Modular Tetrameric Bispecific Antibody Platform

Abstract

The present invention relates to a tetrameric bispecific antibody molecule, a method for producing the tetrameric bispecific antibody molecule, the use of the tetrameric bispecific antibody molecule, and a nucleic acid molecule encoding the tetrameric bispecific antibody molecule.

Description

[0001] Related Applications

[0002] This application claims the benefit and priority of U.S.S.N. 62 / 408,271, filed on October 14, 2016, the content of which is hereby incorporated by reference in its entirety. Field of the Invention

[0003] The present invention generally relates to tetrameric bispecific antibody molecules, methods and systems for producing such tetrameric bispecific antibody molecules.

[0004] Government Interests

[0005] This invention was made with government support under award. The government has certain rights in this invention. Background of the Invention

[0007] Bispecific antibodies (BsAbs) are antibodies or antibody-like molecules with two different binding specificities. BsAbs have extensive applications in biomedicine, particularly in cancer immunotherapy. Currently, the focus of immunotherapy research is on how to utilize 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 effector cells.

[0008] BsAbs can be prepared by methods such as chemical engineering, cell engineering, and genetic engineering. One advantage of genetic engineering is that antibodies can be easily modified, enabling 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. Due to the lack of an IgG Fc domain in those BsAbs, their small size enhances their penetration into tumors, but they have a significantly shorter half-life in vivo and also lack the ADCC effect associated with the constant region of antibodies.

[0009] To improve stability and therapeutic potential, recombinant genetic modifications are made in the heavy chain to promote its heterodimerization and produce a higher yield of Fc-containing IgG-like bispecific antibodies. Several rational design strategies have been used to engineer the antibody CH3 chains for heterodimerization, namely disulfide bonds, salt bridges, and knobs-into-holes. The basis for generating knobs and holes in juxtaposed positions is that knob-hole interactions will favor heterodimer formation, while knob-knob and hole-hole interactions will prevent homodimer formation due to the lack of favorable interactions. Although this knob-into-hole method solves the heavy chain homodimerization problem, it does not address the issue of mismatches between the light chains and heavy chains from two different antibodies. Although it is possible to identify the same light chain for two different antibodies, the possibility of using two antibody sequences that can share a common light chain for BsAb construction is very limited.

[0010] There is a need to provide improved BsAbs that are more easily manufacturable and have better clinical stability and efficacy and / or reduced systemic toxicity. SUMMARY OF THE INVENTION

[0011] The present invention provides tBsAbs that are more easily manufacturable and have better clinical stability and efficacy and / or reduced systemic toxicity.

[0012] One aspect of the invention relates to a tetravalent antibody molecule. The tetravalent antibody can be a dimer of bispecific scFv fragments, the scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen. The two binding sites can be linked together via a linker domain. In an embodiment, the scFv fragment is a tandem scFv and the linker domain comprises the amino acid sequence of an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, and IgG4 hinge region). In an embodiment, the amino acid sequence of the immunoglobulin hinge region can be flanked by flexible linker amino acid sequences such as having the amino acid sequence (GGGS) X1-6 , (GGGGS) X1-6 and GSAGSAAGSGEF. In an embodiment, the linker domain comprises at least a portion of an immunoglobulin Fc domain, e.g., IgG1, IgG2, IgG3, and IgG4 Fc domains. At least a portion of the immunoglobulin Fc domain can be the CH2 domain. The Fc domain can be linked to the C-terminus of the amino acid sequence of an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, and IgG4 hinge region). The linker domain can comprise flexible linker amino acid sequences (e.g., ((GGGS) X1-6 , (GGGGS) X1-6 and GSAGSAAGSGEF) at one or both ends.

[0013] On the other hand, the present invention relates to nucleic acid constructs. The construct may comprise a nucleic acid molecule encoding: a light chain variable region and a heavy chain variable region of an antibody that can specifically bind to a first antigen; a light chain variable region and a heavy chain variable region of an antibody that can specifically bind to a second antigen; and a linker domain. In an embodiment, the linker domain is an amino acid sequence of an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, and IgG4 hinge regions). In an embodiment, the linker domain is at least a portion of an immunoglobulin Fc domain, such as IgG1, IgG2, IgG3, and IgG4 Fc domains. 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 an amino acid sequence of an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, and IgG4 hinge regions). The linker domain may comprise a flexible linker amino acid sequence (e.g., (GGGS)X1-6, (GGGGS)X1-6, and GSAGSAAGSGEF) at one or both ends.

[0014] Another aspect of the present invention is a vector that comprises the nucleic acid construct of the above aspect.

[0015] Another aspect of the present invention is a host cell (e.g., T cell, B cell, follicular T cell, and NK cell) that comprises the vector of the above aspect.

[0016] 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, and the extracellular domain comprises a tetravalent antibody molecule of any of the above aspects or embodiments. In an embodiment, 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 an embodiment, the CAR further comprises one or more additional co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, and OX40) located between the transmembrane domain and the intracellular signaling domain (e.g., CD3ζ chain).

[0017] Another aspect of the present invention is a genetically engineered cell. The genetically engineered cell can express and carry the chimeric antigen receptor of any of the above aspects or embodiments on its cell surface membrane. In an embodiment, 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.

[0018] One aspect of the present invention is a method for treating a disease or disorder. The method may include administering a tetravalent antibody molecule of any of the above aspects or embodiments. In an embodiment, the disease or disorder is a CNS-related disease or disorder, such as a CNS cancer or a neurodegenerative disease. The CNS cancer may be glioblastoma multiforme (GBM). The neurodegenerative disease may be amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, or Huntington's disease. In an embodiment, the tetravalent antibody molecule recognizes and / or binds to a CNS transport receptor, such as the transferrin receptor (TfR), VCAM-1, CD98hc, and the insulin receptor. In this aspect and any of the above aspects or embodiments, the tetravalent antibody molecule enhances transport across the blood-brain barrier.

[0019] Any of the above aspects and embodiments may be combined with any other aspect or embodiment.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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 incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.

[0021] Other features and advantages of the present invention will be apparent from and encompassed by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is an illustration showing the design and formation of a tetrameric bispecific antibody (tBsAb).

[0023] Figure 2 is a schematic illustration of the pcDNA3.1\1scFv-hinge-scFv expression vector.

[0024] Figure 3A is an SDS gel showing the use of NotI and BsiWI digestion and then insertion of a synthetic tetramer linker into a vector expressing a tetramer. Figure 3B is an SDS gel showing the purification of the tBsAb according to the present invention.

[0025] Figure 4A is an illustration showing a method for detecting the antibody binding affinity of the tBsAb. Figures 4B to 4CFigure showing data when plates were coated with CCR4-Fc (B) or with PD-L1-Fc (C) and then incubated with a tetravalent bispecific (anti-CCR4 and anti-PD-L1) and control antibodies. Results show that this tetravalent antibody can bind to both CCR4-Fc and PD-L1-Fc in a dose-dependent manner.

[0026] Figure 5 Figure showing binding of anti-CAIX-PD-L1 bispecific mAb to CAIX-Fc fusion protein.

[0027] Figure 6 Figure showing binding of anti-CAIX-PD-L1 bispecific mAb to PD-L1-Fc fusion protein.

[0028] Figure 7A Illustration of the way in which linker length can be varied to optimize bispecific mAb binding. Figure 7B Schematic diagram of the tBsAb sequence.

[0029] Figure 8A .αGITR-αPD-L1 tBsAb engagement. The tBsAb binds to the GITR protein on T cells and the PD-L1 protein on tumor cells. B. Schematic illustration of the tBsAb form achieved through interchain disulfide bond formation between cysteine residues in the hinge region.

[0030] Figure 9 A. Basic structure of two scFvs linked to form the tBsAb. Figure 9 B. Bispecific dimer taFv against GITR and PD-L1. Each V H and V L pair is linked by a 15-residue linker to form the scFv. Two scFvs are linked by a linker-hinge-linker (55 residues). The hinge region has two cysteine residues, allowing the two taFvs to pair via a disulfide bridge under oxidizing conditions.

[0031] Figure 10 A. Basic structure of tandem scFv. Figure 10 B. Trifunctional tBsAb against GITR and PD-L1 with an additional CH2 domain. Each V H and V L pair is linked by a 15-residue linker. Two scFvs are linked by a linker-hinge-CH2-linker domain. The hinge region has two cysteine residues, allowing the two tandem scFvs to pair via a disulfide bridge under oxidizing conditions. The N-terminus of the CH2 domain can bind to Fc-γ or C1q. The resulting form is a trifunctional tBsAb.

[0032] Figure 11 Mechanism of action of αGITR-αPD-L1 tBsAb. Figure 11 A. Tumor cells overexpress the PD-L1 protein. The PD-1 / PD-L1 interaction inhibits effective T cell activation and promotes immune containment and adaptive immune resistance. Figure 11 B. αGITR-αPD-L1 tBsAb can enhance the immune response. The αPD-L1 arm blocks the PD-1 / PD-L1 pathway and thus can inhibit T cell exhaustion and eliminate Treg suppression. The αGITR arm acts as an agonist of the co-stimulatory GITR receptor, resulting in upregulation of GITR expression, enhancing T cell activation and proliferation.

[0033] Figure 12 Schematic illustration of the αGITR-αPD-L1 cloning process. The donor vector and the pcDNA 3.4 expression vector were digested with SfiI and NotI restriction enzymes. The V H GITR-V L GITR genes were isolated and then ligated to each other. The final plasmid produced the αGITR-αPD-L1 clone.

[0034] Figure 13 Schematic illustration of the cloning process of control plasmid (1). The pcDNA3.1 vector and the expression vector pcDNA 3.4 were digested with SfiI and NotI restriction enzymes. The V H F10-V L F10 gene was isolated and then ligated to the pcDNA 3.4 expression vector. The final plasmid produced the αGITR-αPD-L1 clone.

[0035] Figure 14 Schematic illustration of the cloning processes of control plasmids (2) and (3). The isolated F10V H and V L DNA and the recipient vector pcDNA 3.4 were digested with BsiWI and BamHI restriction enzymes and then ligated to each other. The final plasmid produced the final αGITR1-αPD-L1 and αGITR10-aPD-L1 clones.

[0036] Figure 15 Schematic illustration of the cloning strategy for the αGITR-αPD-L1 construct with CH2. The HindIII restriction site was introduced into the pcDNA 3.4 expression vector by site-directed mutagenesis. Subsequently, the isolated CH2 fragment and the expression vector were digested and ligated to each other to complete the αGITR-αPD-L1 construct with CH2.

[0037] Figure 16 Recipient pcDNA 3.4 vector, six V H GITR-VL GITR insert and a V H F10-V L Restriction enzyme analysis (REA) of the F10 insert. Ethidium bromide-stained 1% agarose gel of DNA electrophoresed in TAE buffer. All plasmids were digested with SfiI and NotI restriction enzymes. Lane 1: shows the 7.5 kb digested recipient pcDNA 3.4 vector. The lower band between 500 and 1000 bp is the previously used scFv insert (from the Marasco laboratory). Lanes 2 - 6: the lower band represents the 800 bp V H GITR-linker-V L GITR insert. The larger band that aggregates at 8 kb is the doubly digested progeny vector. Lane 7: 800 bp V H F10-linker-V L The F10 insert is visualized in the lower band that aggregates between 500 and 1000 bp. Lane "bp" represents the 1 kb DNA ladder (NEB).

[0038] Figure 17 V H F10-linker-V L F10 cDNA and recipient pcDNA 3.4 expression (containing V H GITR1-V L GITR1 or V H GITR10-V L REA of GITR10) shows an ethidium bromide-stained 1% agarose gel of DNA electrophoresed in TAE buffer. The recipient expression vector and insert were digested with BsiWI and BamHI restriction enzymes. Lane: shows a single band that aggregates at 800 bp, which represents the V isolated by PCR H F10-linker-V L F10 (scFv). Lanes 2 and 3: the upper two bands visualize the pcDNA3.4 expression vectors containing V H GITR1-V L GITR1 (lane 2) and V H GITR10-V L GITR10 (lane 3). Both contain 7500 bp and can be detected at the correct ladder band level. The lower bands in lanes 2 and 3 that aggregate between 500 and 1000 bp represent the digested V isolated from their vectors H PD-L1-V L PD-L1 fragment. Lane "bp" corresponds to the 1 kb DNA ladder (NEB).

[0039] Figure 18 Purified tBsAb was analyzed by SDS-PAGE. A Coomassie blue-stained SDS gel of the protein electrophoresed in MES buffer is shown. 3-5 μg of protein samples were loaded onto the gel and separated under (A) reducing and (B) non-reducing conditions. Lanes 1-8: Under non-reducing conditions, SDS PAGE revealed two major bands for each protein. The higher band had an apparent molecular weight between 80 kDa and 115 kDa, and the lower molecular weight band was between 70 and 80 kDa. Some weak but high molecular weight bands (>180 kDa) were observable in non-reducing SDS-gel analysis. SDS-gel analysis under reducing conditions (10% DTT; 70 °C for 10 min) showed only one band with an apparent molecular weight between 70 and 80 kDa. Lane 9: Under non-reducing conditions, a single band with an apparent molecular weight slightly higher than 140 kDa was shown. Visualization of the two bands under reducing conditions emphasized the correct expression of αGITR IgG showing separated heavy and light chains (50 kDa and 25 kDa). Lane kDa represents the BenchMark prestained protein ladder (Invitrogen) run under the corresponding conditions (4%-12% gel concentration in MES buffer).

[0040] Figure 19 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 (0.0001 mg / mL - 1 mg / mL; x-axis) of each αGITR-αPD-L1 was subjected to ELISA on the PD-L1 antigen. Results show the mean and standard deviation of absorbance at 450 nm (y-axis). Each sample was run in triplicate at each concentration. The background signal of the raw signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0041] Figure 20 Cell-based ELISA testing the binding of αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies against acetone-methanol-fixed GITR+-expressing CF2 cells. The F10-αPD-L1 antibody represents the negative control. All antibodies were tested using a series of 1:3 dilutions in the range from 3.3 mg / mL to 0.0046 mg / mL. All antibodies were tested against 1000 GITR+ CF2 cells per well. Each bar represents the mean (deviation represented by the bar) obtained from triplicate samples. The background signal of the raw signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0042] 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. The F10-αPD-L1 antibody represents the negative control. All antibodies were tested using a series of 1:3 dilutions ranging from 3.3 mg / mL to 0.0046 mg / mL. All antibodies were tested against 1000 GITR+ CF2 cells per well. Each bar represents the mean value obtained from triplicate samples (the deviation represented by the bar). The background signal of the raw signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0043] Figure 22 Cell-based ELISA testing the binding of αGITR10-αPD-L1 and commercial αGITR10 mAb antibodies to GITR+-expressing CF2 cells fixed with 8% paraformaldehyde. The F10-αPD-L1 antibody represents the negative control. All antibodies were tested using a series of 1:2 dilutions ranging from 5 mg / mL to 0.078 mg / mL. All antibodies were tested against 10,000 GITR+ CF2 cells per well. Each bar represents the mean value obtained from triplicate samples (the deviation represented by the bar). The background signal of the raw signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0044] Figure 23A . Flow cytometry analysis of fluorescently activated αGITR10-αPD-L1 tBsAb (anti-His Alexa488 (APC)-conjugated) tested on GITR+ CF2 cells. Figure 23B . Flow cytometry analysis of fluorescently activated αGITR10 IgG Ab (anti-human IgG Fc (FITC-conjugated)) tested on GITR+ CF2 cells. The horizontal line indicates the intensity signal of the fluorescence, and the vertical axis indicates the cell count. Each individual picture represents αGITR10-αPD-L1 at different concentrations with a constant number of cells.

[0045] Figure 24A . Flow cytometry analysis of fluorescently activated αGITR1-αPD-L1 tBsAb (anti-His Alexa488 (APC)-conjugated) tested on GITR+ CF2 cells. Figure 24B. Flow cytometry analysis of fluorescently activated αGITR10IgG Ab (anti-human IgG Fc (FITC-conjugated)) tested with GITR+CF2 cells. The horizontal line indicates the intensity signal of fluorescence, and the vertical axis indicates cell count. Each individual picture represents different concentrations of αGITR10-αPD-L1 ( Figure 24A ) and αGITR10 IgG ( Figure 24B ) with a constant number of cells.

[0046] Figure 25 Restriction enzyme analysis (REA) of 16 clones. A 1% agarose gel stained with ethidium bromide of DNA electrophoresed in TAE buffer is shown. All plasmids were digested with HindIII and BamHI restriction enzymes. Two bands are shown in lane 10: the band that aggregates between 6 kb and 8 kb represents the digested recipient pcDNA3.4 vector (7.5 kb). The lower band between 500 and 1000 bp indicates a size very close to the expected theoretical size of 800 bp for the fragment separated by HindIII and BamHI restriction enzymes. The lane "bp" represents a 1 kb DNA ladder.

[0047] Figure 26 REA of clone 10 (GITR10-PDL1 with HindIII) and GITR10-PDL1 (without HindIII restriction site). A 1% agarose gel stained with ethidium bromide of DNA electrophoresed in TAE buffer is shown. The two plasmids were digested with HindIII only (lane 1), NotI only (lane 2), and both HindIII and NotI simultaneously (lane 3). Digestion of clone 10 with a single enzyme (lanes 1 and 2) produced one band that aggregated around 8000 bp. Digestion of clone 10 with both enzymes (lane 3) resulted in the production of two fragments, with the smaller-sized band aggregating below 500 bp. Digestion of αGITR10-αPD-L1 with only the HindIII restriction site (lane 1) revealed supercoiled plasmid DNA.

[0048] Figure 27 Restriction enzyme digestion analysis of vector GITR10-PDL1 (containing HindIII restriction site) and CH2 fragment. A 1% agarose gel stained with ethidium bromide of DNA electrophoresed in TAE buffer is shown. Lane 1: Single digestion of αGITR-αPD-L1 with HindIII. Lane 2: The CH2 fragment digested with HindIII produced a band that aggregated below the 500 bp marker of the ladder. The lane "bp" corresponds to a 1 kb DNA ladder.

[0049] Figure 28Purified αGITR10-αPD-L1 BsAb with CH2 was analyzed by SDS-PAGE. Shown is a Coomassie blue-stained SDS gel of the proteins electrophoresed in MES buffer. 3-5 μg of protein samples were loaded onto the gel and separated under (R) reducing and (NR) non-reducing conditions. Under non-reducing conditions, SDS PAGE revealed two major bands for each protein. The higher band had an apparent molecular weight of approximately 140 kDa, and the lower band had a molecular weight of 80 kDa, correlating with the theoretical sizes of the dimer (150 kDa) and monomer (75 kDa) BsAb. SDS-gel analysis under reducing conditions (10% DTT; 70 °C for 10 minutes) showed only one band with an apparent molecular weight between approximately 80 kDa, and enhanced the correct expression of the tBsAb that could be reduced by its disulfide bridges in the hinge region. Lane "kDa" represents the reference pre-stained protein ladder (Invitrogen) under the corresponding conditions (run on a 4%-12% gel concentration in MES buffer).

[0050] Figure 29 Cell-based ELISA testing the binding of αGITR10-αPD-L1 and αGITR10 IgG antibodies with CH2 against GITR+-expressing CF2 cells fixed with 8% paraformaldehyde. The F10-αPD-L1 antibody represents the negative control. All antibodies were tested using a series of 1:2 dilutions ranging from 5 mg / mL to 0.16 mg / mL. All antibodies were tested against 10,000 GITR+ CF2 cells per well. Each bar represents the mean (deviation represented by the bar) obtained from triplicate samples. The background signal of the raw signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0051] Figure 30 ADCC activity of the αGITR10-αPD-L1 antibody with CH2. The ADCC activity of αGITR10-αPD-L1 with CH2 was measured at different concentrations. All antibodies were serially diluted (1:2), starting at the highest concentration of 20 mg / mL down to 0.02 mg / mL, and tested against 20,000 GITR+ CF2 cells per well. The ratio of effector cells (GITR+ CF2) to target cells (Wils-2) was 5:1. αGITR IgG represents the positive control, and F10-αPD-L1 represents the negative control. The vertical axis represents the raw value of the luciferase activity in the effector cells quantified by a luminescence readout. Each sample was run in triplicate at each concentration; the mean standard deviation is shown in parentheses. The background of the GITR+ CF2 cells in RPMI medium was subtracted from the values obtained.

[0052] Figure 31 The αGITR10-αPD-L1 antibody with CH2 has CDC activity mediated by murine complement. Percentage of lysis of GITR+CF2 cells obtained with serial dilutions of αGITR10-αPD-L1 tBsAb and control αGITR mAb (positive), αGITR10-αPD-L1 (negative) as determined by the CDC assay. All antibodies were serially diluted (1:10) starting at the highest 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. It was calculated as the ratio of the signal of the sample obtained to the signal intensity from fully lysed GITR+CF2 cells. Each sample was run in triplicate at each concentration; the standard deviation of the mean is indicated in parentheses. The background of GITR+CF2 cells in RPMI medium was subtracted from the values obtained. Each bar represents the simple mean (deviation indicated by parentheses) obtained from triplicate samples.

[0053] Figure 32 Cell-based ELISA for the binding of αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies to CF2 cells (no GITR expression) fixed with 8% paraformaldehyde was tested. The F10-αPD-L1 antibody represents the negative control. All antibodies were tested using a series of 1:3 dilutions in the range from 3.3 mg / mL to 0.0046 mg / mL. All antibodies were tested against 1000 GITR-CF2 cells per well. Each bar represents the mean (deviation indicated by the bar) obtained from triplicate samples. The background signal of the original signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0054] Figure 33 Cell-based ELISA for the binding of αGITR10-αPD-L1 and αGITR10 IgG antibodies to CF2 cells (no GITR expression) fixed with 8% paraformaldehyde was tested. The F10-αPD-L1 antibody represents the negative control. All antibodies were tested using a series of 1:2 dilutions in the range from 5 mg / mL to 0.078 mg / mL. All antibodies were tested against 10,000 GITR-CF2 cells per well. Each bar represents the mean (deviation indicated by the bar) obtained from triplicate samples. The background signal of the original signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0055] Figure 34Testing a cell-based ELISA for the binding of αGITR10-αPD-L1 and αGITR10IG antibodies with CH2 against GITR-CF2 cells fixed with 8% paraformaldehyde. The F10-αPD-L1 antibody represents the negative control. All antibodies were tested using a series of 1:2 dilutions ranging from 5 mg / mL to 0.16 mg / mL. All antibodies were tested against 10,000 GITR+CF2 cells per well. Each bar represents the mean (the deviation represented by the bar) obtained from triplicate samples. The background signal of the original signal intensity was corrected by subtracting the mean signal of the wells incubated in the absence of the primary antibody from the wells to which the primary antibody was added.

[0056] Figure 35 Control settings for flow cytometry analysis of fluorescently activated αGITR1-αPD-L1 antibodies. Lanes 1 to 6 show the control settings for GITR+CF2 cells. Lanes 7 to 9 refer to the control settings for GITR-CF2 cells. Detailed Description

[0057] The present invention relates to bispecific antibodies (i.e., tetravalent bispecific antibodies or "tBsAbs") containing two binding sites for each receptor, systems and methods for producing said bispecific antibodies.

[0058] The clinical development of bispecific antibodies (BsAbs) as therapeutic agents has been hampered by the difficulty of preparing sufficient amounts and quality of materials by traditional methods. In recent years, a variety of recombinant methods have been developed for the efficient production of BsAbs as antibody fragments and full-length IgG-like molecules. These recombinant antibody molecules have dual antigen-binding capabilities and are in most cases monovalent for each of their target antigens. The present invention provides an efficient method for producing novel tetravalent BsAbs (tBsAbs) having two antigen-binding sites for each of their target antigens; genetically engineering scFV bispecific antibodies and fusing the two together.

[0059] Compared to bispecific / divalent antibodies, the tBsAbs bind more effectively to their two target antigens and more effectively block the binding of ligands to receptors. Additionally, the expression of the tBsAbs in mammalian cells results in higher production levels and better antibody activity. Importantly, compared to monovalent bispecific antibodies, the tBsAbs exhibit higher stability and longer half-lives. One drawback of monovalent bispecific antibodies is their small size and thus short serum half-lives, which require continuous low-dose administration for several weeks. In contrast, the longer half-lives of the tBsAbs of the present invention address this issue and are thus more suitable for clinical applications. This design and expression of tBsAbs should be applicable to any pair of antigen specificities.

[0060] Preferably, the tBsAb is directed against 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).

[0061] Exemplary antibodies that can be used to construct the tBsAb according to the present invention include antibodies disclosed, for example, in: WO / 2005 / 060520, WO / 2006 / 089141, WO / 2007 / 065027, WO / 2009 / 086514, WO / 2009 / 079259, WO / 2011 / 153380, WO / 2014 / 055897, WO 2015 / 143194, WO 2015 / 164865, WO 2013 / 166500, WO2014 / 144061, WO 2016 / 057488, WO 2016 / 054638, WO / 2016 / 164835, PCT / US2016 / 026232, PCT / US2017 / 050093, PCT / US2017 / 050327, and PCT / US2017 / 043504, the contents of which are hereby incorporated by reference in their entirety.

[0062] PDL1(68)

[0063] Exemplary anti-PDL1 antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO:1485 and a VL nucleotide sequence of SEQ ID NO:1487; a VH nucleotide sequence of SEQ ID NO:1485 and a VL nucleotide sequence of SEQ ID NO:1487; a VH nucleotide sequence of SEQ ID NO:1489 and a VL nucleotide sequence of SEQ ID NO:1491; a VH nucleotide sequence of SEQ ID NO:1493 and a VL nucleotide sequence of SEQ ID NO:1495; a VH nucleotide sequence of SEQ ID NO:1497 and a VL nucleotide sequence of SEQ ID NO:1499; a VH nucleotide sequence of SEQ ID NO:1501 and a VL nucleotide sequence of SEQ ID NO:1503; a VH nucleotide sequence of SEQ ID NO:1505 and a VL nucleotide sequence of SEQ ID NO:1507; a VH nucleotide sequence of SEQ ID NO:1509 and a VL nucleotide sequence of SEQ ID NO:1511; a VH nucleotide sequence of SEQ ID NO:1513 and a VL nucleotide sequence of SEQ ID NO:1515; a VH nucleotide sequence of SEQ ID NO:1517 and a VL nucleotide sequence of SEQ ID NO:1519; a VH nucleotide sequence of SEQ ID NO:1521 and a VL nucleotide sequence of SEQ ID NO:1523; a VH nucleotide sequence of SEQ ID NO:1525 and a VL nucleotide sequence of SEQ ID NO:1527; a VH nucleotide sequence of SEQ ID NO:1529 and a VL nucleotide sequence of SEQ ID NO:1531; a VH nucleotide sequence of SEQ ID NO:1533 and a VL nucleotide sequence of SEQ ID NO:1535; a VH nucleotide sequence of SEQ ID NO:1537 and a VL nucleotide sequence of SEQ ID NO:1539.

[0064] Exemplary anti-PDL1 antibodies include antibodies having: a VH amino acid sequence of SEQ ID NO: 970 and a VL amino acid sequence of SEQ ID NO: 971; a VH amino acid sequence of SEQ ID NO: 1486 and a VL polypeptide sequence of SEQ ID NO: 1488; a VH amino acid sequence of SEQ ID NO: 1490 and a VL polypeptide sequence of SEQ ID NO: 1492; a VH amino acid sequence of SEQ ID NO: 1494 and a VL polypeptide sequence of SEQ ID NO: 1496; a VH amino acid sequence of SEQ ID NO: 1498 and a VL polypeptide sequence of SEQ ID NO: 1500; a VH amino acid sequence of SEQ ID NO: 1502 and a VL polypeptide sequence of SEQ ID NO: 1504; a VH amino acid sequence of SEQ ID NO: 1506 and a VL polypeptide sequence of SEQ ID NO: 1508; a VH amino acid sequence of SEQ ID NO: 1510 and a VL polypeptide sequence of SEQ ID NO: 1512; a VH amino acid sequence of SEQ ID NO: 1514 and a VL polypeptide sequence of SEQ ID NO: 1516; a VH amino acid sequence of SEQ ID NO: 1518 and a VL polypeptide sequence of SEQ ID NO: 1520; a VH amino acid sequence of SEQ ID NO: 1522 and a VL polypeptide sequence of SEQ ID NO: 1524; a VH amino acid sequence of SEQ ID NO: 1526 and a VL polypeptide sequence of SEQ ID NO: 1528; a VH amino acid sequence of SEQ ID NO: 1530 and a VL polypeptide sequence of SEQ ID NO: 1532; a VH amino acid sequence of SEQ ID NO: 1534 and a VL polypeptide sequence of SEQ ID NO: 1536; a VH amino acid sequence of SEQ ID NO: 1538 and a VL polypeptide sequence of SEQ ID NO: 1540.

[0065] In other embodiments, the anti-PDL1 antibody has: a heavy chain having three CDRs comprising amino acid sequences SEQ ID NO: 1541, 1554, 1569, respectively, and a light chain having three CDRs comprising amino acid sequences 1584, 1599, 1610, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1543, 1556, 1571, and a light chain having three CDRs comprising amino acid sequences 1586, 1600, 1612, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1544, 1557, 1572, and a light chain having three CDRs comprising amino acid sequences 1587, 1601, 1613, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1545, 1558, 1573, and a light chain having three CDRs comprising amino acid sequences 1588, 1602, 1614, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1546, 1559, 1574, and a light chain having three CDRs comprising amino acid sequences 1589, 1603, 1615, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1547, 1560, 1575, and a light chain having three CDRs comprising amino acid sequences 1590, 1604, 1616, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1548, 1561, 1576, and a light chain having three CDRs comprising amino acid sequences 1591, 1605, 1617, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1541, 1562, 1577, and a light chain having three CDRs comprising amino acid sequences 1592, 1599, 1618, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1549, 1563, 1578, and a light chain having three CDRs comprising amino acid sequences 1593, 1606, 1619, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1550, 1564, 1579, and a light chain having three CDRs comprising amino acid sequences 1594, 1607, 1620, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1551, 1565, 1580, and a light chain having three CDRs comprising amino acid sequences 1595, 1599, 1621, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1542, 1566, 1581, and a light chain having three CDRs comprising amino acid sequences 1596, 1599, 1622, respectively;or a heavy chain having three CDRs comprising amino acid sequences 1552, 1567, 1582, and a light chain having three CDRs comprising amino acid sequences 1597, 1608, 1623; or a heavy chain having three CDRs comprising amino acid sequences 1553, 1568, 1583, and a light chain having three CDRs comprising amino acid sequences 1598, 1609, 1624.;

[0066] SARS(26)

[0067] Exemplary SARS neutralizing antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO:1626 and a VL nucleotide sequence of SEQ ID NO:1628; a VH nucleotide sequence of SEQ ID NO:1630 and a VL nucleotide sequence of SEQ ID NO:1639; a VH nucleotide sequence of SEQ ID NO:1634 and a VL nucleotide sequence of SEQ ID NO:1640; a VH nucleotide sequence of SEQ ID NO:1632 and a VL nucleotide sequence of SEQ ID NO:1641; a VH nucleotide sequence of SEQ ID NO:1633 and a VL nucleotide sequence of SEQ ID NO:1642; a VH nucleotide sequence of SEQ ID NO:1634 and a VL nucleotide sequence of SEQ ID NO:1643; a VH nucleotide sequence of SEQ ID NO:1635 and a VL nucleotide sequence of SEQ ID NO:1644; a VH nucleotide sequence of SEQ ID NO:1636 and a VL nucleotide sequence of SEQ ID NO:1645; a VH nucleotide sequence of SEQ ID NO:1637 and a VL nucleotide sequence of SEQ ID NO:1646.

[0068] CXCR4(33)

[0069] Exemplary anti-CXCR4 antibodies include antibodies having: a VH amino acid sequence of SEQ ID NO:771 and a VL amino acid sequence of SEQ ID NO:779; a VH amino acid sequence of SEQ ID NO:772 and a VL amino acid sequence of SEQ ID NO:780; a VH amino acid sequence of SEQ ID NO:773 and a VL amino acid sequence of SEQ ID NO:781; a VH amino acid sequence of SEQ ID NO:774 and a VL amino acid sequence of SEQ ID NO:782; a VH amino acid sequence of SEQ ID NO:775 and a VL amino acid sequence of SEQ ID NO:783; a VH amino acid sequence of SEQ ID NO:776 and a VL amino acid sequence of SEQ ID NO:784; a VH amino acid sequence of SEQ ID NO:777 and a VL amino acid sequence of SEQ ID NO:785; or a VH amino acid sequence of SEQ ID NO:778 and a VL amino acid sequence of SEQ ID NO:786.

[0070] In other embodiments, the anti-CXCR4 antibody has: a heavy chain having three CDRs respectively comprising the amino acid sequences SEQ ID NO:803, 804, 805, and a light chain having three CDRs respectively comprising the amino acid sequences 806, 807, 808; or a heavy chain having three CDRs respectively comprising the amino acid sequences 809, 810, 811, and a light chain having three CDRs respectively comprising the amino acid sequences 812, 813, 814; or a heavy chain having three CDRs respectively comprising the amino acid sequences 815, 816, 817, and a light chain having three CDRs respectively comprising the amino acid sequences 818, 819, 820; or a heavy chain having three CDRs respectively comprising the amino acid sequences 827, 828, 829, and a light chain having three CDRs respectively comprising the amino acid sequences 830, 831, 832; or a heavy chain having three CDRs respectively comprising the amino acid sequences 833, 834, 835, and a light chain having three CDRs respectively comprising the amino acid sequences 836, 837, 838; or a heavy chain having three CDRs respectively comprising the amino acid sequences 839, 840, 841, and a light chain having three CDRs respectively comprising the amino acid sequences 842, 843, 844.

[0071] Carbonic anhydrase IX (40)

[0072] Exemplary anti-CAIX antibodies include antibodies having the following: a VH amino acid sequence of SEQ ID NO:845 and a VL amino acid sequence of SEQ ID NO:846; a VH amino acid sequence of SEQ ID NO:847 and a VL amino acid sequence of SEQ ID NO:868; a VH amino acid sequence of SEQ ID NO:848 and a VL amino acid sequence of SEQ ID NO:869; a VH amino acid sequence of SEQ ID NO:849 and a VL amino acid sequence of SEQ ID NO:870; a VH amino acid sequence of SEQ ID NO:850 and a VL amino acid sequence of SEQ ID NO:871; a VH amino acid sequence of SEQ ID NO:851 and a VL amino acid sequence of SEQ ID NO:872; a VH amino acid sequence of SEQ ID NO:852 and a VL amino acid sequence of SEQ ID NO:873; a VH amino acid sequence of SEQ ID NO:853 and a VL amino acid sequence of SEQ ID NO:874; a VH amino acid sequence of SEQ ID NO:854 and a VL amino acid sequence of SEQ ID NO:875; a VH amino acid sequence of SEQ ID NO:855 and a VL amino acid sequence of SEQ ID NO:876; a VH amino acid sequence of SEQ ID NO:856 and a VL amino acid sequence of SEQ ID NO:877; a VH amino acid sequence of SEQ ID NO:857 and a VL amino acid sequence of SEQ ID NO:878; a VH amino acid sequence of SEQ ID NO:858 and a VL amino acid sequence of SEQ ID NO:879; a VH amino acid sequence of SEQ ID NO:859 and a VL amino acid sequence of SEQ ID NO:880; a VH amino acid sequence of SEQ ID NO:860 and a VL amino acid sequence of SEQ ID NO:881; a VH amino acid sequence of SEQ ID NO:861 and a VL amino acid sequence of SEQ ID NO:882; a VH amino acid sequence of SEQ ID NO:862 and a VL amino acid sequence of SEQ ID NO:883; a VH amino acid sequence of SEQ ID NO:863 and a VL amino acid sequence of SEQ ID NO:884; a VH amino acid sequence of SEQ ID NO:864 and a VL amino acid sequence of SEQ ID NO:885; a VH amino acid sequence of SEQ ID NO:865 and a VL amino acid sequence of SEQ ID NO:886;The VH amino acid sequence having SEQ ID NO: 866 and the VL amino acid sequence having SEQ ID NO: 887; the VH amino acid sequence having SEQ ID NO: 867 and the VL amino acid sequence having SEQ ID NO: 888.;

[0073] In other embodiments, the anti-CA IX antibody has: a heavy chain having three CDRs respectively comprising the amino acid sequences SEQ ID NO: 803, 804, 805, and a light chain having three CDRs respectively comprising the amino acid sequences 806, 807, 808; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 905, 906, 952; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 935, 943, 953; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 935, 906, 954; or a heavy chain having three CDRs comprising the amino acid sequences 910, 916, 923, and a light chain having three CDRs comprising the amino acid sequences 936, 944, 955; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 936, 944, 956; or a heavy chain having three CDRs comprising the amino acid sequences 911, 917, 924, and a light chain having three CDRs comprising the amino acid sequences 937, 945, 957; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 935, 946, 958; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 938, 946, 959; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 905, 946, 960; or a heavy chain having three CDRs comprising the amino acid sequences 899, 918, 925, and a light chain having three CDRs comprising the amino acid sequences 937, 947, 955; or a heavy chain having three CDRs comprising the amino acid sequences 899, 918, 926, and a light chain having three CDRs comprising the amino acid sequences 937, 945, 957; or a heavy chain having three CDRs comprising the amino acid sequences 912, 919, 927, and a light chain having three CDRs comprising the amino acid sequences 937, 943, 961;or a heavy chain having three CDRs comprising the amino acid sequences 899, 918, 928, and a light chain having three CDRs comprising the amino acid sequences 937, 906, 960; or a heavy chain having three CDRs comprising the amino acid sequences 899, 918, 928, and a light chain having three CDRs comprising the amino acid sequences 937, 906, 960; or a heavy chain having three CDRs comprising the amino acid sequences 913, 920, 929, and a light chain having three CDRs comprising the amino acid sequences 939, 948, 962; or a heavy chain having three CDRs comprising the amino acid sequences 899, 918, 930, and a light chain having three CDRs comprising the amino acid sequences 935, 944, 955; or a heavy chain having three CDRs comprising the amino acid sequences 899, 921, 931, and a light chain having three CDRs comprising the amino acid sequences 935, 944, 955; or a heavy chain having three CDRs comprising the amino acid sequences 912, 919, 932, and a light chain having three CDRs comprising the amino acid sequences 940, 949, 963; or a heavy chain having three CDRs comprising the amino acid sequences 899, 915, 909, and a light chain having three CDRs comprising the amino acid sequences 935, 943, 960; or a heavy chain having three CDRs comprising the amino acid sequences 914, 922, 933, and a light chain having three CDRs comprising the amino acid sequences 941, 950, 964; or a heavy chain having three CDRs comprising the amino acid sequences 912, 918, 934, and a light chain having three CDRs comprising the amino acid sequences 942, 951, 965.;

[0074] CC - Chemokine Receptor 4 (CCR4) (048)

[0075] Exemplary CC - Chemokine Receptor 4 (CCR4) antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO:969 and a VL nucleotide sequence of SEQ ID NO:971; a VH nucleotide sequence of SEQ ID NO:969 and a V L nucleotide sequence.

[0076] Exemplary CCR4 antibodies include antibodies having: a VH amino acid sequence of SEQ ID NO:970 and a VL amino acid sequence of SEQ ID NO:971.

[0077] In other embodiments, the CCR4 antibody has a heavy chain having three CDRs comprising amino acid sequences SEQ ID NO: 973, 974, and 975, respectively; and a light chain having three CDRs comprising amino acid sequences 976, 977, and 978, respectively.

[0078] Middle East Respiratory Syndrome Coronavirus (MERS-CoV). (85)

[0079] Exemplary anti-Middle East Respiratory Syndrome Coronavirus (MERS-CoV) antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO: 677 and a VL nucleotide sequence of SEQ ID NO: 679; a VH nucleotide sequence of SEQ ID NO: 681 and a VL nucleotide sequence of SEQ ID NO: 683; a VH nucleotide sequence of SEQ ID NO: 685 and a VL nucleotide sequence of SEQ ID NO: 687; a VH nucleotide sequence of SEQ ID NO: 689 and a VL nucleotide sequence of SEQ ID NO: 692; a VH nucleotide sequence of SEQ ID NO: 693 and a VL nucleotide sequence of SEQ ID NO: 695; a VH nucleotide sequence of SEQ ID NO: 697 and a VL nucleotide sequence of SEQ ID NO: 699; and a VH nucleotide sequence of SEQ ID NO: 701 and a VL nucleotide sequence of SEQ ID NO: 703.

[0080] 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 of SEQ ID NO: 680; a VH amino acid sequence of SEQ ID NO: 682 and a VL amino acid sequence of SEQ ID NO: 684; a VH amino acid sequence of SEQ ID NO: 686 and a VL amino acid sequence of SEQ ID NO: 688; a VH amino acid sequence of SEQ ID NO: 690 and a VL amino acid sequence of SEQ ID NO: 692; a VH amino acid sequence of SEQ ID NO: 694 and a VL amino acid sequence of SEQ ID NO: 696; a VH amino acid sequence of SEQ ID NO: 698 and a VL amino acid sequence of SEQ ID NO: 700; and a VH amino acid sequence of SEQ ID NO: 702 and a VL amino acid sequence of SEQ ID NO: 704.

[0081] In other embodiments, the anti-Middle East Respiratory Syndrome Coronavirus (MERS-CoV) antibody has: a heavy chain having three CDRs comprising amino acid sequences 705, 706, and 707, and a light chain having three CDRs comprising amino acid sequences 722, 723, and 724; a heavy chain having three CDRs comprising amino acid sequences 708, 709, and 710, and a light chain having three CDRs comprising amino acid sequences 725, 726, and 727; a heavy chain having three CDRs comprising amino acid sequences 711, 712, and 713, and a light chain having three CDRs comprising amino acid sequences 728, 729, and 730; a heavy chain having three CDRs comprising amino acid sequences 711, 735, and 715, and a light chain having three CDRs comprising amino acid sequences 731, 732, and 733; a heavy chain having three CDRs comprising amino acid sequences 711, 735, and 716, and a light chain having three CDRs comprising amino acid sequences 737, 738, and 739; a heavy chain having three CDRs comprising amino acid sequences 717, 718, and 719, and a light chain having three CDRs comprising amino acid sequences 736, 742, and 743; and a heavy chain having three CDRs comprising amino acid sequences 714, 720, and 721, and a light chain having three CDRs comprising amino acid sequences 740, 729, and 741.

[0082] GITR(93)

[0083] Exemplary anti-human GITR antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO:1361 and a VL nucleotide sequence of SEQ ID NO:1363; a VH nucleotide sequence of SEQ ID NO:1365 and a VL nucleotide sequence of SEQ ID NO:1367; a VH nucleotide sequence of SEQ ID NO:1369 and a VL nucleotide sequence of SEQ ID NO:1371; a VH nucleotide sequence of SEQ ID NO:1381 and a VL nucleotide sequence of SEQ ID NO:1375; a VH nucleotide sequence of SEQ ID NO:1377 and a VL nucleotide sequence of SEQ ID NO:1379; a VH nucleotide sequence of SEQ ID NO:1381 and a VL nucleotide sequence of SEQ ID NO:1383; a VH nucleotide sequence of SEQ ID NO:1385 and a VL nucleotide sequence of SEQ ID NO:1387; a VH nucleotide sequence of SEQ ID NO:1389 and a VL nucleotide sequence of SEQ ID NO:1391; a VH nucleotide sequence of SEQ ID NO:1393 and a VL nucleotide sequence of SEQ ID NO:1395; a VH nucleotide sequence of SEQ ID NO:1397 and a VL nucleotide sequence of SEQ ID NO:1398; or a VH nucleotide sequence of SEQ ID NO:1401 and a VL nucleotide sequence of SEQ ID NO:1403.

[0084] Exemplary anti-human GITR antibodies include antibodies having: a VH amino acid sequence of SEQ ID NO: 1362 and a VL amino acid sequence of SEQ ID NO: 1364; a VH amino acid sequence of SEQ ID NO: 1366 and a VL polypeptide sequence of SEQ ID NO: 1368; a VH amino acid sequence of SEQ ID NO: 1371 and a VL amino acid sequence of SEQ ID NO: 1372; a VH amino acid sequence of SEQ ID NO: 1382 and a VL amino acid sequence of SEQ ID NO: 1376; a VH nucleotide sequence of SEQ ID NO: 1378 and a VL nucleotide sequence of SEQ ID NO: 1380; a VH amino acid sequence of SEQ ID NO: 1382 and a VL polypeptide sequence of SEQ ID NO: 1384; a VH amino acid sequence of SEQ ID NO: 1386 and a VL amino acid sequence of SEQ ID NO: 1388; a VH amino acid sequence of SEQ ID NO: 1390 and a VL amino acid sequence of SEQ ID NO: 1392; a VH amino acid sequence of SEQ ID NO: 1394 and a VL polypeptide sequence of SEQ ID NO: 1396; a VH amino acid sequence of SEQ ID NO: 1399 and a VL amino acid sequence of SEQ ID NO: 1400; or a VH amino acid sequence of SEQ ID NO: 1402 and a VL amino acid sequence of SEQ ID NO: 1404.

[0085] In other embodiments, the anti-human GITR antibody has: a heavy chain having three CDRs comprising amino acid sequences 1405, 1406, and 1407, and a light chain having three CDRs comprising amino acid sequences 1408, 1409, and 1410, respectively; a heavy chain having three CDRs comprising amino acid sequences 1411, 1412, and 1413, and a light chain having three CDRs comprising amino acid sequences 1414, 1415, and 1416, respectively; a heavy chain having three CDRs comprising amino acid sequences 1417, 1418, and 1419, and a light chain having three CDRs comprising amino acid sequences 1420, 1421, and 1422, respectively; a heavy chain having three CDRs comprising amino acid sequences 1423, 1424, and 1425, and a light chain having three CDRs comprising amino acid sequences 1426, 1427, and 1428, respectively; a heavy chain having three CDRs comprising amino acid sequences 1429, 1430, and 1431, and a light chain having three CDRs comprising amino acid sequences 1432, 1433, and 1434, respectively; a heavy chain having three CDRs comprising amino acid sequences 1435, 1436, and 1437, and a light chain having three CDRs comprising amino acid sequences 1438, 1439, and 1440, respectively; a heavy chain having three CDRs comprising amino acid sequences 1441, 1442, and 1443, and a light chain having three CDRs comprising amino acid sequences 1444, 1445, and 1446, respectively; a heavy chain having three CDRs comprising amino acid sequences 1447, 1448, and 1449, and a light chain having three CDRs comprising amino acid sequences 1450, 1451, and 1452, respectively; a heavy chain having three CDRs comprising amino acid sequences 1453, 1454, and 1455, and a light chain having three CDRs comprising amino acid sequences 1456, 1457, and 1458, respectively; a heavy chain having three CDRs comprising amino acid sequences 1459, 1460, and 1461, and a light chain having three CDRs comprising amino acid sequences 1462, 1463, and 1464, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1465, 1466, and 1467, and a light chain having three CDRs comprising amino acid sequences 1468, 1469, and 1470, respectively.

[0086] Flavivirus (73)

[0087] Exemplary anti-West Nile virus envelope protein E (WNE) antibodies include antibodies having a VH nucleotide sequence having: a VH amino acid sequence having SEQ ID NO: 1224; and a VL amino acid sequence having SEQ ID NO: 1226.

[0088] 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.

[0089] In other embodiments, the anti-West Nile virus envelope protein E (WNE) antibody has: a heavy chain having three CDRs comprising the amino acid sequences 1244, 1245, and 1246; and a light chain having three CDRs comprising the amino acid sequences 1247, 1248, and 1249, respectively.

[0090] CCR4(65)

[0091] Exemplary anti-CC chemokine receptor 4 (CCR4) antibodies include antibodies having: a VH nucleotide sequence having SEQ ID NO: 1329 and a VL nucleotide sequence having SEQ ID NO: 1331; a VH nucleotide sequence having SEQ ID NO: 1333 and a V L nucleotide sequence having SEQ ID NO: 1335; a VH nucleotide sequence having SEQ ID NO: 1337 and a V L nucleotide sequence having SEQ ID NO: 1192; a VH nucleotide sequence having SEQ ID NO: 1341 and a VL nucleotide sequence having SEQ ID NO: 1343; or a VH nucleotide sequence having SEQ ID NO: 1357 and a VL nucleotide sequence having SEQ ID NO: 1359.

[0092] Exemplary anti-CC chemokine receptor 4 (CCR4) antibodies include antibodies having: a V having SEQ ID NO: 1330 H amino acid sequence and a V having SEQ ID NO: 1332 L amino acid sequence; a V having SEQ ID NO: 1334 H amino acid sequence and a V having SEQ ID NO: 1336 L amino acid sequence; a V having SEQ ID NO: 1338 H amino acid sequence and a V having SEQ ID NO: 1340 LAmino acid sequence; V having SEQ ID NO: 1342 H Amino acid sequence and V having SEQ ID NO: 1344 L Amino acid sequence; or V having SEQ ID NO: 1358 H Amino acid sequence and V having SEQ ID NO: 1360 L Amino acid sequence.

[0093] In other embodiments, the anti-CC chemokine receptor 4 (CCR4) antibody has: a heavy chain having three CDRs comprising amino acid sequences 1203, 1208, and 1211, and a light chain having three CDRs comprising amino acid sequences 1207, 1209, and 1216, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1204, 1208, and 1212, and a light chain having three CDRs comprising amino acid sequences 1207, 1209, and 1217, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1204, 1208, and 1213, and a light chain having three CDRs comprising amino acid sequences 1207, 1209, and 1217, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1205, 1208, and 1214, and a light chain having three CDRs comprising amino acid sequences 1207, 1209, and 1218, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1206, 1208, and 1210, and a light chain having three CDRs comprising amino acid sequences 1207, 1209, and 1220, respectively; or a heavy chain having three CDRs comprising amino acid sequences 1202, 1208, and 1210, and a light chain having three CDRs comprising amino acid sequences 1207, 1209, and 1219, respectively.

[0094] Human immunoglobulin heavy chain variable region germline gene VH1-69(57)

[0095] 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.

[0096] Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibodies include antibodies having: V having SEQ ID NO: 1154 HAn amino acid sequence and V having SEQ ID NO: 1156 L amino acid sequence; or V having SEQ ID NO: 1164 H amino acid sequence and V having SEQ ID NO: 1156 L amino acid sequence.

[0097] In other embodiments, the anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody has: a heavy chain having three CDRs comprising amino acid sequences 1157, 1158, and 1159; and a light chain having three CDRs comprising amino acid sequences 1160, 1161, and 1162, respectively.

[0098] Influenza (49)

[0099] Exemplary anti-influenza antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO: 981 and a VL nucleotide sequence of SEQ ID NO: 983; a VH nucleotide sequence of SEQ ID NO: 985 and a VL nucleotide sequence of SEQ ID NO: 989; a VH nucleotide sequence of SEQ ID NO: 987 and a VL nucleotide sequence of SEQ ID NO: 991; a VH nucleotide sequence of SEQ ID NO: 993 and a VL nucleotide sequence of SEQ ID NO: 997; a VH nucleotide sequence of SEQ ID NO: 995 and a VK nucleotide sequence of SEQ ID NO: 999; a VH nucleotide sequence of SEQ ID NO: 1001 and a VL nucleotide sequence of SEQ ID NO: 1005; a VH nucleotide sequence of SEQ ID NO: 1003 and a VL nucleotide sequence of SEQ ID NO: 1007; a VH nucleotide sequence of SEQ ID NO: 1009 and a VL nucleotide sequence of SEQ ID NO: 1011; a VH nucleotide sequence of SEQ ID NO: 1013 and a VL nucleotide sequence of SEQ ID NO: 1015; and a VH nucleotide sequence of SEQ ID NO: 1017 and a VK nucleotide sequence of SEQ ID NO: 1019; a VH nucleotide sequence of SEQ ID NO: 1020 and a VL nucleotide sequence of SEQ ID NO: 1022.

[0100] Exemplary anti-influenza antibodies include antibodies having: a VH amino acid sequence of SEQ ID NO:982 and a VL amino acid sequence of SEQ ID NO:984; a VH amino acid sequence of SEQ ID NO:986 and a VL amino acid sequence of SEQ ID NO:988; a VH amino acid sequence of SEQ ID NO:986 and a VL amino acid sequence of SEQ ID NO:990; a VH amino acid sequence of SEQ ID NO:992 and a VL amino acid sequence of SEQ ID NO:994; a VH amino acid sequence of SEQ ID NO:992 and a VK amino acid sequence of SEQ ID NO:996; a VH amino acid sequence of SEQ ID NO:998 and a VL amino acid sequence of SEQ ID NO:1000; a VH amino acid sequence of SEQ ID NO:998 and a VL amino acid sequence of SEQ ID NO:1002; a VH amino acid sequence of SEQ ID NO:1004 and a VL amino acid sequence of SEQ ID NO:1006; a VH amino acid sequence of SEQ ID NO:1008 and a VL amino acid sequence of SEQ ID NO:1010; a VH amino acid sequence of SEQ ID NO:1012 and a VK amino acid sequence of SEQ ID NO:1014; and a VH amino acid sequence of SEQ ID NO:1016 and a VL amino acid sequence of SEQ ID NO:1018.

[0101] In other embodiments, the anti-influenza antibody has: a heavy chain having three CDRs comprising the amino acid sequences 1023, 1031, and 1039, and a light chain having three CDRs comprising the amino acid sequences 1047, 1059, and 1071; a heavy chain having three CDRs comprising the amino acid sequences 1023, 1032, and 1040, and a light chain having three CDRs comprising the amino acid sequences 1048, 1060, and 1072; a heavy chain having three CDRs comprising the amino acid sequences 1025, 1032, and 1040, and a light chain having three CDRs comprising the amino acid sequences 1057, 1069, and 1081; a heavy chain having three CDRs comprising the amino acid sequences 1026, 1033, and 1041, and a light chain having three CDRs comprising the amino acid sequences 1049, 1061, and 1073; a heavy chain having three CDRs comprising the amino acid sequences 1026, 1033, and 1041, and a light chain having three CDRs comprising the amino acid sequences 1054, 1066, and 1078; a heavy chain having three CDRs comprising the amino acid sequences 1027, 1034, and 1042, and a light chain having three CDRs comprising the amino acid sequences 1050, 1062, and 1074; a heavy chain having three CDRs comprising the amino acid sequences 1027, 1034, and 1042, and a light chain having three CDRs comprising the amino acid sequences 1056, 1068, and 1080; a heavy chain having three CDRs comprising the amino acid sequences 1028, 1035, and 1043, and a light chain having three CDRs comprising the amino acid sequences 1051, 1063, and 1065; a heavy chain having three CDRs comprising the amino acid sequences 1028, 1036, and 1044, and a light chain having three CDRs comprising the amino acid sequences 1052, 1064, and 1076; 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.

[0102] Influenza (78)

[0103] Exemplary anti-influenza antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO: 397 and a VL nucleotide sequence of SEQ ID NO: 398; a VH nucleotide sequence of SEQ ID NO: 399 and a V L nucleotide sequence of SEQ ID NO: 400; a VH nucleotide sequence of SEQ ID NO: 401 and a V LNucleotide sequences; a VH nucleotide sequence having SEQ ID NO: 403 and a VL nucleotide sequence having SEQ ID NO: 404; or a VH nucleotide sequence having SEQ ID NO: 405 and a VL nucleotide sequence having SEQ ID NO: 406; or a VH nucleotide sequence having SEQ ID NO: 407 and a VL nucleotide sequence having SEQ ID NO: 408; or a VH nucleotide sequence having SEQ ID NO: 409 and a VL nucleotide sequence having SEQ ID NO: 410; or a VH nucleotide sequence having SEQ ID NO: 411 and a VL nucleotide sequence having SEQ ID NO: 412; or a VH nucleotide sequence having SEQ ID NO: 413 and a VL nucleotide sequence having SEQ ID NO: 414; or a VH nucleotide sequence having SEQ ID NO: 415 and a VL nucleotide sequence having SEQ ID NO: 416; or a VH nucleotide sequence having SEQ ID NO: 417 and a VL nucleotide sequence having SEQ ID NO: 418; or a VH nucleotide sequence having SEQ ID NO: 419 and a VL nucleotide sequence having SEQ ID NO: 420; or a VH nucleotide sequence having SEQ ID NO: 421 and a VL nucleotide sequence having SEQ ID NO: 422; or a VH nucleotide sequence having SEQ ID NO: 423 and a VL nucleotide sequence having SEQ ID NO: 424; or a VH nucleotide sequence having SEQ ID NO: 425 and a VL nucleotide sequence having SEQ ID NO: 426; or a VH nucleotide sequence having SEQ ID NO: 427 and a VL nucleotide sequence having SEQ ID NO: 428; or a VH nucleotide sequence having SEQ ID NO: 429 and a VL nucleotide sequence having SEQ ID NO: 430; or a VH nucleotide sequence having SEQ ID NO: 431 and a VL nucleotide sequence having SEQ ID NO: 432; or a VH nucleotide sequence having SEQ ID NO: 433 and a VL nucleotide sequence having SEQ ID NO: 434; or a VH nucleotide sequence having SEQ ID NO: 435 and a VL nucleotide sequence having SEQ ID NO: 436; or a VH nucleotide sequence having SEQ ID NO: 437 and a VL nucleotide sequence having SEQ ID NO: 438; or a VH nucleotide sequence having SEQ ID NO: 439 and a VL nucleotide sequence having SEQ ID NO: 440; or a VH nucleotide sequence having SEQ ID NO: 441 and a VL nucleotide sequence having SEQ ID NO: 442;or having a VH nucleotide sequence of SEQ ID NO:541 and a VL nucleotide sequence of SEQ ID NO:542; or having a VH nucleotide sequence of SEQ ID NO:543 and a VL nucleotide sequence of SEQ ID NO:544; or having a VH nucleotide sequence of SEQ ID NO:545 and a VL nucleotide sequence of SEQ ID NO:546; or having a VH nucleotide sequence of SEQ ID NO:547 and a VL nucleotide sequence of SEQ ID NO:548; or having a VH nucleotide sequence of SEQ ID NO:549 and a VL nucleotide sequence of SEQ ID NO:550; or having a VH nucleotide sequence of SEQ ID NO:551 and a VL nucleotide sequence of SEQ ID NO:552; or having a VH nucleotide sequence of SEQ ID NO:553 and a VL nucleotide sequence of SEQ ID NO:554; or having a VH nucleotide sequence of SEQ ID NO:555 and a VL nucleotide sequence of SEQ ID NO:556; or having a VH nucleotide sequence of SEQ ID NO:557 and a VL nucleotide sequence of SEQ ID NO:558; or having a VH nucleotide sequence of SEQ ID NO:559 and a VL nucleotide sequence of SEQ ID NO:560; or having a VH nucleotide sequence of SEQ ID NO:561 and a VL nucleotide sequence of SEQ ID NO:562; or having a VH nucleotide sequence of SEQ ID NO:563 and a VL nucleotide sequence of SEQ ID NO:564; or having a VH nucleotide sequence of SEQ ID NO:565 and a VL nucleotide sequence of SEQ ID NO:566; or having a VH nucleotide sequence of SEQ ID NO:567 and a VL nucleotide sequence of SEQ ID NO:568; or having a VH nucleotide sequence of SEQ ID NO:569 and a VL nucleotide sequence of SEQ ID NO:570; or having a VH nucleotide sequence of SEQ ID NO:571 and a VL nucleotide sequence of SEQ ID NO:572; or having a VH nucleotide sequence of SEQ ID NO:573 and a VL nucleotide sequence of SEQ ID NO:574; or having a VH nucleotide sequence of SEQ ID NO:575 and a VL nucleotide sequence of SEQ ID NO:576; or having a VH nucleotide sequence of SEQ ID NO:577 and a VL nucleotide sequence of SEQ ID NO:578; or having a VH nucleotide sequence of SEQ ID NO:579 and a VL nucleotide sequence of SEQ ID NO:580;or having a VH nucleotide sequence of SEQ ID NO:581 and a VL nucleotide sequence of SEQ ID NO:582; or having a VH nucleotide sequence of SEQ ID NO:583 and a VL nucleotide sequence of SEQ ID NO:584; or having a VH nucleotide sequence of SEQ ID NO:585 and a VL nucleotide sequence of SEQ ID NO:586; or having a VH nucleotide sequence of SEQ ID NO:587 and a VL nucleotide sequence of SEQ ID NO:588; or having a VH nucleotide sequence of SEQ ID NO:589 and a VL nucleotide sequence of SEQ ID NO:590; or having a VH nucleotide sequence of SEQ ID NO:591 and a VL nucleotide sequence of SEQ ID NO:592; or having a VH nucleotide sequence of SEQ ID NO:593 and a VL nucleotide sequence of SEQ ID NO:594; or having a VH nucleotide sequence of SEQ ID NO:595 and a VL nucleotide sequence of SEQ ID NO:596; or having a VH nucleotide sequence of SEQ ID NO:597 and a VL nucleotide sequence of SEQ ID NO:598; or having a VH nucleotide sequence of SEQ ID NO:599 and a VL nucleotide sequence of SEQ ID NO:600.;

[0104] Exemplary anti-influenza antibodies include antibodies having the following: a VH amino acid sequence of SEQ ID NO:469 and a VL amino acid sequence of SEQ ID NO:470; a VH amino acid sequence of SEQ ID NO:471 and a VL polypeptide sequence of SEQ ID NO:472; a VH amino acid sequence of SEQ ID NO:473 and a VL amino acid sequence of SEQ ID NO:474; a VH amino acid sequence of SEQ ID NO:475 and a VL amino acid sequence of SEQ ID NO:476; or a VH nucleotide sequence of SEQ ID NO:477 and a VL nucleotide sequence of SEQ ID NO:478; a VH amino acid sequence of SEQ ID NO:479 and a VL amino acid sequence of SEQ ID NO:480; a VH amino acid sequence of SEQ ID NO:481 and a VL amino acid sequence of SEQ ID NO:482; a VH amino acid sequence of SEQ ID NO:483 and a VL amino acid sequence of SEQ ID NO:484; a VH amino acid sequence of SEQ ID NO:485 and a VL amino acid sequence of SEQ ID NO:486; a VH amino acid sequence of SEQ ID NO:487 and a VL amino acid sequence of SEQ ID NO:488; a VH amino acid sequence of SEQ ID NO:489 and a VL amino acid sequence of SEQ ID NO:490; a VH amino acid sequence of SEQ ID NO:491 and a VL amino acid sequence of SEQ ID NO:492; a VH amino acid sequence of SEQ ID NO:493 and a VL amino acid sequence of SEQ ID NO:494; a VH amino acid sequence of SEQ ID NO:495 and a VL amino acid sequence of SEQ ID NO:496; a VH amino acid sequence of SEQ ID NO:497 and a VL amino acid sequence of SEQ ID NO:498; a VH amino acid sequence of SEQ ID NO:499 and a VL amino acid sequence of SEQ ID NO:500; a VH amino acid sequence of SEQ ID NO:501 and a VL amino acid sequence of SEQ ID NO:502; a VH amino acid sequence of SEQ ID NO:503 and a VL amino acid sequence of SEQ ID NO:504; a VH amino acid sequence of SEQ ID NO:505 and a VL amino acid sequence of SEQ ID NO:506; a VH amino acid sequence of SEQ ID NO:507 and a VL amino acid sequence of SEQ ID NO:508;The VH amino acid sequence having SEQ ID NO: 509 and the VL amino acid sequence having SEQ ID NO: 510; the VH amino acid sequence having SEQ ID NO: 511 and the VL amino acid sequence having SEQ ID NO: 512; the VH amino acid sequence having SEQ ID NO: 513 and the VL amino acid sequence having SEQ ID NO: 514; the VH amino acid sequence having SEQ ID NO: 515 and the VL amino acid sequence having SEQ ID NO: 516;; the VH amino acid sequence having SEQ ID NO: 517 and the VL amino acid sequence having SEQ ID NO: 518; the VH amino acid sequence having SEQ ID NO: 519 and the VL amino acid sequence having SEQ ID NO: 520; the VH amino acid sequence having SEQ ID NO: 521 and the VL amino acid sequence having SEQ ID NO: 522; the VH amino acid sequence having SEQ ID NO: 523 and the VL amino acid sequence having SEQ ID NO: 524; the VH amino acid sequence having SEQ ID NO: 525 and the VL amino acid sequence having SEQ ID NO: 526; the VH amino acid sequence having SEQ ID NO: 527 and the VL amino acid sequence having SEQ ID NO: 528; the VH amino acid sequence having SEQ ID NO: 529 and the VL amino acid sequence having SEQ ID NO: 530; the VH amino acid sequence having SEQ ID NO: 531 and the VL amino acid sequence having SEQ ID NO: 532; the VH amino acid sequence having SEQ ID NO: 533 and the VL amino acid sequence having SEQ ID NO: 534; the VH amino acid sequence having SEQ ID NO: 535 and the VL amino acid sequence having SEQ ID NO: 536; the VH amino acid sequence having SEQ ID NO: 537 and the VL amino acid sequence having SEQ ID NO: 538; the VH amino acid sequence having SEQ ID NO: 539 and the VL amino acid sequence having SEQ ID NO: 540; the VH amino acid sequence having SEQ ID NO: 601 and the VL amino acid sequence having SEQ ID NO: 602; the VH amino acid sequence having SEQ ID NO: 603 and the VL amino acid sequence having SEQ ID NO: 604; the VH amino acid sequence having SEQ ID NO: 605 and the VL amino acid sequence having SEQ ID NO: 606; the VH amino acid sequence having SEQ ID NO: 607 and the VL amino acid sequence having SEQ ID NO: 608;The VH amino acid sequence having SEQ ID NO: 609 and the VL amino acid sequence having SEQ ID NO: 610; the VH amino acid sequence having SEQ ID NO: 611 and the VL amino acid sequence having SEQ ID NO: 612; the VH amino acid sequence having SEQ ID NO: 613 and the VL amino acid sequence having SEQ ID NO: 614; the VH amino acid sequence having SEQ ID NO: 615 and the VL amino acid sequence having SEQ ID NO: 616; the VH amino acid sequence having SEQ ID NO: 617 and the VL amino acid sequence having SEQ ID NO: 618; the VH amino acid sequence having SEQ ID NO: 619 and the VL amino acid sequence having SEQ ID NO: 620; the VH amino acid sequence having SEQ ID NO: 621 and the VL amino acid sequence having SEQ ID NO: 622; the VH amino acid sequence having SEQ ID NO: 623 and the VL amino acid sequence having SEQ ID NO: 624; the VH amino acid sequence having SEQ ID NO: 625 and the VL amino acid sequence having SEQ ID NO: 626; the VH amino acid sequence having SEQ ID NO: 627 and the VL amino acid sequence having SEQ ID NO: 628; the VH amino acid sequence having SEQ ID NO: 629 and the VL amino acid sequence having SEQ ID NO: 630; the VH amino acid sequence having SEQ ID NO: 631 and the VL amino acid sequence having SEQ ID NO: 632; the VH amino acid sequence having SEQ ID NO: 633 and the VL amino acid sequence having SEQ ID NO: 634; the VH amino acid sequence having SEQ ID NO: 635 and the VL amino acid sequence having SEQ ID NO: 636; the VH amino acid sequence having SEQ ID NO: 637 and the VL amino acid sequence having SEQ ID NO: 638; the VH amino acid sequence having SEQ ID NO: 639 and the VL amino acid sequence having SEQ ID NO: 640; the VH amino acid sequence having SEQ ID NO: 641 and the VL amino acid sequence having SEQ ID NO: 642; the VH amino acid sequence having SEQ ID NO: 643 and the VL amino acid sequence having SEQ ID NO: 644; the VH amino acid sequence having SEQ ID NO: 645 and the VL amino acid sequence having SEQ ID NO: 646; the VH amino acid sequence having SEQ ID NO: 647 and the VL amino acid sequence having SEQ ID NO: 648;The VH amino acid sequence having SEQ ID NO: 649 and the VL amino acid sequence having SEQ ID NO: 650; the VH amino acid sequence having SEQ ID NO: 651 and the VL amino acid sequence having SEQ ID NO: 652; the VH amino acid sequence having SEQ ID NO: 653 and the VL amino acid sequence having SEQ ID NO: 654; the VH amino acid sequence having SEQ ID NO: 655 and the VL amino acid sequence having SEQ ID NO: 656; the VH amino acid sequence having SEQ ID NO: 657 and the VL amino acid sequence having SEQ ID NO: 658; the VH amino acid sequence having SEQ ID NO: 659 and the VL amino acid sequence having SEQ ID NO: 660.;

[0105] In other embodiments, the anti-influenza antibody has: a heavy chain having three CDRs comprising amino acid sequences SEQ ID NO: 1, 37, 73, respectively, and a light chain having three CDRs comprising amino acid sequences 109, 145, 181, respectively; or a heavy chain having three CDRs comprising amino acid sequences 2, 38, 74, respectively, and a light chain having three CDRs comprising amino acid sequences 110, 146, 182, respectively; or a heavy chain having three CDRs comprising amino acid sequences 3, 39, 75, respectively, and a light chain having three CDRs comprising amino acid sequences 111, 147, 183, respectively; or a heavy chain having three CDRs comprising amino acid sequences 4, 40, 76, respectively, and a light chain having three CDRs comprising amino acid sequences 112, 148, 184, respectively; or a heavy chain having three CDRs comprising amino acid sequences 5, 41, 77, respectively, and a light chain having three CDRs comprising amino acid sequences 113, 149, 185, respectively; or a heavy chain having three CDRs comprising amino acid sequences 6, 42, 78, respectively, and a light chain having three CDRs comprising amino acid sequences 114, 150, 186, respectively; or a heavy chain having three CDRs comprising amino acid sequences 7, 43, 79, respectively, and a light chain having three CDRs comprising amino acid sequences 115, 151, 187, respectively; or a heavy chain having three CDRs comprising amino acid sequences 8, 44, 80, respectively, and a light chain having three CDRs comprising amino acid sequences 116, 152, 188, respectively; or a heavy chain having three CDRs comprising amino acid sequences 9, 45, 81, respectively, and a light chain having three CDRs comprising amino acid sequences 117, 153, 189, respectively; or a heavy chain having three CDRs comprising amino acid sequences 9, 45, 81, respectively, and a light chain having three CDRs comprising amino acid sequences 117, 153, 189, respectively; or a heavy chain having three CDRs comprising amino acid sequences 10, 46, 82, respectively, and a light chain having three CDRs comprising amino acid sequences 118, 154, 190, respectively; or a heavy chain having three CDRs comprising amino acid sequences 11, 47, 83, respectively, and a light chain having three CDRs comprising amino acid sequences 119, 155, 191, respectively; or a heavy chain having three CDRs comprising amino acid sequences 12, 48, 84, respectively, and a light chain having three CDRs comprising amino acid sequences 120, 156, 192, respectively;or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 13, 49, 85, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 121, 157, 193; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 14, 50, 86, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 122, 158, 194; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 15, 51, 87, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 123, 159, 195; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 16, 52, 88, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 124, 160, 196; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 17, 53, 89, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 125, 161, 197; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 18, 54, 90, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 126, 162, 198; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 19, 55, 91, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 127, 163, 199; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 20, 56, 92, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 128, 164, 200; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 21, 57, 93, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 129, 165, 201; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 22, 58, 94, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 130, 166, 202; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 23, 59, 95, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 131, 167, 203; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 24, 60, 96, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 132, 168, 204; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 25, 61, 95, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 133, 169, 205; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 26, 62, 96, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 134, 170, 206;or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 27, 63, 97, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 135, 171, 207; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 28, 64, 98, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 136, 172, 208; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 29, 65, 99, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 137, 173, 209; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 30, 66, 100, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 138, 174, 210; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 31, 67, 101, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 139, 175, 211; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 32, 68, 102, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 140, 176, 212; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 33, 69, 103, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 141, 177, 213; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 34, 70, 104, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 142, 178, 214; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 35, 71, 105, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 143, 179, 215; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 36, 72, 106, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 144, 180, 216; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 217, 247, 277, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 307, 337, 367; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 218, 248, 278, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 308, 338, 368; or a heavy chain, the heavy chain having three CDRs respectively comprising amino acid sequences 219, 249, 279, and a light chain, the light chain having three CDRs respectively comprising amino acid sequences 309, 339, 369;or a heavy chain having three CDRs respectively comprising amino acid sequences 220, 250, 280, and a light chain having three CDRs respectively comprising amino acid sequences 310, 340, 370; or a heavy chain having three CDRs respectively comprising amino acid sequences 221, 251, 281, and a light chain having three CDRs respectively comprising amino acid sequences 311, 341, 371; or a heavy chain having three CDRs respectively comprising amino acid sequences 222, 252, 282, and a light chain having three CDRs respectively comprising amino acid sequences 312, 342, 372; or a heavy chain having three CDRs respectively comprising amino acid sequences 223, 253, 283, and a light chain having three CDRs respectively comprising amino acid sequences 313, 343, 373; or a heavy chain having three CDRs respectively comprising amino acid sequences 224, 254, 284, and a light chain having three CDRs respectively comprising amino acid sequences 314, 344, 374; or a heavy chain having three CDRs respectively comprising amino acid sequences 225, 255, 285, and a light chain having three CDRs respectively comprising amino acid sequences 315, 345, 375; or a heavy chain having three CDRs respectively comprising amino acid sequences 226, 256, 286, and a light chain having three CDRs respectively comprising amino acid sequences 316, 346, 376; or a heavy chain having three CDRs respectively comprising amino acid sequences 227, 257, 287, and a light chain having three CDRs respectively comprising amino acid sequences 317, 347, 377; or a heavy chain having three CDRs respectively comprising amino acid sequences 228, 258, 288, and a light chain having three CDRs respectively comprising amino acid sequences 318, 348, 378; or a heavy chain having three CDRs respectively comprising amino acid sequences 229, 259, 289, and a light chain having three CDRs respectively comprising amino acid sequences 319, 349, 379; or a heavy chain having three CDRs respectively comprising amino acid sequences 230, 260, 290, and a light chain having three CDRs respectively comprising amino acid sequences 320, 350, 380; or a heavy chain having three CDRs respectively comprising amino acid sequences 231, 261, 291, and a light chain having three CDRs respectively comprising amino acid sequences 321, 351, 381; or a heavy chain having three CDRs respectively comprising amino acid sequences 232, 262, 292, and a light chain having three CDRs respectively comprising amino acid sequences 322, 352, 382;or a heavy chain having three CDRs respectively comprising amino acid sequences 233, 263, 293, and a light chain having three CDRs respectively comprising amino acid sequences 323, 353, 383; or a heavy chain having three CDRs respectively comprising amino acid sequences 234, 273, 294, and a light chain having three CDRs respectively comprising amino acid sequences 324, 354, 384; or a heavy chain having three CDRs respectively comprising amino acid sequences 235, 274, 295, and a light chain having three CDRs respectively comprising amino acid sequences 325, 355, 385; or a heavy chain having three CDRs respectively comprising amino acid sequences 236, 275, 296, and a light chain having three CDRs respectively comprising amino acid sequences 326, 356, 386; or a heavy chain having three CDRs respectively comprising amino acid sequences 237, 276, 297, and a light chain having three CDRs respectively comprising amino acid sequences 327, 357, 387; or a heavy chain having three CDRs respectively comprising amino acid sequences 237, 277, 298, and a light chain having three CDRs respectively comprising amino acid sequences 328, 358, 388; or a heavy chain having three CDRs respectively comprising amino acid sequences 238, 278, 299, and a light chain having three CDRs respectively comprising amino acid sequences 329, 359, 389; or a heavy chain having three CDRs respectively comprising amino acid sequences 239, 279, 300, and a light chain having three CDRs respectively comprising amino acid sequences 330, 360, 390; or a heavy chain having three CDRs respectively comprising amino acid sequences 240, 280, 301, and a light chain having three CDRs respectively comprising amino acid sequences 331, 361, 391; or a heavy chain having three CDRs respectively comprising amino acid sequences 241, 281, 302, and a light chain having three CDRs respectively comprising amino acid sequences 332, 362, 392; or a heavy chain having three CDRs respectively comprising amino acid sequences 242, 282, 303, and a light chain having three CDRs respectively comprising amino acid sequences 333, 363, 393; or a heavy chain having three CDRs respectively comprising amino acid sequences 243, 283, 304, and a light chain having three CDRs respectively comprising amino acid sequences 334, 364, 394; or a heavy chain having three CDRs respectively comprising amino acid sequences 244, 284, 305, and a light chain having three CDRs respectively comprising amino acid sequences 335, 365, 395;or a heavy chain having three CDRs comprising amino acid sequences 245, 285, and 306, respectively, and a light chain having three CDRs comprising amino acid sequences 336, 366, and 396, respectively.;

[0106] Other anti-influenza antibodies include those having the amino acid or nucleic acid sequences shown in Table 1 below.

[0107]

[0108] 3I14 and 3I14V L The amino acid sequences of the heavy and light chain complementarity determining regions of the D94N neutralizing influenza antibody are shown in Table 2 below.

[0109] Table 2

[0110]

[0111] CC-chemokine receptor 4 CCR4 (94)

[0112] Exemplary anti-CCR4 antibodies include antibodies having: a VH nucleotide sequence of SEQ ID NO: 1678 and a VL nucleotide sequence of SEQ ID NO: 1679; or a VH nucleotide sequence of SEQ ID NO: 1680 and a VL nucleotide sequence of SEQ ID NO: 1681; or a VH nucleotide sequence of SEQ ID NO: 1682 and a VL nucleotide sequence of SEQ ID NO: 1683; or a VH nucleotide sequence of SEQ ID NO: 1684 and a VL nucleotide sequence of SEQ ID NO: 1685; or a VH nucleotide sequence of SEQ ID NO: 1686 and a VL nucleotide sequence of SEQ ID NO:: 1687; or a VH nucleotide sequence of SEQ ID NO: 1688 and a VL nucleotide sequence of SEQ ID NO: 1689.

[0113] Exemplary anti-CCR4 antibodies include antibodies having: the VH amino acid sequence of SEQ ID NO: 1690 and the VL amino acid sequence of SEQ ID NO: 1691; or the VH amino acid sequence of SEQ ID NO: 1692 and the VL amino acid sequence of SEQ ID NO: 1693; or the VH amino acid sequence of SEQ ID NO: 1694 and the VL amino acid sequence of SEQ ID NO: 1695; or the VH amino acid sequence of SEQ ID NO: 1696 and the VL amino acid sequence of SEQ ID NO: 1697; or the VH amino acid sequence of SEQ ID NO: 1698 and the VL amino acid sequence of SEQ ID NO:: 1699; or the VH amino acid sequence of SEQ ID NO: 1700 and the VL amino acid sequence of SEQ ID NO: 1701.

[0114] In other embodiments, the anti-influenza antibody has: a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1702, 1703, and 1704 respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1705, 1706, and 1707 respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1708, 1709, and 1710 respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1711, 1712, and 1713 respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1714, 1715, and 1716 respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1717, 1718, and 1719 respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1720, 1721, and 1722 respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1723, 1724, and 1725 respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1726, 1727, and 1728 respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1729, 1730, and 1731 respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1732, 1733, and 1734 respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1735, 1736, and 1737 respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1738, 1739, and 1740 respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1741, 1742, and 1743 respectively.

[0115] Human immunoglobulin heavy chain variable region germline gene (VH1-69) (133)

[0116] 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.

[0117] Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibodies comprise an antibody having a VH amino acid sequence of SEQ ID NO:1746 and a VL amino acid sequence of SEQ ID NO:1747; or an antibody having a VH amino acid sequence of SEQ ID NO:1750 and a VL amino acid sequence of SEQ ID NO:1751; or an antibody having a VH amino acid sequence of SEQ ID NO:1754 and a VL amino acid sequence of SEQ ID NO:1755.

[0118] In other embodiments, the anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody has: a heavy chain having three CDRs with amino acid sequences of SEQ ID NOs:1756, 1757, 1758, respectively; and a light chain having three CDRs with amino acid sequences of SEQ ID NOs:1759, 1760, 1761, respectively.

[0119] Zika virus antibody (140)

[0120] Exemplary antibodies that target and neutralize Zika virus include antibodies having: a VH nucleotide sequence of SEQ ID NO:1762 and a VL nucleotide sequence of SEQ ID NO:1763.

[0121] Exemplary antibodies that target and neutralize Zika virus include antibodies having: a VH amino acid sequence of SEQ ID NO:1764 and a VL amino acid sequence of SEQ ID NO:1765.

[0122] In other embodiments, the antibody that targets and neutralizes Zika virus has a heavy chain having three CDRs with amino acid sequences of SEQ ID NOs:1766, 1767, 1768, respectively; and a light chain having three CDRs with amino acid sequences of SEQ ID NOs:1769, 1770, 1771, respectively.

[0123] Glucocorticoid-induced tumor necrosis factor receptor (GITR) (141)

[0124] Exemplary glucocorticoid-induced tumor necrosis factor receptor (GITR) antibodies include: having a VH nucleotide sequence of SEQ ID NO: 1772 and a VL nucleotide sequence of SEQ ID NO: 1773; or having a VH nucleotide sequence of SEQ ID NO: 1774 and a VL nucleotide sequence of SEQ ID NO: 1775; or having a VH nucleotide sequence of SEQ ID NO: 1776 and a VL nucleotide sequence of SEQ ID NO: 1777; or having a VH nucleotide sequence of SEQ ID NO: 1778 and a VL nucleotide sequence of SEQ ID NO: 1779; or having a VH nucleotide sequence of SEQ ID NO: 1780 and a VL nucleotide sequence of SEQ ID NO: 1781; or having a VH nucleotide sequence of SEQ ID NO: 1782 and a VL nucleotide sequence of SEQ ID NO: 1783; or having a VH nucleotide sequence of SEQ ID NO: 1784 and a VL nucleotide sequence of SEQ ID NO: 1785; or having a VH nucleotide sequence of SEQ ID NO: 1786 and a VL nucleotide sequence of SEQ ID NO: 1787; or having a VH nucleotide sequence of SEQ ID NO: 1788 and a VL nucleotide sequence of SEQ ID NO: 1789; or having a VH nucleotide sequence of SEQ ID NO: 1790 and a VL nucleotide sequence of SEQ ID NO: 1791; or having a VH nucleotide sequence of SEQ ID NO: 1792 and a VL nucleotide sequence of SEQ ID NO: 1793; or having a VH nucleotide sequence of SEQ ID NO: 1794 and a VL nucleotide sequence of SEQ ID NO: 1795; or having a VH nucleotide sequence of SEQ ID NO: 1796 and a VL nucleotide sequence of SEQ ID NO: 1797.

[0125] Exemplary glucocorticoid-induced tumor necrosis factor receptor (GITR) antibodies comprise 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 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.

[0126] In other embodiments, the anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody has a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1824, 1825, 1826, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1827, 1828, 1829, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1830, 1831, 1832, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1833, 1834, 1835, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1836, 1837, 1838, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1839, 1840, 1841, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1842, 1843, 1844, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1845, 1846, 1847, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1848, 1849, 1850, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1851, 1852, 1853, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1854, 1855, 1856, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1857, 1858, 1859, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1860, 1861, 1862, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1863, 1864, 1865, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1866, 1867, 1868, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1869, 1870, 1871, respectively; or a heavy chain having three CDRs with amino acid sequences having SEQ ID NOs: 1872, 1873, 1874, respectively, and a light chain having three CDRs with amino acid sequences having SEQ ID NOs: 1875, 1876, 1877, respectively;or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1878, 1879, and 1880, respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1881, 1882, and 1883, respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1884, 1885, and 1886, respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1887, 1888, and 1889, respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1890, 1891, and 1892, respectively, and a light chain having three CDRs with amino acid sequences of SEQ ID NO: 1893, 1894, and 1895, respectively; or a heavy chain having three CDRs with amino acid sequences of SEQ ID NO: 1896, 1897, and 1898, respectively, and a light chain having three CDRs with amino acid sequences comprising SEQ ID NO: 1899, 1900, and 1901, respectively.

[0127] The tetravalent antibody is a dimer of bispecific scFv fragments, the 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 an IgG1, IgG2, IgG3, or IgG4 hinge region. Exemplary hinge region amino acid sequences include EPKSCDKTHTCPPCP (SEQ ID NO: 1902); ERKCCVECPPCP (SEQ ID NO: 1903); and ESKYGPPCPSCP (SEQ ID NO: 1904).

[0128] In some embodiments, the linker domain further comprises at least a portion of an immunoglobulin Fc domain. The at least a portion of the immunoglobulin Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. The at least a portion of the immunoglobulin Fc domain is linked to the C-terminus of the hinge region. The at least a portion of the immunoglobulin Fc domain refers to, 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.

[0129] Including at least a portion of the immunoglobulin Fc domain (e.g., the CH2 domain) provides a third functional binding site (i.e., Fc effector function), thereby generating a trifunctional bispecific antibody. Thus, it may be desirable to modify at least a portion with respect to effector function in order to enhance, for example, 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 internalization ability and / or increased complement-mediated cell killing and antibody-dependent cell cytotoxicity (ADCC). Alternatively, at least a portion of the immunoglobulin Fc domain is 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 asparagine corresponding to amino acid position 297. While glycosylation is important for stability, defucosylation of the CH2 carbohydrate can also increase the binding affinity for FcγR and result in further enhancement of ADCC.

[0130] In certain embodiments, the tBsAb of the present invention may comprise an Fc variant that contains an amino acid substitution that alters the antigen-independent effector function of the antibody, particularly the circulating half-life of the antibody. Such antibodies exhibit increased or decreased binding to FcRn when compared to antibodies lacking these substitutions and thus have an increased or decreased serum half-life, respectively. Fc variants with improved affinity for FcRn are expected to have a longer serum half-life, and such molecules have useful applications in methods of treating mammals where a long half-life of the antibody to be administered is desired, e.g., to treat chronic diseases or conditions. In contrast, Fc variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such molecules can also be used, e.g., for administration to a mammal where a shortened circulation time may be advantageous, e.g., for in vivo diagnostic imaging or in cases where the antibody has toxic side effects when present in the circulation for an extended period. In one embodiment, the Fc domain has 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 - 299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication No. WO05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, the antibody or fragment thereof of the present invention comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0131] Preferably, at least a portion of the Fc domain is the CH2 domain amino acid sequence. Exemplary CH2 domain amino acid sequences include APELLGGPDVFLF (SEQ ID NO: 1905).

[0132] In other aspects, the immunoglobulin hinge region amino acid sequence or immunoglobulin hinge region / Fc domain amino acid sequence flanks a flexible linker amino acid sequence. Flexible linker amino acid sequences include, for example, (GGGS) X＝1-6 , (GGGGS) X＝1-6 or GSAGSAAGSGEF.

[0133] Increasing the linker by adding multiple repeats (e.g., four or more) will predominantly yield monomeric scFv, and thus it can increase accessibility to the epitope. The length and composition of the linker can be selected to optimize stability and functional activity, taking into account the topography of the epitope on the target protein.

[0134] The invention also includes a nucleic acid construct comprising a nucleic acid molecule encoding: the light and heavy chain variable regions of an antibody that specifically binds to a first antigen; the light and heavy chain variable regions of an antibody that specifically binds to a second antigen; and a linker domain.

[0135] In another aspect, the invention provides a genetically engineered cell that expresses and attaches to the cell surface membrane tBsAb of the invention. The cell is a T cell, B cell, follicular T cell, or NK cell. The T cell is CD4+ or CD8+. The cell is a mixed population of CD4+ and CD8+ cells. The cell is further engineered to express and secrete tBsAb.

[0136] The vector contains the nucleic acid construct according to the invention, and a host cell (e.g., a mammalian cell) expresses the vector of the invention.

[0137] Chimeric antigen receptor

[0138] The tBsAb of the invention can be used to generate a chimeric antigen receptor (CAR). The CAR generally comprises at least one transmembrane polypeptide, the at least one transmembrane polypeptide comprising at least one extracellular ligand-binding domain comprising the tBsAb of the invention; and a transmembrane polypeptide comprising at least one intracellular signaling domain.

[0139] 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" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region serves to provide greater flexibility and accessibility to the extracellular ligand-binding domain. The stalk region domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 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 part of the human CD8α chain

[0140] The signal transduction domain or intracellular signaling domain of the CAR of the present invention is responsible for intracellular signaling after binding of the extracellular ligand-binding domain to the target, thereby generating activation of immune cells and an immune response. In other words, the signal transduction domain is responsible for activating at least one normal effector function of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytotoxic activity or helper activity, including the secretion of cytokines. Thus, the term "signaling domain" refers to the portion of a protein that transduces an effector signal function signal and directs the cell to perform a specialized function.

[0141] The signal transduction domain comprises two different classes of cytoplasmic signaling sequences, namely those that initiate antigen-dependent primary activation and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequences may comprise signaling motifs, the immunoreceptor tyrosine-based activation motifs known as ITAMs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of a variety of receptors, which serve as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention may include, as non-limiting examples, those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signal transduction domain of the CAR may comprise a CD13ζ signal transduction domain, or the cytoplasmic domain of the FcεRIβ or γ chain. In another preferred embodiment, signaling is provided by CD3ζ together with co-stimulation provided by CD28 and / or tumor necrosis factor receptor (TNFr) (such as, for example, 4-1BB or OX40).

[0142] In certain embodiments, the intracellular signaling domain of the CAR of the present invention comprises a co-stimulatory signaling molecule. In some embodiments, the intracellular signaling domain contains two, three, four or more co-stimulatory molecules in tandem. A co-stimulatory molecule is a cell surface molecule required for an effective immune response in addition to an antigen receptor or its ligand.

[0143] "Co-stimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which signal mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc. Co-stimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also particularly encompass antibodies that specifically bind to co-stimulatory 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, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0144] "Co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory 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, etc. In another specific embodiment, the signal transduction domain is a TNFR-associated factor 2 (TRAF2) binding motif that co-stimulates the cytoplasmic tail of members of the TNFR family. The cytoplasmic tails of co-stimulatory 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.

[0145] Distinguishing features of suitable transmembrane polypeptides include the ability to be expressed on the surface of immune cells, particularly on the surface of lymphocytes or natural killer (NK) cells, and the ability to interact together to direct the cellular response of immune cells against a predetermined target cell. The different transmembrane polypeptides of the CARs of the present invention that contain extracellular ligand-binding domains and / or signal transduction domains interact together to participate in signal transduction and induce an immune response after binding to a target ligand. The transmembrane domain can be derived from a natural source or a synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein, although certain transmembrane domains that are optimally adapted to the chimeric body are preferred, for example, those that promote self-aggregation or an increase in the basal activation of CAR T cells in the absence of target binding, which may lead to premature exhaustion.

[0146] As used herein, the term "a portion of" refers to any subset of a molecule, i.e., a shorter peptide. Alternatively, functional variants of the amino acid sequence of a polypeptide 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 obtain the final construct, provided that the final construct has the desired activity, particularly exhibiting specific anti-target cell immune activity. The functionality of the CARs of the present invention within a host cell can be detected in an assay suitable for demonstrating the signal transduction potential when the CAR binds to a specific target. Such assays are available to those skilled in the art. For example, such an assay allows detection of signal transduction pathways triggered upon target binding, such as assays involving measurement of an increase in calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate conversion, or the production of interleukin (IL)-2, interferon γ, GM-CSF, IL-3, IL-4 thereby achieved.

[0147] Method of Use

[0148] The tBsAbs, cells expressing the tBsAbs, or CARs according to the present invention can be used to treat cancer, viral infections, or autoimmune disorders in a patient in need thereof. In another embodiment, the tBsAbs, cells expressing the tBsAbs, or CARs according to the present invention can be used to manufacture a medicament for treating cancer, viral infections, or autoimmune disorders in a patient in need thereof.

[0149] The treatment can be amelioration, cure, or prevention. It can be part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment. Autologous means that the cells, cell lines, or cell populations used to generate the tBsAb or to express the tBsAb are derived from the patient or from a human leukocyte antigen (HLA)-compatible donor. Allogeneic means that the cells or cell populations used to generate the tBsAb or to express the tBsAb are not derived from the patient but from a donor.

[0150] The treatment can be used to treat patients diagnosed with cancer, viral infection, autoimmune disorder, or graft-versus-host disease (GvHD). Treatable cancers include non-vascularized or not yet substantially vascularized tumors as well as vascularized tumors. Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or can include 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 and lymphoid malignancies, benign and malignant tumors, and malignancies such as sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0151] It can be a treatment in combination with one or more cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiation therapy.

[0152] In another embodiment, the compositions of the invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, T cell depletion therapy using chemotherapeutic agents (such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide) or antibodies (such as OKT3 or CAM PATH).

[0153] Definitions

[0154] It should be noted that the term "a / an" entity means one or more of such entities; for example, "bispecific antibody" is understood to represent one or more bispecific antibodies. Thus, the terms "a / an" (or "one / a"), "one or more" and "at least one" are used interchangeably herein.

[0155] As used herein, the term "polypeptide" is intended to cover both the singular "polypeptide" and the plural "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any one or more chains having two or more amino acids, and does not refer to the specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to one or more chains having two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the post-expression modification products of polypeptides, and such modifications include, but are not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant techniques, but need not be translated from a designated nucleic acid sequence. It can be produced in any manner, including by chemical synthesis.

[0156] As used herein, the term "isolated" when used in connection with cells, nucleic acids such as DNA or RNA means that the molecule is separated from other DNA or RNA that are present in the natural source of the macromolecule. The term "isolated" as used herein also means that a nucleic acid or peptide is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or is substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" means a nucleic acid fragment that is not naturally occurring as a fragment and would not exist in nature in that state. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to cover purified polypeptides and recombinant polypeptides.

[0157] As used herein, the term "recombinant" in relation to a polypeptide or polynucleotide means a form of polypeptide or polynucleotide that is not naturally occurring, and non-limiting examples thereof can be produced by combining polynucleotides or polypeptides that do not normally occur naturally.

[0158] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in the respective sequences that are aligned for comparison purposes. When the positions in the sequences being compared are occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences varies with the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, but preferably less than 25% identity, with one of the sequences of the present disclosure.

[0159] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that, when comparing two sequences, a certain percentage of the bases (or amino acids) are the same upon alignment. Such alignment and the percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Preferably, default parameters are used for alignment. One alignment program is BLAST, which uses default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the World Wide Web (www) ncbi.nlm.nih.gov / blast / Blast.cgi last accessed on May 21, 2008. Biologically equivalent polynucleotides are those polynucleotides having the specified percentage homology as described above and encoding polypeptides having the same or similar biological activity.

[0160] 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. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence with a certain degree of homology with their complement. In one aspect, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a certain degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0161] Hybridization reactions can be carried out under conditions of different "stringency". Generally, low stringency hybridization reactions are carried out at about 40 in a solution of about 10x SSC or equivalent ionic strength / temperature. Medium stringency hybridizations are typically carried out at about 50 in about 6x SSC, and high stringency hybridizations are typically carried out at about 60 in about 1x SSC. Hybridization reactions can also be carried out under "physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are Mg typically found in cells2 The temperature, ionic strength, pH, and concentration of +.

[0162] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and when the polynucleotide is RNA, uracil (U) replaces thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into a database in a computer with a central processing unit and is used in bioinformatics applications such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one form of a gene or a part thereof. The presence of at least two different forms, i.e., a part of a gene with two different nucleotide sequences, is called a "polymorphic region of the gene". A polymorphic region can be a single nucleotide whose identity differs among different alleles.

[0163] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. 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. A polynucleotide can contain modified nucleotides such as methylated nucleotides and nucleotide analogs. If present, the modification to the nucleotide structure can be given before or after the assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the present disclosure that is a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0164] The term "encoding" as applied to a polynucleotide refers to a polynucleotide that, if the polynucleotide is transcribed and / or translated to produce an mRNA of a polypeptide and / or a fragment thereof when in its native state or when manipulated by methods well known to those skilled in the art, is said to "encode" the polypeptide. The antisense strand is the complement of such a nucleic acid molecule, and the coding sequence can be inferred therefrom.

[0165] As used herein, the term "detectable label" means a compound or composition that is directly or indirectly detectable and that is directly or indirectly conjugated to a composition to be detected, such as a polynucleotide or a protein such as an antibody, to produce 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). A label can be detectable (e.g., radioisotope-labeled or fluorescently labeled) or, in the case of an enzyme label, can catalyze a chemical change in a detectable substrate compound or composition. Labels can be suitable for small-scale assays 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. A label can be simply detected or can be quantified. A response for simple detection typically includes just a response that confirms the presence, while a response for quantification typically includes a response having a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other attributes. In a luminescence or fluorescence assay, a luminophore or fluorophore associated with the assay component actually involved in binding can be used directly or a luminophore or fluorophore associated with another (e.g., reporter molecule or indicator) component can be used indirectly to produce a detectable response.

[0166] 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 and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Examples of such include, but are not limited to, complementarity determining regions (CDRs) of a heavy or light chain or ligand-binding portions thereof, variable regions of a heavy or light chain, constant regions of a heavy or light chain, framework (FR) regions or any portion thereof, or at least a portion of a binding protein.

[0167] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a part of an antibody, such as F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen as the whole antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0168] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V H ) and light chain (V LA fusion protein of the variable region of ( ). In some aspects, the region is linked to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of V H to the C-terminus of V L , and vice versa. Despite removing the constant region and introducing a linker, this protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0169] "Tandem scFv" consists of two scFv's linked by a short linker that allows the two separate antigen-binding units to rotate freely, resulting in a flexible structure.

[0170] The term antibody encompasses a wide variety of biochemically distinguishable classes of polypeptides. Those skilled in the art will appreciate that heavy chains are classified as γ, μ, α, δ, or ε, with some subclasses among them (e.g., γ1 to γ4). The nature of such chains determines the "class" of the antibody to be IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5 etc. are well-characterized and are known to confer functional specialization. Given the present disclosure, those skilled in the art can readily discern the respective modified forms of these classes and isotypes and are thus within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion will generally relate to immunoglobulin molecules of the IgG class. With respect to IgG, a standard immunoglobulin molecule comprises 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 to 70,000. These four chains are typically linked together by disulfide bonds in a "Y" configuration, where the light chains start from the opening of the "Y" and are bracketed with the heavy chains through the variable regions throughout.

[0171] The antibodies or antigen-binding polypeptides, variants, or derivatives of the present disclosure include, but are not limited to: polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab) 2 , Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), comprising V K or V HFragments of domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies of the LIGHT antibodies disclosed herein). The immunoglobulins 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 molecules.

[0172] Light chains are classified as κ or λ. Each heavy chain class can bind to either a κ or λ light chain. Generally, the light and heavy chains are covalently bonded to each other, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent associations. In the heavy chain, 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.

[0173] Both the light and heavy chains are partitioned into regions having structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light (V K ) and heavy (V H ) chain portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CK) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the constant region domains are numbered 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 constant region is at the C-terminal portion; the CH3 domain and the CK domain actually contain the carboxyl-terminus of the heavy and light chains, respectively.

[0174] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the V K domain and the V H domain or a subset of the complementarity-determining regions (CDRs) combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site at the end of each arm of the Y. More specifically, the antigen-binding site is composed of V H and V KThree CDRs are defined on each of the chains, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some examples, for instance, certain immunoglobulin molecules (intact immunoglobulin molecules) derived from camelid species or engineered based on camelid immunoglobulins may consist only of heavy chains and lack light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0175] In naturally occurring antibodies, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain, called "framework" regions, exhibit less intermolecular variability. The framework regions predominantly adopt a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. Thus, the framework regions function to form a scaffold that provides the correct orientation of the CDRs through interchain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunologically reactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. The amino acids that constitute the CDRs and framework regions, respectively, can be readily identified by one of ordinary skill in the art for any given heavy or light chain variable region, as they have been precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entirety).

[0176] Where there are two or more definitions of a term used and / or accepted within the art, the definition of the term as used herein is intended to include all such meanings, unless expressly stated to the contrary. A specific example is the use of the term "complementary determining region" ("CDR") to describe the non - contiguous antigen - combining sites found in the variable regions of both heavy and light chain polypeptides. Such specific regions have been described by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901 - 917 (1987), which are hereby incorporated by reference in their entirety. The CDR definitions according to Kabat and Chothia include an overlap or subset of amino acid residues when compared to each other. However, the application of either definition to refer to the CDR of an antibody or its variant is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues covering the CDR as defined by each of the above - cited references are listed in the following table for comparison. The exact residue numbers covering a particular CDR will vary depending on the sequence and size of the CDR. One of ordinary skill in the art can generally determine what residues comprise the specific CDR of the variable region amino acid sequence of a given antibody.

[0177] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. One of ordinary skill in the art can unambiguously assign such a "Kabat numbering" system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system elucidated by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0178] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tyrosine residue. CDR-H2 starts at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts at approximately the thirty-third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 starts at approximately residue 24 (i.e., after the cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 starts at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 starts at approximately the thirty-third residue after the end of CDR-L2 (i.e., after the cysteine residue); includes approximately 7-11 residues and ends at the sequence W-G-X-G, where X is any amino acid.

[0179] The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another embodiment, the variable regions can be derived from a chondrichthoid (e.g., from a shark).

[0180] As used herein, the term "heavy chain constant region" includes amino acid sequences derived from immunoglobulin heavy chains. A polypeptide containing a heavy chain constant region includes at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or variants or fragments thereof. For example, an antigen-binding polypeptide used in the present disclosure can include: a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptides of the present disclosure include a polypeptide chain containing a CH3 domain. Additionally, the antibodies used in the present disclosure can lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As set forth above, one of ordinary skill in the art will understand that the heavy chain constant regions can be modified such that their amino acid sequences differ from those of naturally occurring immunoglobulin molecules.

[0181] The heavy chain constant region 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 IgG 3 molecule. In another example, the heavy chain constant region can comprise a portion derived from an IgG molecule and a hinge region derived from an IgG 3 molecule. In another example, the heavy chain portion can comprise a portion derived from an IgG molecule and a hinge region derived from an IgG 4 molecule. The chimeric hinge of the molecule.

[0182] As used herein, the term "light chain constant region" comprises an amino acid sequence derived from an immunoglobulin light chain. Preferably, the light chain constant region comprises at least one of the constant κ domain or the constant λ domain.

[0183] A "light chain-heavy chain pair" refers to a collection of a light chain and a heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0184] As previously indicated, the subunit structure and three-dimensional conformation of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "V H 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. The CH1 domain is adjacent to the V H domain and is the amino terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0185] As used herein, the term "CH2 domain" includes the portion of the heavy chain molecule that extends from about residue 244 to residue 360 of the antibody using a conventional numbering scheme (residues 244 to residue 360, Kabat numbering system; and residues 231 to residue 340, EU numbering system; see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983). The CH2 domain is unique in that it does not pair tightly with another domain. Two N-linked branched carbohydrate chains are interposed between the two CH2 domains of the intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0186] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further divided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).

[0187] 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 disulfide bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 region and the CK region are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

[0188] As used herein, the term "chimeric antibody" will mean any antibody in which the immunoreactive region or site is obtained from or derived from a first species and the constant region (which may be full-length, partial, or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target-binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is from human.

[0189] 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, subtracting that number from the total number of amino acids, and then dividing by the total number of amino acids and multiplying by 100.

[0190] "Specifically binds" or "is specific for" generally means that an antibody binds to an epitope via its antigen-binding domain, and the binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity by which a given antibody binds to a given epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B", or it may be said that antibody "A" binds to epitope "C" with higher specificity than it binds to a related epitope "D".

[0191] As used herein, the terms "treat" or "treatment" refer to therapeutic treatment and prophylactic or preventive measures, wherein the aim is to prevent or slow down (alleviate) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminishment of the extent of the disease, stabilization (i.e., non-worsening) of the disease state, delay or slowing of the progression of the disease, improvement or palliation of the disease state, and remission (whether partial or total), whether detectable or not. "Treatment" can also mean prolongation of survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already suffering from a condition or disorder and those prone to or intending to prevent a condition or disorder.

[0192] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject in need of diagnosis, prognosis or treatment, particularly mammalian subjects. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, bears, etc.

[0193] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from administration (e.g., for detection, for diagnostic procedures and / or for treatment) of the antibodies or compositions of the present disclosure.

[0194] The present disclosure describes the development of bispecific antibodies that can be applied to cancer therapy. To achieve this goal, two tetrameric bispecific antibodies (tBsAbs) that are dual-specific for the GITR protein and the PD-L1 protein were generated. An advantage of these constructs is that they will enhance the anti-tumor response by activating T cells and eliminating regulatory T cell suppression. Also described are anti-CCR4-anti-PDL1 tBsAbs (Figures 3 and 4) and anti-CAIX-anti-PDL1 ( Figure 5 and 6 ).

[0195] Two different forms 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 parental antibodies. The αGITR scFv and αPD-L1 scFv are tandemly linked via the IgG1 hinge region between two flexible linkers. The first construct has the structure of 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 form was constructed equivalently to the first form. However, it includes an additional domain, such as the CH2 domain, introduced between the hinge and one of the linker regions. The second construct has the structure of VH GITR-linker-VL GITR-linker-hinge-CH2-linker-VH PD-L1-linker-VL PD-L1. Compared with the first construct, the second construct contains an Fc domain and may generate a trifunctional tBsAb.

[0196] Both tBsAbs were successfully expressed by transient transfection in HEK cells and purified by affinity chromatography using Ni-NTA agarose.

[0197] The purified proteins were evaluated by SDS-PAGE, and the results showed that under reducing conditions, all protein profiles of the tBsAbs exhibited a single band and were consistent with the theoretical values of the 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 the αGITR-αPD-L1 (130 kDa) and αGITR-αPD-L1 with CH2 (150 kDa) tBsAbs matched the apparent molecular weights (see Figure 18 ).

[0198] ELISA and flow cytometry demonstrated the biological activity of the newly designed tBsAb. In ELISA, the retained binding activity of the αPD-L1 arm of the generated BsAb was retained in vitro and showed similar binding activity to αPD-L1 mAb. Since CCR4, which is not bound by the αGITR-αPD-L1 antibody, did not show any signal, non-specific binding of the tBsAb was excluded. In addition, similar binding activity of the αGITR arm to the GITR protein was observed for the generated BsAbs (αGITR-αPD-L1 and αGITR-αPD-L1 with CH2). Non-specific binding was also excluded from this arm, as the αGITR-αPD-L1 antibody did not show any binding specificity to GITR-CF2 cells. When αGITR IgG was compared with each BsAb, αGITR IgG showed higher binding in the ELISA experiment, indicating a lower affinity of the tBsAb. However, depending on the spatial arrangement of the antigen-binding sites and the antigen surface distribution, the bivalency of the tBsAb can increase avidity, which can compensate for the weak binding.

[0199] Flow cytometry analysis of αGITR-αPD-L1 tested with GITR+CF2 cells showed that the novel tBsAb recognized the GITR protein in its native conformation when expressed on the cells. Similar binding affinity of the αGITR-αPD-L1 tBsAb compared to the GITR mAb was observed. Thus, ELISA and flow cytometry analysis demonstrated the ability of αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2 to specifically recognize the corresponding antigens when expressed on the cells, and the same was true in vivo. These characterization studies showed similar binding behavior of αGITR-αPD-L1 and αGITR-αPD-L1 with CH2.

[0200] An important aspect of αGITR10-αPD-L1 with CH2 lies in its function in inducing complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC). These further exerted the beneficial effects of the tBsAb when targeting tumor cells for destruction.

[0201] In the ADCC assay, 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 unexpected results. For the αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2 antibodies, the original signal of luciferase activity decreased at higher antibody concentrations and was significantly lower than the signal of single target and effector cells (see Figure 30 ).

[0202] The methods described herein allow for the generation of tBsAbs that involve only one cloning step. Such tBsAbs maintain dual affinity for GITR protein and PD-L1 antigen in a small-sized molecule of only about 150 kDa. Such tBsAbs are crucial for effective cancer therapeutics.

[0203] Examples

[0204] Example 1: Cloning of αGITR-αPD-L1 tetravalent bispecific antibody (tBsAb).

[0205] Cloning strategy

[0206] The goal was to clone a plasmid containing two recombinant single-chain variable fragments (scFvs) derived from different parental antibodies and linked by a flexible linker. While one of the scFvs was directed against the GITR protein, the other was directed against PD-L1. This plasmid would produce two scFvs that were covalently linked by a linker-hinge-linker domain and produced a tetravalent bispecific antibody (αGITR-αPD-L1 tBsAb).

[0207] The mammalian expression vector pcDNA3.4 plasmid was the basis of the construct (V H GITR-linker-V L GITR-linker-hinge-linker-V H PD-L1-linker-V L PD-L1). The basic structure of the pcDNA 3.4 expression vector pre-contained V fused to an N-terminal 6x-His tag H X -linker-V L X -linker-hinge-linker-V H PD-L1-linker-V L PD-L1 gene ( Figure 12 ).

[0208] Restriction enzyme digestion and ligation

[0209] Six αGITR scFv gene sequences were individually cloned into the pcDNA 3.4 expression vector. The six V H GITR-linker-V L GITR gene sequences were labeled as V H GITRL1-V L GITRL1, V H GITRL10-V L GITRL10, V H GITRL11-V LGITRL11, V H GITRL14-V L GITRL14, V H GITRL15-V L GITRL15 and V H GITRL17-V L GITRL17. All six αGITR gene sequences are flanked by SfiI and NotI restriction sites and are isolated from the corresponding donor plasmids by digestion (Table 1). Similarly, the pcDNA3.4 expression vector is also digested with SfiI and NotI restriction enzymes. The digested vectors and fragments are analyzed on a 1% agarose gel and purified using the QIAquick Gel Extraction Kit. The sticky inserts from the SfiI and NotI digestions are ligated into the corresponding vector pcDNA 3.4 at a 5-fold molar ratio using the T4 Ligation Kit. 50 ng of the recipient vector is used for each ligation reaction. The ligation products generate V H GITR-linker-V L GITR-linker-hinge-linker-V H PD-L1-linker-V L The final configuration of PD-L1( Figure 12 ).

[0210] Three additional clones are constructed separately to generate control antibodies. The F10 gene is selected as the "control group"; because V H F10-V L The F10 binding domain does not have binding affinity for either the GITR and PD-L1 proteins. Therefore, this domain is defined as a negative control. F10 is a validated antibody against the influenza HA protein. To maintain the same antibody format, its gene sequence is only substituted for V H GITR1-linker-V L GITR1 or V H PD-L1-linker-V L PD-L1. The three control plasmids exhibit the following sequencing order:

[0211] (1) V H F10-linker-V L F10-linker-hinge-linker-V H PD-L1-linker-V L PD-L1 (F10-αPD-L1)

[0212] (2) V H GITR1-linker-V L GITR1-linker-hinge-linker-V H F10-linker-V LF10(αGITR1-F10)

[0213] (3)V H GITR10-Linker-V L GITR10-Linker-Hinge-Linker-V H F10-Linker-V L F10(αGITR10-F10)

[0214] Construction of plasmid (1):

[0215] V was isolated from the pcDNA 3.1 vector by digestion with SfiI and NotI RE H F10-Linker-V L F10 gene. For the expression vector, the same pcDNA3.4 vector ( Figure 13 ) was used. It contains SfiI and NotI restriction sites at the desired insertion sites and was thus digested with the corresponding restriction enzymes. The final plasmid was obtained by ligating the two digestion products to each other. Ligation was carried out overnight at 16 using a T4 ligation kit.

[0216] Steps for constructing plasmids (2) and (3):

[0217] To replace αPD-L1scFv in the previously constructed construct, forward and reverse primers were designed and synthesized to introduce BsiWI and BamHI restriction sites and V H F10-Linker-V L 5' and 3' of the F10 fragment. After PCR amplification, the PCR product containing V H F10-Linker-V L F10 and the pcDNA3.4 expression vector were digested with BsiWI and BamHI and used to replace 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) encoding V H PD-L1-Linker-V LDNA fragment of PD-L1. The digested expression vector and insert were gel purified using the QIAquick Gel Extraction Kit (1% agarose), and then ligated to each other by rapid ligation (5 minutes, at room temperature). This procedure produced plasmids (2) and (3)( Figure 14 ).

[0218] Primer design for constructing control plasmid constructs

[0219] As described above, two primers were designed for control plasmids (2) and (3) to isolate V H F10-linker-V L F10. Forward primers (5'-3') were designed to bind to the 3' end of the DNA complementary strand; reverse primers (3'-5') were designed to bind to the 3' end of the main DNA strand and be reverse complementary. The primers were approximately 20 bp in length, with an optimal melting temperature between 62 and 65, and a deviation of no more than ±1. The forward primer (containing the BsiWI restriction site (No. 1) and the reverse primer (containing the BamHI restriction site (No. 2)) were synthesized by Genewiz. For the PCR reaction, 100 ng of DNA template (pcDNA3.1) was used in the thermal cycling. The PCR products were purified using the QIAquick PCR Purification Kit according to the manufacturer's protocol and analyzed on a 1% agarose gel.

[0220] Example 2: Cloning of αGITR10-αPD-L1 tetrameric bispecific antibody (tBsAb) containing the CH2 domain

[0221] Cloning strategy

[0222] The purpose of this example was to introduce the CH2 domain from IgG1 into the previously constructed plasmid, thereby generating V H GITR-linker-V L GITR-linker-hinge-CH2-linker-V H PD-L1-linker-V L The basic structure of PD-L1. The addition of CH2 increased the effector function, thereby generating a trifunctional tBsAb.

[0223] The pcDNA3.4 expression vector containing αGITR10-αPDL1 was used as a template for constructing a new plasmid. A new 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 chosen for several reasons. The HindIII restriction site is unique in the plasmid, and its genomic sequence is not similar to the adjacent coding regions. However, HindIII has some drawbacks, such as its relatively long length (6 nucleotides) which may reduce the mutagenesis efficiency.

[0224] 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 pcDNA 3.4 expression vector (V H GITR 10 -linker-V L GITR 10 -linker-hinge-HindIII * -V H PD-L1-linker-V L PD-L1) and the amplified CH2 fragment were digested with the corresponding restriction enzyme. The digested vector and fragment were gel purified (1% agarose) using the QIAquick Gel Extraction Kit. The sticky inserts from the HindIII digest were ligated into the vector (pcDNA3.4) at a twenty-fold insert to vector ratio using the Quick Ligation Kit, resulting in the generation of a new plasmid V H GITR 10 -linker-V L GITR 10 -linker-hinge-CH2-linker-V H PD-L1-linker-V L construction of PD-L1.

[0225] Site-directed mutagenesis

[0226] Using the QuikChange Lightning Site-Directed Mutagenesis Kit according to the manufacturer's protocol the mutagenesis of the GITR10-PDL1 vector was completed. Two oligonucleotide primers were synthesized, each complementary to the opposite strand of the vector. Both primers contained HindIII as the desired mutation.

[0227] Primers were designed to display HindIII mutations in the middle of primers flanked by 7 to 10 bases. During the temperature cycling, oligonucleotide primers were used for extension by PfuUltra HF DNA polymerase. This method was able to generate mutant plasmids containing staggered nicks. During the next temperature cycle, the product was treated with DpnI to digest the parental DNA template containing methylated and hemimethylated DNA. As a control, a 4.5-kp pWhitescript plasmid was used to test the mutant plasmid. The pWhitescript plasmid encodes a stop codon (TAA) at the position where the glutamine codon would appear in the β-galactosidase gene of pBluescript II, usually eliminating the blue color of colonies on LB-ampicillin agar plates containing IPTG and Xgal. However, the oligonucleotide control primers generated point mutations on the pWhitescript 4.5-kb control plasmid that reverted the T residue of the stop codon to C, resulting in a blue phenotype on media containing IPTG and X-gal. After cycling, 2 μL of DpnI restriction enzyme (37, 5 minutes) was added to digest the parental dsDNA. Then the mutagenized plasmid was transformed into supercompetent cells and spread on LB-ampicillin agar plates containing 80 μg / ml X-gal and 20 mM IPTG (37; >16 hours). On the next day, 16 clones were picked from the LB-ampicillin plates, purified using the QIAprep Spin Miniprep Kit, and digested with HindIII and NotI restriction enzymes to identify successfully mutated clones. The positive clone No. 10 (GITR10-PDL1 with HindIII) was digested another time to compare it with the original plasmid GITR10-PDL1 (without HindIII). Each sample was digested separately with HindIII or BamHI, and simultaneously digested with HindIII and BamHI-HF together, resulting in a total of six digestions (see Table 1 below).

[0228] Table 1 | Parameters and volumes for a total of six restriction enzyme digestions

[0229]

[0230] The six samples were incubated at 37 for 2 hours and analyzed on a 1% agarose gel.

[0231] Bacteria containing the positive mutant clone No. 10 were amplified overnight at 37 in 120 mL of YT medium and then plasmid DNA was purified using the QIAGEN Plasmid Maxi Kit. By sequencing ( Confirm the correct construct containing the HindIII restriction site using a pre-designed primer. Prepare a glycerol stock and store it at -80. Digest the recipient plasmid containing the HindIII domain and the CH2 fragment with HindIII and then ligate them to each other. Transform the ligation product into super-competent cells by heat pulse as described herein. Verify the correct plasmid by sequencing

[0232] transformation

[0233] Transform the ligation product into super-competent cells by heat pulse. Thaw these cells gently on ice. For each transformation, mix 45 μL of cells with 2 μL of β-mercaptoethanol and 1.5 μL of the DNA of interest. Incubate the transformation reaction for 30 minutes and then heat pulse at 42 °C water bath for 40 seconds. Add 0.5 mL of S.O.C medium (Life ) and incubate at 37 °C for 1 hour. The transformation reaction grows overnight at 37 °C on an LB-ampicillin plate.

[0234] Pick several colonies from each ligation sample individually and grow them in 1.5 mL of 2-YT medium for 8 hours. Purify the plasmid of the picked clones using the QIAprep Spin Miniprep Kit as specified by the manufacturer. Verify the correct plasmid by sequencing The bacteria of the positive clones are grown overnight in 120 mL of YT medium (37 °C, 240 rpm) and the plasmid DNA is purified using the QIAGEN Plasmid Maxi Kit (according to the manufacturer's protocol). Prepare a glycerol stock by adding 400 μL of glycerol and 600 μL of the culture to a cryotube vial and then store it at -80.

[0235] Cell culture and transfection

[0236] For protein expression, the 293F human cell line 293F is obtained from Life and the 293T adherent cell line is obtained from the ATCC cell bank. For the cell-based ELISA assay, the CF2-GITR cell line is generated in the Marasco laboratory to express GITR on the cell surface.

[0237] Suspended 293F cells are used for protein expression

[0238] The suspension culture of 293F cells (derived from human embryonic kidney cells; HEK cells) is maintained at 37 °C and 5% CO 2 in an Erlenmeyer flask and 293 Freestyle medium (Life ) During the logarithmic growth phase, the cells were passaged and diluted to the optimal density (200,000 cells / mL) with fresh medium to continue growth.

[0239] 293T and CF2-GITR adherent cells

[0240] The adherent 293T or CF2-GITR cells were maintained at 37 in 5% CO 2 in a 75 cm² flask (Cellstar) and DMEM medium (Life ) supplemented with 10% FBS (fetal bovine serum) (Life ) and 1% SP (sodium pyruvate) (Life ). The cells were passaged at 80%-100% confluence and diluted to the optimal seeding density (2x10 6 cells) with fresh medium to continue growth.

[0241] Transfection

[0242] To generate tetrameric bispecific antibodies (tBsAb) (αGITR1-αPD-L1, αGITR 10-αPD-L1, αGITR11-αPD-L1, αGITR14-αPD-L1, αGITR15-αPD-L1, αGITR17-αPDL1 and αGITR10-αPD-L1 (with CH2)) and control antibodies (αGITR 1-F10, αGITR10-F10, F1-αPD-L1, αGITR IgG), 293F or 293T cells were transfected with the corresponding plasmids.

[0243] Polyethylenimine (PEI)-mediated transient transfection in 293F HEK cells

[0244] One day before transfection, the cells were made to a final concentration of 6x10 5 cells / mL with a total volume of 300 mL. On the day of transfection, the cell density was between 1.0x10 6 and 1.4x10 6between cells / mL. Prepare the corresponding plasmid for transfection. The total charge of the transfection complex is determined by the ratio of transfection reagent to DNA. The negative charge contributed by the phosphate groups in the DNA backbone is neutralized by the positive charge of the transfection reagent. This allows for good complex formation and neutralizes the electrostatic repulsion imparted to the DNA by the negatively charged cell membrane. A 1:1 ratio of plasmid:PEI allows for complete binding of the polymer to the DNA and complete condensation to protect the cargo; however, an excess of PEI is crucial for overcoming the inhibitory effects of the anionic cell surface. For every million cells, transfect using 1 μg of plasmid and 3 μg of PEI, and dilute each separately into 15 mL of Opti-MEM (reduced serum medium) (Life )). Add the diluted PEI to the plasmid and incubate at room temperature for 20 minutes. The neutralization efficiency increases with the time of exposure to the PEI-DNA complex; however, prolonged exposure to the lipid reagent can be toxic. Pour the PEI / plasmid complex into 293F suspension cells (1x10 6 cells / mL; 300 mL per flask) and incubate at 37 and 140 rpm for 6 days.

[0245] Polyethylenimine (PEI)-mediated transient transfection in 293T HEK cells

[0246] Transfecting 293T HEK cells using PEI follows the same protocol as described above for 293F suspension HEK cells, with a few minor changes. Transfection is performed on 293T cells growing at 80% confluence in DMEM medium supplemented with 10% FBS in a tissue culture dish (200 mm). For 40 μg of DNA, use 200 μg of PEI (1:5 ratio), and dilute each separately in 1 mL of Opti-MEM (reduced serum medium) (Life )). Add the diluted PEI to the plasmid and store at room temperature for 20 minutes. Gently add the DNA / PEI complex dropwise to the culture dish to prevent cell detachment and death. Then incubate the cells at 37 for 2 days.

[0247] Example 3: Protein purification

[0248] Ni-NTA purification of bispecific antibodies

[0249] Harvest the suspension of 293HEK cells and centrifuge it at 5000 rpm and 4 for 35 minutes. To purify the bispecific antibody via its N-terminal 6xHis-tag, filter the supernatant (0.22 μm PEV, ) Incubate with 1 mL of Ni-NTA agarose (Qiagen) for 2 hours (240 rpm, RT). Pass the supernatant through a 15 ml Ni-NTA agarose gravity flow column twice. After washing, wash the column containing the beads with four column volumes of Ni-NTA wash buffer (0.02 M imidazole, 0.3 M NaCl, 1 M TrisHCl (pH = 7.0)), and slowly elute the protein with 13 mL of Ni-NTA elution buffer (0.5 M imidazole, 0.3 NaCl, 0.02 Tris HCl (pH = 7.0)). Use a centrifugal filter unit with a 100,000 MW Exchange the buffer of the eluted protein through PBS buffer. Measure the yield of tBsAb using a NanoDrop ND-1000.

[0250] Protein A purification of αGITR IgG antibody

[0251] Harvest the αGITR IgG antibody from the suspension of 293HEK cells and centrifuge it at 5000 rpm at 4 °C for 35 minutes. To purify the αGITR IgG antibody via the Fc domain, incubate the filtered supernatant with 1 mL of Protein A (GE Lifesciences) (room temperature, shaking) for 2 hours, then pass it through a 15 ml gravity flow column (Biorad) twice, followed by 10 mL of PBS for washing. Elute the αGITR IgG with 2 ml of TEA (100 nM), and add 200 μL of Tris-HCl (1 M, (pH = 7)) to the eluate to neutralize the TEA. Add another 2 mL of PBS to the column and collect the eluted protein into a tube.

[0252] Example 4: Protein characterization

[0253] SDS-PAGE analysis

[0254] According to the NuPAGE Technical Guide (Invitrogen), SDS-PAGE analysis is used to verify the purity of the protein. NuPAGE Bis-Tris gel (4% - 12%) (Novex) is used in MES SDS running buffer, and the total protein mass is between 3 μg and 5 μg. Mix the protein sample with 4x LDS sample buffer (Novex) containing dodecyl sulfate to denature the protein. Under reducing conditions, boil the protein sample at 100 °C for 10 minutes. Then load the sample onto a Novex Bis-Tris gel in MES SDS running buffer. Run the gel in an Xcell SureLock Mini-Cell at 200 V for 35 minutes, and then use simplyBlueTM Processed with Coomassie G-250 staining using Safe Stain (Novex).

[0255] Direct ELISA of αGITR-αPD-L1 on passively adsorbed soluble PD-L1 antigen. Coat a Maxisorb 96-well plate overnight at room temperature with 100 μL of 5 μg / mL PD-L1 rabbit Fc antigen and CCR4 protein (negative control) in PBS. On the next day, wash the plate 3 times with PBS and block for 2 hours at room temperature with 200 μL of blocking solution (2% BSA in PBS). Wash the plate 3 times with PBS. Prepare the 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 commercial anti-mouse PD-L1 mAb (Biolegend) in 1X PBS with variable concentrations and add to the wells (100 μL), incubate for 2 hours at room temperature. The highest antibody concentration tested is 1 μg / mL and then serially diluted 10-fold until a 1x 10 -5 μg / mL dilution. Each sample is run in triplicate at each concentration. A number of controls are set up and listed in the following table (Table 2). Wash the 96-well plate (Costar) three times with 1X PBS buffer. Dilute the secondary antibodies (6xHis-HRP (Thermoscientific) and goat anti-mouse IgGFc, HRP conjugate (Thermoscientific)) in 1X PBS (1:2000 and 1:5000). Add the secondary antibody (100 μL) to each well and incubate for 2 hours at room temperature. Finally, wash each well 4 times with PBS. Develop the 96-well plate with 100 μL of TBM substrate solution (Thermoscientific); add 100 μL of phosphoric acid stop solution (Thermoscientific) after development. Record the endpoint OD data at 450 nm using a Bio-Rad BenchmarkPlus and analyze with Microplate Manager 5.2.1 software.

[0256] Table 2 | Experimental overview of test samples and controls for direct ELISA of αGITR-αPD-L1 on passively adsorbed PD-1 antigen

[0257]

[0258] GITR+ Cell-based ELISA of αGITR-αPD-L1 BsAb on CF2

[0259] For the cell-based ELISA, the retained binding capabilities of αGITR1-αPD-L1, αGITR10-αPD-L1, and αGITR10-αPD-L1 antibody with CH2 on GITR+CF2 cells were tested. A total of four ELISA experiments were established.

[0260] The first cell-based ELISA of αGITR1-F10 and αGITR10-F10 tetravalent bispecific antibodies (tBsAb) was analyzed. For GITR+CF2 and GITR-CF2 cells (negative control) seeding, 1,000 cells per well were added in 200 μL of 1% DNEM medium and incubated overnight to allow attachment. On the next day, the 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 general assay procedures and development were carried out according to the ELISA protocol mentioned in Chapter 2.6.2. The primary antibodies αGITR1-αPD-L1 and αGITR10-αPD-L1 were tested at variable concentrations. The tBsAb was serially diluted one-third in 1X incubation buffer; the highest concentration was 3.33 mg / mL, and the lowest concentration was 0.0411 mg / mL. A number of controls were set up and listed in the following table (Table 3).

[0261] 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

[0262]

[0263] After evaluating the results of the cell-based ELISA ( Figure 20 ), the experiment of the second cell-based ELISA was repeated using the same procedure as above, except that the cells were fixed with 8% paraformaldehyde.

[0264] The third cell-based ELISA was performed to compare αGITR10-αPD-L1 tBsAb with commercial human αGITR mAb. For GITR + CF2 and GITR -CF2 cells (negative control) were seeded. 10,000 cells per well were added to 200 μL of 1% DNEM medium and incubated overnight to allow attachment. On the next day, the cells were fixed with 100 μL of 8% paraformaldehyde and incubated for 20 minutes at room temperature. The paraformaldehyde solution was aspirated from the plate and the cells were washed three times with 1X PBS. The general assay procedures and development were carried out according to the ELISA protocol mentioned herein. Primary antibodies αGITR10-αPD-L1 and αGITR mAb were tested at variable concentrations. The antibodies were serially diluted (1:2) in 1X incubation buffer; the highest concentration was 5 mg / mL and the lowest concentration was 0.078 mg / mL. A number of controls were set up and are listed in the following table (Table 4).

[0265] Table 4 | Experimental overview of test samples and controls for cell-based ELISA of αGITR10-αPD-L1 and αGITR mAb on GITR+CF2 cells

[0266]

[0267] A fourth ELISA was performed to compare αGITR10-αPD-L1 tBsAb with CH2 to commercial αGITR mAb. The assay procedure was the same as the third ELISA (mentioned above).

[0268] Flow cytometry analysis of αGITR1-αPD-L1 and αGITR10-αPD-L1

[0269] The biological activity of αGITR on GITR+CF2 cells was analyzed by fluorescence-activated cell sorting FACS analysis. The cells GITR+CF2 cells and GITR-CF2 were cultured in 75 cm 2Grow in flasks (Cellstar) until they reach approximately 80% confluence. Detach and resuspend them by adding 1:10 diluted trypsin in PBS with 0.25% trypsin-EDTA (Life Technologies), and then add them to a 96-well round bottom plate in FACS buffer (PBS, 1% FBS, 2 mM EDTA). In the next step, add αGITR1-αPD-L1 and αGITR10-αPD-L1 at variable concentrations for 1 hour at 4°C. The highest antibody concentration tested was 100 μg / mL, and then serially diluted 2-fold until a 0.05 μg / mL dilution. Detect the primary antibody with His-tag Alexa Fluor 488-conjugated (Biotechne). Dilute the secondary antibody in PBS (Life Technologies) and add it to each well for 30 minutes. Then wash the cells three times with PBS buffer and resuspend them in FACS buffer. Analyze a total of 10,000 events with a FACS Calibur. Analyze the results with FlowJo 10.1 software. Several controls were performed and are listed in the following table. (Table 5)

[0270] 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

[0271]

[0272] Example 5: Functional studies

[0273] ADCC assay of αGITR-αPDL1 with CH2 on GITR+CF2 cells

[0274] The antibody-dependent cell-mediated cytotoxicity of αGITR-αPD-L1 with CH2 on GITR+CF2 cells was analyzed using an ADCC Reporter Gene Bioluminescence Assay Kit (WIL2-S) (Promega) and performed according to the manufacturer's protocol. The aim was to test the ADCC of αGITR10-αPD-L1 with CH2. The assay was performed using ADCC reporter cells (WIL2-S) with an Fcγ receptor and a luciferase gene driven by a response element.

[0275] The cells GITR + CF2 cells and GITR - CF2 in 75 cm 2Grown in flasks (Cellstar) until they reached approximately 80% confluence. They were detached by adding 1:10 diluted 0.25% trypsin-EDTA (Life Technologies) in PBS and their viability was tested. GITR + CF2 cells were used as target cells and plated at a density of 2x10 cells per well diluted in RPMI 1640 medium (Life 4 serum-free) in 96-well cell culture plates (PerkinElmer). 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. In a concentration-dependent manner, starting at 20 mg / mL (highest concentration), then 2 mg / mL, 0.2 mg / mL, and 0.02 mg / mL (1:10 serial dilutions) respectively, four antibodies were added and incubated at room temperature for 5 minutes. After incubation, effector cells WIL2-S were suspended in ADCC assay medium and added to the target cell / antibody mixture at a density of 10x10 6 cells per well, and the effector-to-target cell ratio was set to 5:1 (E / T). After incubation at 37 (5% CO 2 2) for approximately 6 hours, an equal volume of Bio-Glo luciferase assay reagent (Promega) was added to the wells and incubated (room temperature, 10 minutes). The luminescence of the cells was measured using a Polarstar Omega. The assays were performed in triplicate. All data were plotted using Excel.

[0276] GITR + CDC assay of αGITR-α-PDL1 with CH2 on GITR

[0277] To test the complement-dependent cytotoxicity (CDC) of αGITR10-α-PDL1 tBsAb with CH2, the CellTox Green dye (Promega) that binds to the DNA contained in cells was used in the CellTox TM Green Cytotoxicity Assay (Promega) using young rabbit complement (Cedarlane Laboratories). The fluorescence signal generated by the binding of the dye to the DNA of dead cells is proportional to the cytotoxicity. The assay was performed according to the manufacturer's protocol. The experimental procedures and settings for testing complement-dependent cytotoxicity were similar to the above CDC test, except that CellTox TMAssays were developed and analyzed using the Green Cytotoxicity Assay (Promega). Antibodies testing complement-dependent cytotoxicity were αGITR10-αPDL1 and αGITR10-α-PDL1 tetramer bispecific antibodies (tBsAb) with CH2. αGITR mAb was used as a positive control, and F10-αPD-L1 was used as a negative control.

[0278] After incubation at 37 (5% CO 2 ) for approximately 4 hours, 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. The assays were performed in triplicate three times. All data were plotted using Excel.

[0279] Example 6: Isolation and Characterization of αGITR-αPD-L1 BsAb

[0280] Generation of Expression Vectors

[0281] A total of six vectors (αGITR-αPD-L1) were constructed to generate the desired tBsAb and three additional vectors to generate control Abs (αGITR1-F10, αGITR10-F10, and F10-αPD-L1). Expression vectors were generated according to the above cloning strategy.

[0282] The recipient expression vector pcDNA3.4 and all donor vectors (6 V H GITR-linker-V L GITR inserts and 1 V H F10-V L F10 inserts) were digested with SfiI and NotI restriction enzymes, and the fragments were separated on a 1% agarose gel and stained with ethidium bromide. The seven digestion patterns of SfiI and NotI were consistent with the theoretical calculated values. The digested recipient vector pcDNA3.4 vector contained 7500 bp and could be detected at the correct level of the ladder (lane; 8000 bp). The smaller fragment in lane 1 was shown to be between 500 and 1000 bp and corresponded to the V H X -linker-V L X . The GITR inserts (lanes 2-6) and F10 inserts (lane 7) were clustered between 500 and 1000 bp. The larger bands observed at the 8000 bp (lanes 2-7) level represented the corresponding progeny vectors.

[0283] Two additional control plasmids (2) and (3) were constructed. The recipient expression vectors pcDNA3.4 encoding αGITR1-αPDL1 and αGITR10-αPDL1scFv were digested with BsiWI and BamHI Res to replace the V H F10-linker-V L F10 fragment for V H PD-L1-linker-V L PD-L1 fragment. To isolate the V H F10-linker-V L F10 fragment from the pcDNA3.1 donor vector, forward and reverse primers (No. 1 and No. 2) containing BsiWI and BamHI restriction sites were designed. After isolating the cDNA using PCR, it was digested with BsiWI and BamHI RE. Gel analysis of all three digests was consistent with the theoretical numbers. As expected, PCR of the F10 fragment showed only one band at the correct position relative to the ladder. The two digested recipient vectors (containing V H GITR1-V L GITR1 or V H GITR10-V L GITR10) were approximately 8000 bp in size and matched the theoretical size of the vector (7500 bp).

[0284] All digested fragments were extracted and purified from the agarose gel and corresponding ligation reactions were performed. The resulting plasmids were successfully constructed and confirmed by sequencing (Genewiz).

[0285] Expression of GITR-PDL1 bispecific antibody and αGITR-IgG

[0286] αGITR-αPD-L1 protein was expressed in 293F HEK cells and isolated by Ni-NTA purification. αGITR IgG protein was expressed in HEK 293F cells and isolated by protein A purification. The yields measured by NanoDrop spectrophotometer are listed in Table 6.

[0287] Table 6 | Antibody yields expressed in 293F HEK

[0288]

[0289] SDS-PAGE analysis

[0290] The purity of tBsAbαGITR1-αPDL1, αGITR10-αPDL1, αGITR11-αPDL1, αGITR14-αPDLl, αGITR15-αPDL1, αGITR17-αPDL1 and F10-αPD-L1 was analyzed by SDS-PAGE. Protein samples between 3 μg - 5 μg were loaded onto the gel, electrophoretically separated and stained with Coomassie blue.

[0291] Notably, under non-reducing conditions, there were two bands that particularly caught attention. The upper band was in the range of 115 kDa and 140 kDa. The quantitative predominance of this band in each protein profile and its apparent molecular size close to that of the αGITR-αPD-L1 tetrameric bispecific antibody (tBsAb) (130 kDa) indicated successful antibody production. The lower band was between 70 and 80 kDa and could thus be interpreted as a large amount of monomeric tandem scFv (65 kDa). In addition, some weaker bands above 140 kDa were observable, indicating aggregate formation.

[0292] Under reducing conditions, only one band was observable between 70 and 80 kDa, indicating the reduction of the disulfide bonds of the tBsAb to tandem scFv (65 kDa). The deviation in the apparent molecular weight from the theoretical calculated value could stem from post-translational modifications such as glycosylation and phosphorylation, as well as protein conformation while it was passing through SDS PAGE. The difference in the amount loaded onto the gel could account for the difference in band intensity between αGITR-αPD-L1 tBsAbs.

[0293] In addition, the purity of αGITR-IgG was also analyzed by SDS-PAGE. Under non-reducing conditions, the analysis showed one band with an apparent molecular weight of 140 kDa, and approximately equal to the theoretically calculated molecular weight of αGITR IgG (150 kDa). Reducing SDS analysis revealed two bands, which suggested successful disulfide bond reduction of αGITR IgG, thus producing a heavy chain (50 kDa) and a light chain (25 kDa).

[0294] Direct ELISA of αGITR-αPD-L1 BsAb on passively adsorbed PD-L1 antigen

[0295] Direct ELISA of αGITR-αPD-L1 BsAb was performed to characterize their reactivity against the PD-L1 antigen. As Figure 19As shown, reactivity to the PD-L1 antigen was observed in all αGITR-αPD-L1 tBsAbs, and no non-specific sticking to CCR4 was 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. In addition, the absorbance values of αGITR-αPD-L1 tBsAb binding were comparable to those of commercial αPD-L1 mAb, and the intensity signal decreased at lower concentrations. ELISA did not show saturation at higher concentrations and had a very weak signal at concentrations below 0.01 mg / mL.

[0296] Cell-based ELISA of αGITR-αPD-L1 tBsAb on GITR+CF2

[0297] In previous studies of αGITR IgG, αGITR1 IgG and αGITR10 IgG were shown to have the best characteristics, which is the reason for the project here to narrow down the following experiments to αGITR10-αPD-L1 and αGITR1-αPD-L1 tBsAbs. Cell-based enzyme-linked immunosorbent assay (ELISA) was used to test different concentrations of αGITR1-αPD-L1 and αGITR10-αPD-L1 against GITR+CF2 cells to analyze their reactivity. As Figure 20 shown, reactivity to GITR+CF2 was observed for 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; then they gradually weakened as the concentration decreased.

[0298] At all concentrations, the signal intensity of αGITR10-αPD-L1 was better than that of αGITR1-αPD-L1. Unexpectedly, the negative control F10-αPD-L1 antibody not only showed absorbance but also seemed to behave in a concentration-dependent manner. For αGITR1-αPD-L1 and F10-αPD-L1, no readout signal was detected below the threshold of 0.1235 mg / mL. In summary, the standard deviation of the mean was abnormally high.

[0299] Due to the unexpected results of the previous ELISA (see Figure 20), Repeated experiments. The settings were kept the same, except that GITR+CF2 cells were fixed with 8% paraformaldehyde instead of the acetone-methanol solution. The results of this second method 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 still depended on the concentration used. When incubated with GITR-CF2 cells, the tBsAb did not show binding. See Figure 32 .

[0300] A third cell-based ELISA was performed to compare the αGITR10-αPD-L1 antibody with commercial αGITR IgG. Reactivity of both antibodies was observed in GITR+CF2 cells ( Figure 22 ), but not in GITR-CF2 cells (see Figure 33 ). Again, the results of αGITR10-αPD-L1 matched the previously recorded data. At all concentrations, the signal intensity of the αGITR mAb was superior to that of the αGITR10-αPD-L1. Unexpectedly, saturation of the signal readout might not be 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 .

[0301] Flow cytometry analysis of αGITR-αPD-L1 BsAb on GITR+ cells

[0302] Flow cytometry analysis was performed to evaluate the binding of the αGITR1-αPD-L1 and αGITR10-αPD-L1 antibodies to GITR+CF2 cells (Figures 23 and 24). The results showed that both antibodies (co-stained with APC-labeled His-tag Alexa Fluor 488) could specifically bind to GITR+CF2. In addition, the tBsAb did not have reactivity against GITR-CF2 ( Figure 34 ). Note that some non-specific binding was caused by the secondary antibody as shown in the control ( Figure 34 ). Comparison of the two antibodies with each other showed that they exhibited similar binding under the same conditions. Therefore, only the αGITR10-αPD-L1 tBsAb was selected for further characterization. Standard measurements of αGITR1 IgG and αGITR10 IgG revealed similar binding characteristics when compared with the tBsAb.

[0303] Example 7: Isolation and Characterization of αGITR-αPD-L1 bsAb with CH2

[0304] Generation of Bacterial Expression Vectors

[0305] In previous studies, αGITR10 mAb was shown to exhibit the best characteristics, which was the reason for choosing αGITR10-αPD-L1 as the expression vector for engineering a new construct containing the CH2 domain. Vectors were generated according to the above cloning strategy, resulting in the gene order V H GITR-Linker-V L GITR-Linker-Hinge-CH2-Linker-V H PD-L1-Linker-V L PD-L1.

[0306] Site-directed mutagenesis enabled the introduction of a HindIII restriction site into the recipient pcDNA 3.4 vector between the IgG1 hinge region and the linker (GGGGS) 6 After transformation into Escherichia coli strain super-competent cells, 16 clones were picked and then DNA purification was performed. Restriction enzyme digestion analysis using HindIII and BamHI restriction enzymes was shown on a 1% agarose gel to test for the correct introduction of the HindIII restriction site (see Figure 25 ). Among the 16 clones, only clone No. 10 showed two bands. The size of the smaller band that aggregated between 500 and 1000 bp corresponded to the theoretical size (800 bp) of the HindIII and BamHI digestion. Since HindIII and BamHI represent unique restriction sites in the plasmid, this result indicated that HindIII was successfully introduced into the cellular DNA of clone No. 10.

[0307] Additional gel analysis was performed on clone No. 10 to compare it with the original GITR10-PDL1 (without the HindIII restriction site); see Figure 26Clone 10 (GITR10-PDL1 with HindIII) and GITR10-PDL1 (without HindIII) each underwent three digestions separately. The first digestion was performed only with the HindIII restriction enzyme, the second digestion was performed only with the NotI restriction enzyme, and the third digestion was performed with both the HindIII and NotI restriction enzymes. Digestion of Clone 10 with a single enzyme produced an open circular conformation and aggregated around 8000 bp. In contrast, double digestion of Clone 10 with the HindIII and NotI restriction enzymes produced two bands. The lower band aggregated below 500 bp and corresponded to the theoretical calculated value of the HindIII / NotI digestion fragment (117 bp). The αGITR10-αPD-L1 plasmid does not contain a HindIII restriction site, which is the reason for the gel analysis of the single HindIII digestion revealing (as expected) supercoiled plasmid DNA. These results strongly suggest the correct introduction of the HindIII restriction site.

[0308] Sequencing results of Clone 10 (Genewiz) confirmed the correct introduction of HindIII between the hinge and linker domains. However, during site-directed mutagenesis, five of the six (GGGGS) repeat sequences in the linker were deleted. Thus, the new construct only exhibited one repeat of the linker sequence instead of six linker repeats. However, it was decided to continue plasmid construction with this newly generated plasmid, which contained the hinge region followed by a single linker repeat sequence (GGGGS).

[0309] 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 singly digested with the HindIII restriction enzyme and analyzed on a 1% agarose gel ( Figure 27 ). Both digestions produced fragment sizes that matched the theoretical calculated values: 7.5 bp for the recipient vector GITR10-PDL1 with HindIII and 350 bp for the CH2 fragment.

[0310] Thus, the pcDNA 3.4 expression vector αGITR10-αPD-L1 with CH2 was successfully constructed and confirmed by sequencing (Genewiz).

[0311] SDS-PAGE analysis

[0312] The αGITR10-αPD-L1 protein with CH2 was expressed in 293T HEK cells and purified and isolated via Ni-NTA. A total of 100 mL of culture medium yielded a protein production of 200 ng (NanoDrop analysis). The purity of the GITR10-PDL1 tBsAb with CH2 was analyzed by SDS-PAGE ( Figure 28 ). A total of 3 μg of protein sample was loaded onto the gel, electrophoretically separated, and stained with Coomassie blue. Notably, under non-reducing conditions, there were two bands that particularly caught attention. The upper band was slightly higher than 140 kDa. The quantitative predominance of this band and its apparent molecular size being close to the apparent molecular size of the αGITR10-αPD-L1 tBsAb with CH2 (150 kDa) indicated successful antibody production. The lower band had an apparent molecular weight of 80 kDa and could thus be interpreted as containing a large amount of tandem scFv with non-dimerized CH2 (75 kDa). Under reducing conditions, only one band was visible at 80 kDa, indicating that the disulfide bonds of the tBsAb were reduced to tandem scFv (75 kDa).

[0313] GITR + Cell-based ELISA of the αGITR-αPD-L1 tetravalent bispecific antibody (tBsAb) with CH2 on CF2

[0314] Cell-based enzyme-linked immunosorbent assay (ELISA) was performed to test different concentrations of αGITR10-αPD-L1 with CH2 against GITR + CF2 to analyze their signal intensities.

[0315] As Figure 29 shown, reactivity against GITR + CF2 was observable in the αGITR10-αPD-L1 antibody with CH2, while no non-specific adhesion to GITR - CF2 was noted; see Figure 35 . The OD values of the αGITR10-αPD-L1 with CH2 depended on their respective concentrations. Consistent with expectations, the strongest signal was measured at the highest concentration; then it gradually weakened as the concentration decreased. At most concentrations, the signal intensity of αGITR IgG was superior to that of the αGITR10-αPD-L1 with CH2. Unexpectedly, saturation of the signal readout might not be observed at higher concentrations. The control antibody (F10-αPD-L1) was tested at the highest concentration (5 μg / mL) and had some reactivity as previously seen ( Figure 22 and 21 ).

[0316] Example 8: Functional study of αGITR-αPD-L1 BsAb with CH2

[0317] In an initial attempt to establish functional data, the complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC) of αGITR-αPD-L1 BsAb with CH2 were tested. However, the results were inconclusive.

[0318] ADCC reporter gene assay of αGITR-αPD-L1 BsAb with CH2 on GITR+CF2

[0319] The ADCC activity of αGITR10-αPD-L1 BsAb with CH2 was tested using GITR+CF2 cells (target cells) and WIL2-S (effector cells) (E / T = 5:1). The antibody bioactivity in ADCC was quantified by luciferase produced as a result of NFAT pathway activation, and its activity in effector cells was quantified with a luminescence readout. In the ADCC assay, αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2 showed unexpected results ( Figure 30 ). Compared with the target cells and effector cells alone, the negative control F10-αPD-L1 showed a similar signal intensity for ADCC and was unbiased for variable concentrations. On the other hand, the positive control αGITR IgG showed an increased value at higher concentrations as expected. Unexpectedly, for αGITR10-αPD-L1 and αGITR10-αPD-L1 with CH2, the ADCC signal intensity decreased at higher concentrations and was significantly lower than the signal of only target cells and effector cells at 20 μg / mL tBsAb.

[0320] The ADCC activity of ααGITR10-αPD-L1 with CH2 was measured at variable concentrations. All antibodies were serially diluted (1:2), starting at the highest concentration of 20 mg / mL down to 0.02 mg / mL, and tested against 20,000 GITR+CF2 cells per well. The ratio of effector cells (GITR+CF2) to target cells (Wils-2) was 5:1. αGITR IgG represented the positive control, and F10-αPD-L1 represented the negative control. The vertical axis represents the raw value of luciferase activity in effector cells quantified with a luminescence readout. Each sample was run in triplicate at each concentration; the standard deviation of the mean is shown in parentheses. The background of GITR+CF2 cells in RPMI medium was subtracted from the values obtained.

[0321] CDC assay of αGITR10-αPD-L1 BsAb with CH2 on GITR+CF2 cells

[0322] The complement-dependent cytotoxicity of the αGITR10-αPD-L1 antibody with CH2 against GITR-expressing CF2 cells was tested by measuring the amount of fluorescent CellTox Green bound to the incorporated DNA. The percentage of lysis was calculated as the ratio of the signal intensity obtained from the sample to the signal intensity from fully lysed GITR+CF2 cells ( Figure 31 ).

[0323] The negative control F10-αPD-L1 BsAb exhibited a cytotoxicity percentage similar to that of the positive control αGITR IgG. The αGITR10-αPD-L1 with CH2 showed similar cytotoxicity levels at all concentrations between 65% and 70% and did not appear to be concentration-dependent. Neither of the two measured antibodies had a significantly higher percentage of cytotoxicity. These findings largely contradicted the expected results; a possible reason for these inconsistencies was the potential low viability of the GITR+CF2 cells used.

[0324] Other embodiments

[0325] While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative rather than limiting the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A tetravalent antibody molecule, wherein the tetravalent antibody molecule is a dimer of bispecific scFv fragments, wherein, The bispecific scFv fragment comprises a first binding site for a first antigen, a second binding site for a second antigen, and a linker-hinge-linker domain, wherein the first binding site and the second binding site are linked together via the linker-hinge-linker domain, wherein the first binding site comprises an antibody or antibody fragment specific for GITR, the second binding site comprises an antibody or antibody fragment specific for PD-L1, wherein the linker-hinge-linker domain consists of an immunoglobulin hinge region amino acid sequence according to SEQ ID NO: 1902 flanked by linker amino acid sequences at both ends, and wherein the linker amino acid sequence independently comprises the amino acid sequence (GGGS) 1 , (GGGS) 4 or (GGGS) 6 , and wherein the tetravalent antibody molecule does not comprise an antibody constant region or a portion thereof; wherein the antibody or antibody fragment specific for GITR is a sequence having the VH amino acid sequence of SEQ ID NO: 1378 and the VL amino acid sequence of SEQ ID NO: 1380; the antibody or antibody fragment specific for PD-L1 is a sequence having the VH amino acid sequence of SEQ ID NO: 1510 and the VL amino acid sequence of SEQ ID NO: 1512.

2. The tetravalent antibody molecule according to claim 1, wherein the scFv fragment is a tandem scFv.

3. A nucleic acid construct for obtaining the tetravalent antibody molecule according to any one of claims 1 to 2, which comprises a nucleic acid molecule encoding: the light chain variable region and the heavy chain variable region of an antibody that can specifically bind to a first antigen, wherein, the first antigen includes GITR, wherein the light chain variable region has the VL amino acid sequence of SEQ ID NO: 1380, and the heavy chain variable region has the VH amino acid sequence of SEQ ID NO: 1378; the light chain variable region and the heavy chain variable region of an antibody that can specifically bind to a second antigen, wherein the second antigen includes PD-L1, wherein the light chain variable region has the VL amino acid sequence of SEQ ID NO: 1512, and the heavy chain variable region has the VH amino acid sequence of SEQ ID NO: 1510; and Linker-Hinge-Linker domain, wherein the Linker-Hinge-Linker domain consists of an immunoglobulin hinge region amino acid sequence according to SEQ ID NO: 1902 flanked by linker amino acid sequences at both ends, wherein the linker amino acid sequences independently comprise the amino acid sequence (GGGS) 1 、(GGGS) 4 or (GGGS) 6 , and wherein the tetravalent antibody molecule does not comprise an antibody constant region or a portion thereof.

4. A vector comprising the nucleic acid construct according to claim 3.

5. A host cell comprising the vector according to claim 4.

6. The host cell according to claim 5, wherein the cell is a T cell, a B cell, or an NK cell.

7. The host cell according to claim 6, wherein the T cell is a follicular T cell.

8. A chimeric antigen receptor, the chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, the extracellular domain comprising the tetravalent antibody molecule according to claim 1.

9. The chimeric antigen receptor according to claim 8, wherein the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain.

10. The chimeric antigen receptor according to claim 8, wherein the transmembrane domain comprises CD28.

11. The chimeric antigen receptor according to claim 8, which further comprises one or more additional co-stimulatory molecules located between the transmembrane domain and the intracellular signaling domain.

12. The chimeric antigen receptor according to claim 11, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40.

13. The chimeric antigen receptor according to claim 8, wherein the intracellular signaling domain comprises the CD3ζ chain.

14. A genetically engineered cell that expresses and carries on its cell surface membrane the chimeric antigen receptor according to any one of claims 8-13.

15. The genetically engineered cell according to claim 14, wherein the cell is a T cell or an NK cell.

16. The genetically engineered cell according to claim 15, wherein the T cell is CD4+ or CD8+.

17. The genetically engineered cell according to claim 16, which comprises a mixed population of CD4+ and CD8+ cells.

18. Use of a tetravalent antibody molecule according to any one of claims 1 to 2 in the preparation of a medicament for the treatment of a disease or disorder selected from glioblastoma (GBM), amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease.

19. The use according to claim 18, wherein the tetravalent antibody molecule recognizes and / or binds to a CNS transport receptor.

20. The use according to claim 19, wherein the CNS transport receptor is transferrin receptor (TfR), VCAM-1, CD98hc, or insulin receptor.

21. The use according to claim 18, wherein the tetravalent antibody molecule enhances transport across the blood-brain barrier.

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