriction enzyme and analyzed on a 1% agarose gel (Figure 27). Both digestions yielded fragment sizes consistent with the theoretical calculations: 7.5 bp for the recipient vector GITR10-PDL1 containing HindIII and 350 bp for the CH2 fragment.

[0210] Therefore, the pcDNA3.4 expression vector αGITR10-αPD-L1 carrying CH2 was successfully constructed and confirmed by sequencing (Genewiz).

[0211] SDS-PAGE analysis αGITR10-αPD-L1 protein with CH2 was expressed in 293T HEK cells and isolated by Ni-NTA purification. A total of 100 mL of culture medium resulted in a protein yield of 200 ng (NanoDrop analysis). The purity of GITR10-αPD-L1 tBsAb with CH2 was analyzed by SDS-PAGE (Figure 28). A total of 3 μg of protein sample was loaded onto the gel, separated by electrophoresis, and stained with Coomassie blue. Notably, under non-reducing conditions, two bands are particularly noteworthy. The upper band is slightly above 140 kDa. The quantitative dominance of this band and its apparent molecular size, which is similar to that of αGITR10-αPD-L1 tBsAb with CH2 (150 kDa), suggest successful antibody production. The lower band has an apparent molecular weight of 80 kDa and is therefore likely to consist of a significant amount of tandem scFv, including undimerized CH2 (75 kDa). Under reducing conditions, only one band of 80 kDa was observed, suggesting reduction of the disulfide bond of tBsAb to tandem scFv (75 kDa).

[0212] GITR + Cell-based ELISA of αGITR-αPD-L1 tetrameric bispecific antibody (tBsAb) with CH2 against CF2 Cell-based enzyme-linked immunosorbent assay (ELISA) is + A study was conducted to test various concentrations of αGITR10-αPD-L1 with CH2 against CF2 and analyze their signal intensities.

[0213] As shown in Figure 29, GITR + Reactivity to CF2 could be observed with the αGITR10-αPD-L1 antibody with CH2, but not with GITR10. -No nonspecific adhesion to CF2 was observed. See Figure 35. The OD values ​​of αGITR10-αPD-L1 with CH2 depended on their respective concentrations. Consistent with expectations, the maximum signal was measured at the highest concentration, which then gradually decreased as the concentration decreased. The signal intensity of αGITR IgG was superior to αGITR10-αPD-L1 with CH2 at most concentrations. Surprisingly, no saturation of the signal readout was observed at higher concentrations. The control antibody (F10-αPD-L1) was tested at the highest concentration (5 μg / mL) and had some reactivity, as shown previously (Figures 22 and 21).

[0214] Example 8: Functional testing of αGITR-αPD-L1 BsAb with CH2 In an initial attempt to establish functional data, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested for the CH2-bearing αGITR-αPD-L1 BsAb, but the results were inconclusive.

[0215] ADCC reporter assay of αGITR-αPD-L1 BsAb with CH2 against GITR+ CF2 The αGITR10-αPD-L1 BsAb with CH2 was tested for ADCC activity using GITR+ CF2 cells (target cells) and WIL2-S (effector cells) (E / T=5:1). The antibody's biological activity in ADCC was quantified through luciferase produced as a result of NFAT pathway activation, and its activity in effector cells was quantified by luminescence readout. In the ADCC analysis, αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2 showed surprising results (Figure 30). The negative control F10-αPD-L1 showed similar signal intensity for ADCC compared to target cells and effector cells alone, and was unbiased across various concentrations. Meanwhile, the positive control αGITR IgG showed increasing values ​​at higher concentrations, as expected. Surprisingly, for αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2, the ADCC signal intensity decreased at higher concentrations and was substantially lower than the signals from target cells and effector cells alone at 20 μg / mL of tBsAb.

[0216] The ADCC activity of αGITR10-αPD-L1 with CH2 was measured at various concentrations. All antibodies were serially diluted (1:2) from a highest concentration of 20 mg / mL down to 0.02 mg / mL and tested against 20,000 GITR+ CF2 cells per well. The effector cell (GITR+ CF2) to target cell (Wils-2) ratio was 5:1. αGITR IgG represents the positive control, and F10-αPD-L1 is the negative control. The vertical axis represents the raw value of luciferase activity in effector cells, quantified by luminescence readout. Each sample was tested in triplicate at each concentration, and the mean standard deviation is shown in parentheses. The background of GITR+ CF2 cells in RPMI medium was subtracted from the obtained values.

[0217] CDC analysis of αGITR10-αPD-L1 BsAb with CH2 on GITR+ CF2 cells The αGITR10-αPD-L1 antibody with CH2 was tested for complement-dependent cytotoxicity against GITR-expressing CF2 cells by measuring the amount of fluorescent CellTox Green bound to the constitutive DNA. The percentage of lysis was calculated as the ratio of the intensity of the signal obtained from the sample to the intensity of the signal from fully lysed GITR+ CF2 cells (Figure 31).

[0218] The negative control F10-αPD-L1 BsAb exhibited a similar rate of cytotoxicity as the positive control αGITR IgG. αGITR10-αPD-L1 with CH2 exhibited similar levels of cytotoxicity at all concentrations, ranging from 65% to 70%, and did not appear to be concentration-dependent. None of the antibodies measured had a substantially higher rate of cytotoxicity. These findings are largely at odds with expected results, and a possible reason for these discrepancies is the possible low viability of the GITR+ CF2 cells used.

[0219] Other embodiments While the present invention has been described in conjunction with its detailed description, the above description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0220] Sequence information SEQUENCE LISTING <110> Dana-Farber Cancer Institute, Inc. <120> MODULAR TETRAMERIC BISPECIFIC ANTIBODY PLATFORM <150> US 62 / 408,271 <151> 2016-10-14 <160> 1905 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 1 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 2 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 3 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 4 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 5 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 6 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 7 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 8 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 9 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 10 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 11 Gly Val Thr Phe Arg Ser Tyr Ala 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 12 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 13 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 14 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 15 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 16 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 17 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 18 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 19 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 20 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 21 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 22 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 23 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 24 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 25 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 26 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 27 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 28 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 29 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 30 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 31 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 32 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 33 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 34 Gly Val Thr Phe Arg Ser Tyr Ala 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 35 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 36 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 37 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 38 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 39 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 40 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 41 Ile Met Pro Met Phe Gly Thr Ala 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 42 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 43 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 44 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 45 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 46 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 47 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 47 Ile Ser Gly Ile Phe Gly Thr Ala 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 48 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 49 Ile Ser Gly Ile Phe Gly Thr Ala 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 50 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 51 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 52 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 53 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 53 Ile Thr Pro Met Phe Gly Thr Ala 1 5 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 54 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 55 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 55 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 56 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 56 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 57 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 58 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 59 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 60 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 61 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 62 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 63 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 63 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 64 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 64 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 65 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 65 Ile Thr Pro Ile Phe Gly Thr Ala 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 66 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 67 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 67 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 68 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 68 Ile Thr Pro Met Phe Gly Thr Ala 1 5 <210> 69 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 69 Ile Ser Ala Met Phe Gly Thr Ala 1 5 <210> 70 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 70 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 71 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 71 Ile Thr Pro Met Phe Gly Thr Ala 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 72 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 73 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 73 Ala Arg Asp Asp Gly Tyr Ala Pro Ser Gly Gly Leu Arg Glu Phe Asp 1 5 10 15 Val <210> 74 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 74 Ala Arg Gly Arg Gly Ala Tyr Met Gly Pro Ser Met Asp Val 1 5 10 <210> 75 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 75 Ala Arg Gly Ala Arg Tyr Tyr Ala Gly Gly Tyr Phe Asp Val 1 5 10 <210> 76 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 76 Ala Arg Asp Ser Gly Asn Tyr Asp Gly Tyr Gly Pro Gly Ser Arg Phe 1 5 10 15 Asp Val <210> 77 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 77 Ala Arg Glu Arg Gly Ser Trp Ser Phe Gly Tyr Phe Asp Val 1 5 10 <210> 78 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 78 Ala Arg Ser Arg Thr Tyr Ala Asp Gly Arg Thr Phe Asp Val 1 5 10 <210> 79 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 79 Ala Arg Glu Leu Gly Tyr Leu Ala Gly Ser Pro Ser Pro Gly Phe Asp 1 5 10 15 Tyr <210> 80 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 80 Ala Arg Ser Arg Arg Tyr Trp Ala Asp Gly Gly Phe Asp Tyr 1 5 10 <210> 81 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 81 Ala Arg Glu Gly Gly Tyr Ser Pro Gly Gly Val Asp Phe Asp Tyr 1 5 10 15 <210> 82 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 82 Ala Arg Gly Thr Thr Tyr Ser Thr Ala Arg Tyr Phe Asp Val 1 5 10 <210> 83 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 83 Ala Arg Ser Pro Ala Tyr Tyr Phe Gly Pro Asn Met Asp Val 1 5 10 <210> 84 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 84 Ala Arg Ser Ser Arg Tyr Ala Pro Ser Asp Ser Thr Asn Phe Asp Gln 1 5 10 15 <210> 85 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 85 Ala Arg Gly Asp Arg Phe Tyr Val Gly Glu Arg Phe Asp Val 1 5 10 <210> 86 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 86 Ala Arg Gly Gly Gly Val Gly Arg Ile Trp Ile Ala Gly Tyr Gly Phe 1 5 10 15 Asp Gln <210> 87 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 87 Ala Arg Gly Pro Gly Tyr His Pro Ala Gly Ala Ser Gly Gln Phe Phe 1 5 10 15 Asp Leu <210> 88 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 88 Ala Arg Gly Arg Gly Tyr Ala Pro Asp Ala Leu Thr Asn Phe Asp Val 1 5 10 15 <210> 89 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 89 Ala Arg Gly Arg Gly Tyr Ile Ala Val Ala Gly Asp Met Asp Val 1 5 10 15 <210> 90 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 90 Ala Arg Gly Asp Ala Tyr Tyr Val Gly Gly Gly Ala Arg Pro Phe Asp 1 5 10 15 Leu <210> 91 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 91 Ala Arg Gly Tyr Ser Tyr Tyr Pro Gly Gly Gly Gly Gly Arg Asn Phe 1 5 10 15 Asp Tyr <210> 92 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 92 Ala Arg Ala Pro Thr Tyr Tyr Ala Ser Arg Asp Ser Tyr Asn Phe Asp 1 5 10 15 Tyr <210> 93 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 93 Ala Arg Asp Thr Thr Tyr Ile Ala Gly Gly His Phe Asp Val 1 5 10 <210> 94 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 94 Ala Arg Ala Ser Gly Tyr Phe Thr Gly Trp Gly Thr Phe Asp Tyr 1 5 10 15 <210> 95 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 95 Ala Arg Gly Arg Tyr Tyr Tyr Thr Val Gly Val Tyr Asp Val 1 5 10 <210> 96 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 96 Ala Arg Gly Gly Gly Tyr Ser Ala Asp Gly Gly Ala Gly Asn Asn Thr 1 5 10 15 Ile Phe Asp Val 20 <210> 97 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 97 Ala Arg Glu Arg Gly Tyr Thr Val Gly Gly Gly Gly Met Asp Val 1 5 10 15 <210> 98 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 98 Ala Arg Glu Tyr Leu Gly Asp Asp Tyr Ser Ser Gly Ser Tyr Phe Asp 1 5 10 15 Val <210> 99 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 99 Ala Arg Glu Ser Gly Tyr Ser Gly Thr Gly Gln Phe Asp Val 1 5 10 <210> 100 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 100 Ala Arg Ser Gly Gly Tyr Tyr Asp Tyr Gly Val Gly Tyr Asp Gln 1 5 10 15 <210> 101 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 101 Ala Arg Ser Gly Gly Tyr Ser Pro Ser Ile Gly Gly Phe Asp Val 1 5 10 15 <210> 102 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 102 Ala Arg Gly Pro Gly Tyr Asp Pro Ser Ser Leu Arg Gly Phe Asp Val 1 5 10 15 <210> 103 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 103 Ala Arg Gly Glu Glu Ala Tyr Tyr Asp Leu 1 5 10 <210> 104 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 104 Ala Arg Gly Thr Ser Tyr Leu Pro Gly Arg Ser Gly Phe Asp Val 1 5 10 15 <210> 105 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 105 Ala Arg Gly Arg Gly Tyr Asp Pro Ser Val Gly Gly Phe Asp Val 1 5 10 15 <210> 106 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 106 Ala Arg Asp Ser Thr Pro Ser Val Thr Ser Ser Leu Tyr Arg Ile Pro 1 5 10 15 Ala Phe Asp Val 20 <210> 107 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 107 Ala Arg Gly Pro Gly Tyr Tyr Pro Asp Ser Asn Asn Tyr Asp Leu 1 5 10 15 <210> 108 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 108 Ala Arg Gly Gly Thr Tyr Ser Pro Gly Gly Thr Tyr Phe Asp Val 1 5 10 15 <210> 109 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 109 Arg Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 110 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 110 Asn Ile Gly Ser Lys Ser 1 5 <210> 111 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 111 Gln Thr Val Ser Asn Tyr 1 5 <210> 112 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 112 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 113 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 113 His Ile Gly Ser Lys Ser 1 5 <210> 114 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 114 Ser Ser Asp Val Gly Gly Tyr Asn His 1 5 <210> 115 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 115 Ser Ser Asp Val Gly Gly Tyr Asn Tyr 1 5 <210> 116 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 116 Ser Ser Asn Met Gly Arg Asn Thr 1 5 <210> 117 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 117 Ser Gly Ser Ile Ala Ser Thr Tyr 1 5 <210> 118 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 118 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 119 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 119 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 120 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 120 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 121 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 121 Asp Ile Gly Ser Lys Ser 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 122 Ser Gly Ser Val Ser Thr Ser Asn Tyr 1 5 <210> 123 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 123 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 124 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 124 Arg Ser Leu Phe Asp Ser Ser Asp Asn Lys Asn Tyr 1 5 10 <210> 125 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 125 Arg Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 126 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 126 Ser Ser Asn Ile Gly Val Asn Tyr 1 5 <210> 127 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 127 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 128 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 128 Gln Ser Val Asp Arg Gly Tyr 1 5 <210> 129 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 129 Ser Ser Asp Ile Gly Ala Tyr Asn Tyr 1 5 <210> 130 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 130 Ala Leu Pro Lys Gln Tyr 1 5 <210> 131 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 131 Ser Asp Asn Val Gly Asn Gln Gly 1 5 <210> 132 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 132 Asn Asn Asn Val Gly Asn Gln Gly 1 5 <210> 133 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 133 Ser Ser Asp Val Gly Ala His Asn Phe 1 5 <210> 134 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 134 Gln Ser Val Asp Ser His 1 5 <210> 135 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 135 Thr Ser Asn Val Gly Arg Asn Thr 1 5 <210> 136 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 136 Arg Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 137 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 137 Arg Ser Asn Ile Gly Arg Asn Thr 1 5 <210> 138 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 138 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 139 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 139 Gln Ser Val Ser Ser Phe 1 5 <210> 140 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 140 Ala Leu Pro Lys Gln Tyr 1 5 <210> 141 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 141 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 142 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 142 Asn Gly Pro Ser Asn Tyr Ile 1 5 <210> 143 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 143 Gln Ser Val Ser Ser Ser Tyr 1 5 <210> 144 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 144 Ser Ser Asn Ile Gly Val Ser Phe 1 5 <210> 145 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 145 Trp Ala Ser 1 <210> 146 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 146 Asp Asp Ser 1 <210> 147 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 147 Ala Ala Ser 1 <210> 148 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 148 Ser Asn Asn 1 <210> 149 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 149 Ser Asn Asn 1 <210> 150 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 150 Asp Val Ser 1 <210> 151 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 151 Glu Val Thr 1 <210> 152 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 152 Asp Asn Asp 1 <210> 153 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 153 Glu Asp His 1 <210> 154 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 154 Glu Asp Asn 1 <210> 155 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 155 Asp Asn Ser 1 <210> 156 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 156 Glu Asp Asn 1 <210> 157 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 157 Asp Asp Ile 1 <210> 158 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 158 Ser Thr Asn 1 <210> 159 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 159 Arg Asn Asn 1 <210> 160 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 160 Trp Ala Ser 1 <210> 161 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 161 Trp Ala Ser 1 <210> 162 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 162 Arg Asn Asn 1 <210> 163 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 163 Ser Asn Asn 1 <210> 164 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 164 Gly Ala Ser 1 <210> 165 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 165 Glu Val Ser 1 <210> 166 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 166 Lys Asp Ser 1 <210> 167 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 167 Arg Asp Asn 1 <210> 168 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 168 Arg Asn Asn 1 <210> 169 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 169 Glu Val Asn 1 <210> 170 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 170 Gly Ala Ser 1 <210> 171 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 171 Asn Asp Asn 1 <210> 172 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 172 Ser Asn Asn 1 <210> 173 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 173 Ser Asn Asn 1 <210> 174 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 174 Lys Asn Asn 1 <210> 175 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 175 Asp Ala Ser 1 <210> 176 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 176 Lys Asp Thr 1 <210> 177 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 177 Glu Asp Asn 1 <210> 178 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 178 Leu Asn Ser Asp Gly Ser His 1 5 <210> 179 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 179 Gly Ala Ser 1 <210> 180 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 180 Arg Asp Asp 1 <210> 181 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 181 Gln Gln Tyr Tyr Ser Gly Ser Trp Thr 1 5 <210> 182 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 182 Gln Val Trp Asp Arg Ser Ser Asp His Val Val 1 5 10 <210> 183 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 183 Gln Gln Tyr Asp Asn Leu Pro Pro Val Thr 1 5 10 <210> 184 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 184 Ser Ala Trp Asp Asp Ser Leu Gly Gly Glu Val 1 5 10 <210> 185 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 185 Gln Val Trp Asp Ser Ser Asn Asp His Pro Val 1 5 10 <210> 186 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 186 Thr Ser Tyr Ala Gly Ser Asn Ser Leu Val 1 5 10 <210> 187 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 187 Ser Ser Tyr Ala Gly Gly Lys Trp Val 1 5 <210> 188 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 188 Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 189 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 189 Gln Ser Phe Asp Ala Ser Thr Leu Val 1 5 <210> 190 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 190 Gln Ser Tyr Asp Ser Asp Asn His Glu Val Ile 1 5 10 <210> 191 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 191 Gln Ser Tyr Asp Ser Ser Leu Ser Val Val Val 1 5 10 <210> 192 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 192 Gln Ser Tyr Asp Thr Ser Asn Arg Lys Val 1 5 10 <210> 193 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 193 Gln Val Trp Asp Thr Asn Ser Asp Pro Val Phe Val 1 5 10 <210> 194 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 194 Val Leu Tyr Met Gly Ser Gly Ile Ser Met 1 5 10 <210> 195 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 195 Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 196 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 196 Gln Gln Tyr Phe Ser Ser Pro Pro Ile Phe Thr 1 5 10 <210> 197 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 197 Gln Gln Tyr Tyr Ser Thr Pro Pro Thr 1 5 <210> 198 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 198 Gly Val Trp Asp Asp Ser Leu Asn Gly His Trp Val 1 5 10 <210> 199 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 199 Ala Ala Trp Asp Asp Ser Leu Lys Gly Arg Val 1 5 10 <210> 200 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 200 Gln Gln Tyr Gly Ser Ser Arg Leu Ser 1 5 <210> 201 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 201 Ser Ser Tyr Ala Gly Ser Asn Asn Val Val 1 5 10 <210> 202 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 202 Gln Ala Trp Asp Ser Ser Thr Ala Val 1 5 <210> 203 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 203 Ser Ala Trp Asp Ser Ser Leu Thr Ala Val Val 1 5 10 <210> 204 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 204 Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 205 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 205 Ala Ala Trp Asp Asp Ser Leu Asp Gly Pro Val 1 5 10 <210> 206 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 206 Gln Gln Arg Ser Met Trp Pro Leu Thr 1 5 <210> 207 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 207 Ser Ser Trp Asp Asp Asp Leu Asn Gly Pro Val 1 5 10 <210> 208 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 208 Gln Ser Tyr Asp Ser Ser Val Val 1 5 <210> 209 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 209 Ala Ala Trp Asp Val Ser Leu Asn Gly Gln Val 1 5 10 <210> 210 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 210 Ser Ala Trp Asp Ser Ser Leu Ser Asp Trp Val 1 5 10 <210> 211 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 211 Gln Gln Arg Phe Asn Trp Pro Pro Thr 1 5 <210> 212 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 212 Gln Ser Ala Asp Ala Ser Glu Asn Ser Val 1 5 10 <210> 213 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 213 Gln Ser Tyr Asp Thr Ser Asn Leu Val 1 5 <210> 214 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 214 Glu Thr Trp Asp Ser Asn Thr His Val Val 1 5 10 <210> 215 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 215 Gln Gln Val Asn Ser Phe Pro Arg Thr 1 5 <210> 216 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 216 Ser Ala Trp Asp Glu Ser Leu Ser Ser Val Leu 1 5 10 <210> 217 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 217 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 218 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 218 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 219 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 219 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 220 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 220 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 221 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 221 Gly Val Ile Phe Ser Ser Tyr Ala 1 5 <210> 222 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 222 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 223 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 223 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 224 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 224 Gly Gly Pro Phe Arg Ser Tyr Ala 1 5 <210> 225 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 225 Gly Val Pro Phe Ser Ser Tyr Ala 1 5 <210> 226 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 226 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 227 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 227 Gly Val Ile Phe Ser Ser Tyr Ala 1 5 <210> 228 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 228 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 229 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 229 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 230 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 230 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 231 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 231 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 232 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 232 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 233 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 233 Gly Gly Ile Phe Arg Ser Tyr Ala 1 5 <210> 234 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 234 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 235 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 235 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 236 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 236 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 237 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 237 Gly Gly Thr Phe Arg Ser Tyr Ala 1 5 <210> 238 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 238 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 239 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 239 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 240 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 240 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 241 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 241 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 242 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 242 Gly Val Thr Phe Arg Ser Tyr Ala 1 5 <210> 243 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 243 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 244 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 244 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 245 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 245 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 246 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 246 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 247 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 247 Ile Ile Thr Ile Phe Gly Thr Ala 1 5 <210> 248 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 248 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 249 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 249 Ile Ser Ala Ile Phe Gly Thr Ala 1 5 <210> 250 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 250 Ile Thr Pro Ile Phe Gly Thr Ala 1 5 <210> 251 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 251 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 252 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 252 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 253 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 253 Ile Ile Thr Ile Phe Gly Thr Ala 1 5 <210> 254 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 254 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 255 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 255 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 256 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 256 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 257 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 257 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 258 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 258 Ile Ser Pro Val Phe Gly Thr Ala 1 5 <210> 259 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 259 Ile Val Pro Leu Phe Gly Thr Ala 1 5 <210> 260 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 260 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 261 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 261 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 262 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 262 Ile Ser Ala Ile Phe Gly Thr Ala 1 5 <210> 263 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 263 Ile Ile Pro Met Phe Gly Thr Ala 1 5 <210> 264 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 264 Ile Asn Pro Ile Phe Gly Thr Ala 1 5 <210> 265 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 265 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 266 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 266 Ile Thr Pro Leu Phe Gly Thr Ala 1 5 <210> 267 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 267 Ile Met Pro Ile Phe Gly Thr Ala 1 5 <210> 268 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 268 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 269 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 269 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 270 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 270 Ile Thr Pro Ile Phe Gly Thr Ala 1 5 <210> 271 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 271 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 272 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 272 Ile Ser Gly Ile Phe Gly Thr Ala 1 5 <210> 273 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 273 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 274 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 274 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 275 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 275 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 276 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 276 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 277 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 277 Ala Arg Gly Ala Thr Gly Phe Tyr Asp Val 1 5 10 <210> 278 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 278 Ala Arg Gly Arg Glu Tyr Tyr Ala Ser Asn Gly Asp Ser Phe Asp Val 1 5 10 15 <210> 279 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 279 Ala Arg Asp Leu Ser Arg Asp Ser Leu Asn Leu Pro Gly Ser Ser Pro 1 5 10 15 Gly Tyr Asp Leu 20 <210> 280 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 280 Ala Arg Gly Ser Gly Tyr Tyr Val Ala Ala Ser Gly Ala Phe Asp Val 1 5 10 15 <210> 281 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 281 Ala Arg Ser Arg Gly Tyr Ala Pro Gly Thr Ser Phe His Tyr Asp Val 1 5 10 15 <210> 282 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 282 Ala Arg Asp Gln Gly Gly Thr Arg Gly Asn Tyr Phe Asp Val 1 5 10 <210> 283 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 283 Ala Arg Gly Gly Gly Gly Arg Phe Asp Val 1 5 10 <210> 284 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 284 Ala Arg Gly Gly Val Tyr Ser Phe Asp Val 1 5 10 <210> 285 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 285 Ala Arg Gly Leu Gly Thr Tyr Ser Pro Ser Leu Tyr Pro Arg Gly Met 1 5 10 15 Asp Val <210> 286 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 286 Ala Arg Gly Arg Ala Tyr Leu Ser Val Arg Gly Ser Phe Asp Val 1 5 10 15 <210> 287 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 287 Ala Arg Gly Gly Ser Gly Ser Phe Asp Val 1 5 10 <210> 288 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 288 Ala Arg Ser Arg Gly Tyr Thr Val Ser Ser Leu Ala Gly Arg Tyr Phe 1 5 10 15 Asp Gln <210> 289 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 289 Ala Arg Gly Leu Gly Leu Tyr Phe Asp Val 1 5 10 <210> 290 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 290 Ala Arg Val Arg Gly Gly Tyr Gly Pro Tyr Gly Asp Phe Asp Val 1 5 10 15 <210> 291 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 291 Ala Arg Gly Arg Ser Tyr Ile Val Ser Val Ser Pro Gly Phe Asp Val 1 5 10 15 <210> 292 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 292 Ala Arg Asp Ser Gly Ile Ala Ser Gly Tyr Thr Ala Tyr Met Asp Tyr 1 5 10 15 <210> 293 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 293 Ala Arg Gly Ala Gly Ser Thr Phe Asp Val 1 5 10 <210> 294 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 294 Ala Arg Gly Glu Ser Ala Tyr Tyr Ser Arg Asn Tyr Asp Val 1 5 10 <210> 295 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 295 Ala Arg Gly Gly Gly Tyr Tyr Pro Ala Gly Val Gly Arg Tyr Asp Val 1 5 10 15 <210> 296 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 296 Ala Arg Gly Pro Thr Leu Tyr Ser Pro Pro Val Phe Asp Val 1 5 10 <210> 297 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 297 Ala Arg Gly Ala Gly Val Ser Ala Gly Pro Ser Trp Pro Phe Asp Val 1 5 10 15 <210> 298 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 298 Ala Arg Ser Arg Gly Tyr Asn Val Ala Ala Ser Phe Gly Phe Asp Val 1 5 10 15 <210> 299 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 299 Ala Arg Gly Thr Asp Tyr Ser Gly Tyr Arg Gly Phe Asp Val 1 5 10 <210> 300 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 300 Ala Arg Gly Gly Gly Val Phe Asp Val 1 5 <210> 301 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 301 Ala Arg Glu Gly Gly Tyr Ser Pro Gly Gly Val Asp Phe Asp Tyr 1 5 10 15 <210> 302 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 302 Ala Arg Ser Pro Ala Tyr Tyr Phe Gly Pro Asn Met Asp Val 1 5 10 <210> 303 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 303 Ala Arg Gly Pro Gly Tyr His Pro Ala Gly Ala Ser Gly Gln Phe Phe 1 5 10 15 Asp Leu <210> 304 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 304 Ala Arg Gly Arg Gly Tyr Ala Pro Asp Ala Leu Thr Asn Phe Asp Val 1 5 10 15 <210> 305 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 305 Ala Arg Gly Tyr Ser Tyr Tyr Pro Gly Gly Gly Gly Gly Arg Asn Phe 1 5 10 15 Asp Tyr <210> 306 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 306 Ala Arg Ser Gly Gly Tyr Tyr Asp Tyr Gly Val Gly Tyr Asp Gln 1 5 10 15 <210> 307 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 307 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 308 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 308 Asn Ile Ala Thr Lys Ser 1 5 <210> 309 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 309 Asn Ile Ala Thr Lys Ser 1 5 <210> 310 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 310 Ile Asn Asn Val Gly Asp Gln Gly 1 5 <210> 311 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 311 Asn Ile Gly Ser Lys Ser 1 5 <210> 312 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 312 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 313 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 313 Thr Ser Asn Ile Gly Asn Asn Ala 1 5 <210> 314 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 314 Gly Ser Asn Val Gly Ser Asn Val 1 5 <210> 315 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 315 Ser Ser Asn Ile Gly Arg Asn Asp 1 5 <210> 316 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 316 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 317 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 317 Ser Ser Asn Leu Gly Ser Asn Tyr 1 5 <210> 318 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 318 Glu Ser Leu Cys Ser Thr Cys 1 5 <210> 319 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 319 Thr Gly Ala Val Thr Ser Gly Tyr Tyr 1 5 <210> 320 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 320 Ser Ser Asn Ile Gly Ser His Ser 1 5 <210> 321 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 321 Ser Ser Asp Val Gly Gly Tyr Asn Tyr 1 5 <210> 322 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 322 Ser Leu Arg Thr Ser Tyr 1 5 <210> 323 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 323 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 324 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 324 Ser Ser Asn Val Gly Asn Gln Gly 1 5 <210> 325 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 325 Gln Asn Val Leu Tyr Ser Ser Asn Asn Lys Asn Asn 1 5 10 <210> 326 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 326 Ser Gly Ser Val Ser Thr Thr Asn Tyr 1 5 <210> 327 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 327 Ser Asn Asn Val Gly Lys Gln Gly 1 5 <210> 328 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 328 Gln Tyr Ile Asp Arg Ser 1 5 <210> 329 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 329 Ser Gly Ser Val Ser Ser Phe Asn Tyr 1 5 <210> 330 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 330 Ser Ser Asn Ile Gly Asn Asn Ala 1 5 <210> 331 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 331 Ser Gly Ser Ile Ala Ser Thr Tyr 1 5 <210> 332 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 332 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 333 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 333 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 334 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 334 Arg Ser Leu Phe Asp Ser Ser Asp Asn Lys Asn Tyr 1 5 10 <210> 335 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 335 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 336 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 336 Arg Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 337 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 337 Ser Asn Asn 1 <210> 338 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 338 His Asp Ser 1 <210> 339 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 339 Arg Asn Ser 1 <210> 340 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 340 Gly Ala Ser 1 <210> 341 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 341 Tyr Asp Ser 1 <210> 342 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 342 Arg Asn Asn 1 <210> 343 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 343 Ser Leu Asn 1 <210> 344 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 344 Arg Asn Asn 1 <210> 345 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 345 Gly Arg Asp 1 <210> 346 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 346 Ser Asn Asn 1 <210> 347 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 347 Arg Asn Ser 1 <210> 348 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 348 Gly Ala Thr 1 <210> 349 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 349 Ser Thr Ser 1 <210> 350 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 350 Gly Asn Ser 1 <210> 351 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 351 Glu Val Ser 1 <210> 352 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 352 Gln Ser Thr 1 <210> 353 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 353 Ser Asn Asn 1 <210> 354 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 354 Arg Asn Asp 1 <210> 355 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 355 Trp Ala Ser 1 <210> 356 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 356 Asn Thr Asn 1 <210> 357 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 357 Arg Asn Asn 1 <210> 358 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 358 Tyr Ala Ser 1 <210> 359 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 359 Asn Thr Asn 1 <210> 360 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 360 Tyr Asp Asp 1 <210> 361 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 361 Glu Asp His 1 <210> 362 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 362 Asp Asn Ser 1 <210> 363 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 363 Arg Asn Asn 1 <210> 364 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 364 Trp Ala Ser 1 <210> 365 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 365 Ser Asn Asn 1 <210> 366 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 366 Ser Asn Asn 1 <210> 367 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 367 Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 368 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 368 Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Trp Val 1 5 10 <210> 369 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 369 Ser Ala Trp Asp Ser Ser Leu Ser Asp Trp Val 1 5 10 <210> 370 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 370 Gln Gln Tyr Ser Ser Ser Pro Tyr Thr 1 5 <210> 371 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 371 Gln Leu Trp Asp His Thr Asn Ser His Val Val 1 5 10 <210> 372 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 372 Ser Ala Trp Asp Asn Thr Val Ser Gly Trp Val 1 5 10 <210> 373 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 373 Glu Ala Trp Asp Asp Ser Leu Ser Gly Pro Val 1 5 10 <210> 374 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 374 Ala Ala Trp Asp Asp Arg Leu Asn Gly Phe Val 1 5 10 <210> 375 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 375 Ala Ala Trp Asp Ala Ser Leu Met Ile Tyr Val 1 5 10 <210> 376 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 376 Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val 1 5 10 <210> 377 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 377 Ala Ala Trp Asp Asp Ser Leu Asn Gly Val Val 1 5 10 <210> 378 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 378 Gln Gln Tyr Gly Ser Ser Pro Gln Thr 1 5 <210> 379 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 379 Leu Leu Tyr Tyr Gly Gly Pro Trp Val 1 5 <210> 380 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 380 Ala Ala Trp Asp Asp Gly Leu Ser Gly Trp Val 1 5 10 <210> 381 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 381 Ala Ser Trp Asp Asp Ser Leu Asn Ala Tyr Val 1 5 10 <210> 382 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 382 Asn Ser Arg Gly Ser Gly Gly Asn Pro Tyr Val 1 5 10 <210> 383 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 383 Ala Ala Trp Asp Asp Ser Leu Asn Gly Arg Val 1 5 10 <210> 384 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 384 Ser Ala Trp Asp Asn Ser Leu Ser Ala Trp Val 1 5 10 <210> 385 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 385 Gln Gln Tyr Tyr Gly Lys Pro Phe Thr 1 5 <210> 386 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 386 Val Leu Tyr Met Gly Arg Gly Ile Tyr Val 1 5 10 <210> 387 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 387 Ser Ala Trp Asp Ser Ser Leu Ser Val Trp Val 1 5 10 <210> 388 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 388 His Gln Thr Ser Ser Leu Pro Trp Thr 1 5 <210> 389 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 389 Ala Leu Tyr Val Gly Gly Gly Ile Ser Val 1 5 10 <210> 390 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 390 Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Val 1 5 10 <210> 391 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 391 Gln Ser Phe Asp Ala Ser Thr Leu Val 1 5 <210> 392 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 392 Gln Ser Tyr Asp Ser Ser Leu Ser Val Val Val 1 5 10 <210> 393 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 393 Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 394 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 394 Gln Gln Tyr Phe Ser Ser Pro Pro Ile Phe Thr 1 5 10 <210> 395 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 395 Ala Ala Trp Asp Asp Ser Leu Lys Gly Arg Val 1 5 10 <210> 396 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 396 Gln Dear Tyr Asp Dear Dear Val Val 1 5 <210> 397 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 397 caggttcaat tagtgcagtc tggtgctgaa gtgaaaaagc ccggctcaag tgttaaagta 60 agctgtaagg cgagcggtgg cccattcagc tcatacgcca ttagctgggt gcgacaggct 120 cctggtcagg gcctcgaatg gatgggcggc attagcccaa tgtttggcac tgcaaattat 180 gcccagaaat ttcagggtag agtcacaatt accgcagaca agagcacctc aaccgcctac 240 atggaactga gtagcctgcg ttccgaagat acagctgtgt attactgtgc gcgcgacgac 300 ggttacgctc ctagtggtgg tctgcgtgag tttgacgttt ggggccaggg gaccttagtc 360 actgtgtcta gc 372 <210> 398 <211> 340 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 398 gacatccaga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagccg gagtgtttta tacagctcca acaacaagaa ctacttagct 120 tggtaccaac aaaaaccggg acagcctcct aagttgctca tttattgggc ttctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggcg gtttattact gtcagcaata ttatagtggt 300 tcctggacat tcggccaagg gaccaaggtg gaaatcaaac 340 <210> 399 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 399 caggttcaat tagtgcagtc tggtgctgaa gtgaaaaagc ccggctcaag tgttaaagta 60 agctgtaagg cgagcggagg catctttagc tcatacgcca ttagctgggt gcgacaggct 120 cctggtcagg gcctcgaatg gatgggcggc attagcccta tctttggcac tgcaaattat 180 gcccagaaat ttcagggtag agtcacaatt accgcagata aaagcacgaa taccgcctac 240 atggaactga gtagcctgcg ttccgaagat acagctgtgt attactgtgc gcgcggtcgt 300 ggtgcttaca tgggtcctag tatggatgtg tggggccagg ggaccttagt cactgtgtct 360 agc 363 <210> 400 <211> 325 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 400 cagcctgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120 caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaggccggg 240 gatgaggccg actactactg tcaggtgtgg gataggagta gtgatcatgt ggtgttcggc 300 ggagggacca agctgaccgt cctag 325 <210> 401 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 401 caggttcaat tagtgcagtc tggtgctgaa gtgaaaaagc ccggctcaag tgttaaagta 60 agctgtaagg cgagcggagg taccttcagc tcatacgcca ttagctgggt gcgacaggct 120 cctggtcagg gcctcgaatg gatgggcggc atcagcccaa tctttggcac tgcaaattat 180 gcccagaaat ttcagggtag agtcacaatt accgcagata aaagcacgtc aactgtgtat 240 atggaactga gtagcctgcg ttccgaagat acagctgtgt attactgtgc gcgcggtgct 300 cgttactacg ctggtggtta cttcgatgtg tggggccagg ggaccttagt cactgtgtct 360 agc 363 <210> 402 <211> 325 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 402 Gly Ala Ala Ala Thr Thr Gly Thr Gly Cys Thr Gly Ala Cys Thr Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys Ala Gly Gly Cys Ala Cys Cys Cys Thr 20 25 30 Gly Thr Cys Thr Thr Thr Gly Thr Cys Thr Cys Cys Ala Gly Gly Gly 35 40 45 Gly Ala Ala Ala Gly Ala Gly Cys Cys Ala Cys Cys Cys Thr Cys Thr 50 55 60 Cys Cys Thr Gly Cys Ala Gly Gly Gly Cys Cys Ala Gly Thr Cys Ala 65 70 75 80 Gly Ala Cys Thr Gly Thr Thr Ala Gly Cys Ala Ala Cys Thr Ala Cys 85 90 95 Thr Thr Ala Gly Cys Cys Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys 100 105 110 Ala Gly Ala Gly Ala Cys Cys Thr Gly Gly Cys Cys Ala Gly Gly Cys 115 120 125 Thr Cys Cys Cys Ala Gly Gly Cys Thr Cys Cys Thr Cys Ala Thr Cys 130 135 140 Thr Ala Cys Gly Cys Thr Gly Cys Ala Thr Cys Cys Ala Cys Gly Cys 145 150 155 160 Gly Gly Gly Cys Cys Ala Cys Thr Gly Gly Thr Gly Thr Cys Cys Cys 165 170 175 Ala Gly Cys Cys Ala Gly Gly Thr Thr Cys Ala Gly Thr Gly Gly Cys 180 185 190 Ala Gly Cys Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Cys Ala Gly 195 200 205 Ala Gly Thr Thr Cys Ala Cys Thr Cys Thr Cys Ala Cys Cys Ala Thr 210 215 220 Cys Ala Gly Cys Ala Gly Cys Cys Thr Gly Cys Ala Ala Thr Cys Thr 225 230 235 240 Gly Ala Ala Gly Ala Thr Thr Thr Thr Gly Cys Ala Ala Thr Thr Thr 245 250 255 Ala Thr Thr Ala Cys Thr Gly Thr Cys Ala Ala Cys Ala Gly Thr Ala 260 265 270 Thr Gly Ala Thr Ala Ala Cys Thr Thr Gly Cys Cys Thr Cys Cys Gly 275 280 285 Gly Thr Cys Ala Cys Thr Thr Thr Cys Gly Gly Cys Cys Cys Thr Gly 290 295 300 Gly Gly Ala Cys Cys Ala Cys Ala Gly Thr Gly Gly Ala Thr Ala Thr 305 310 315 320 Cys Ala Ala Ala Cys 325 <210> 403 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 403 caggttcaat tagtgcagtc tggtgctgaa gtgaaaaagc ccggctcaag tgttaaagta 60 agctgtaagg cgagcggtgt tacctttagc tcatacgcca ttagctgggt gcgacaggct 120 cctggtcagg gcctcgaatg gatgggcggc atcagcccaa tcttcggtac tgcaaattat 180 gcccagaaat ttcagggtag agtcacaatt accgcagatc agagtaccaa cactgtctac 240 atggaactga gtagcctgcg ttccgaagat acagctgtgt attactgtgc gcgcgacagt 300 ggtaattacg acggttacgg tcctggtagt cgtttcgacg tgtggggcca ggggacctta 360 gtcactgtgt ctagc 375 <210> 404 <211> 331 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 404 cagcctgggc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc caacatcgga agtaatactg taaactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240 tctgaggatg aggctgatta tttctgttca gcttgggatg acagcctggg tggcgaggtc 300 ttcggaactg ggaccaaggt caacgtccta g 331 <210> 405 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 405 caggttcaat tagtgcagtc tggtgctgaa gtgaaaaagc ccggctcaag tgttaaagta 60 agctgtaagg cgagcggagt gacctttagt tcttacgcca ttagctgggt gcgacaggct 120 cctggtcagg gcctcgaatg gatgggcggc attatgccta tgttcggcac tgcaaattat 180 gcccagaaat ttcagggta...

Claims

1. A composition for enhancing an immune response, comprising a tetravalent antibody molecule which is a dimer of a bispecific scFv fragment, wherein the bispecific scFv fragment comprises an scFv that specifically binds to the GITR protein, an scFv that specifically binds to the PD-L1 protein, and a linker-hinge-linker domain, wherein the two scFvs are bound together via the linker-hinge-linker domain, and the linker-hinge-linker domain consists of an immunoglobulin hinge region amino acid sequence of IgG1, IgG2, IgG3, or IgG4 adjacent to a flexible linker amino acid sequence at both ends, and the flexible linker amino acid sequence is an amino acid sequence (GGGS) X1-6 (GGGGS) X1-6 The composition comprising , or GSAGSAAGSGEF.

2. A nucleic acid construct comprising the following nucleic acid molecules, wherein the nucleic acid construct encodes the tetravalent antibody molecule described in Claim 1: A nucleic acid molecule encoding scFv, which specifically binds to GITR proteins; Nucleic acid molecules encoding scFv that specifically bind to the PD-L1 protein; and A nucleic acid molecule that encodes a linker-hinge-linker domain.

3. A vector comprising the nucleic acid construct described in Claim 2.

4. A host cell comprising the vector described in Claim 3.

5. The host cell according to claim 4, which is a T cell, a B cell, a follicular T cell, or an NK cell.

6. A chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing the tetravalent antibody molecule described in Claim 1.

7. The CAR according to claim 6, wherein the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain.

8. The CAR according to claim 6 or 7, wherein the transmembrane domain comprises a CD28 transmembrane domain.

9. The CAR according to any one of claims 6 to 8, further comprising one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain.

10. The CAR according to claim 9, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40.

11. The CAR according to any one of claims 6 to 10, wherein the intracellular signaling domain comprises a CD3 zeta chain intracellular signaling domain.

12. A genetically modified cell that expresses and carries the CAR described in any one of claims 6 to 11 on its cell surface membrane.

13. The genetically modified cell according to claim 12, which is a T cell or an NK cell.

14. The genetically modified cell according to claim 13, wherein the T cell is CD4+ or CD8+.

15. The genetically modified cells according to claim 14, comprising a mixed population of CD4+ cells and CD8+ cells.

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