Anti-alpha-4-beta-7 antibody

Abstract

To provide anti-α4β7 antibodies that bind human α4β7, methods of making them, and uses thereof to treat patients with HIV infection.SOLUTION: An anti-human α4β7 antibody comprises (i) a VH chain region comprising three CDRs having respective specific amino acid sequences; and (ii) a VL chain region comprising three CDRs having respective specific amino acid sequences.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on July 14, 2021, is named 483WO_SL.txt, and is 106,797 bytes in size.

[0002] This application relates, inter alia, to novel anti-α4β7 antibodies, polynucleotides encoding the antibodies, methods of producing the antibodies, and methods of using these antibodies. [Background technology]

[0003] Today, more than 37 million people worldwide are infected with the human immunodeficiency virus (HIV), and the epidemic continues to grow. Significant advances in HIV management have been made with the advent of combination antiretroviral therapy (cART). cART must be taken consistently throughout the life of a person living with HIV. cART has a risk-benefit margin and, furthermore, does not reduce the HIV latent viral reservoir. There is a critical need for improved treatments that can maintain undetectable viral loads without requiring lifelong treatment.

[0004] During acute human HIV infection, both high levels of viral replication and the profound depletion of infected CD4+ T cells are thought to play central roles in the development of immunodeficiency associated with HIV infection. α4β7 is a gut-homing integrin expressed on T cells (CD4+ or CD8+), B cells, and other immune cells, and plays an important role in the pathogenesis of HIV infection. α4β7 has also been reported to be incorporated into the HIV envelope upon budding of virions from infected host cells (Guzzo et al., Sci. Immunol., 2017).

[0005] The α4β7 integrin is a heterodimeric receptor expressed on T cell subsets, B cells, NK cells, and other immune cells. This integrin mediates lymphocyte trafficking into the gut-associated lymphoid tissue (GALT) by binding to its ligand, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which is expressed on endothelial venules of the intestinal mucosa. High α4β7-expressing CD4+ T cells are targets of HIV infection in vitro (Cicala et al., PNAS 2009), and these cells become infected and therefore depleted during acute HIV infection (Sivro et al., Sci Transl Med 2018). α4β7, present on HIV-infected CD4+ cells as well as on HIV virions, is thought to mediate their trafficking into the GALT (Guzzo et al., Sci Immunol 2017). CD4+ T cells homing to the GALT constitute the largest HIV reservoir in the body, even during antiretroviral therapy (Brenchley and Douek, Mucosal Immunol 2008). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Guzzo et al., Sci. Immunol., 2017 [Non-patent document 2] Cicala et al., PNAS 2009 [Non-patent document 3] Sivro et al., Sci Transl Med 2018 [Non-patent document 4] Brenchley and Douek, Mucosal Immunol 2008 Summary of the Invention [Means for solving the problem]

[0007] (Summary of the Invention) The present disclosure provides anti-α4β7 antibodies and binding fragments thereof that specifically bind to human α4β7. The amino acid sequences of exemplary CDRs of the heavy and light chains of exemplary anti-α4β7 antibodies, as well as V H and V L The amino acid sequences of the regions are provided below in the detailed description.

[0008] Polynucleotides comprising nucleotide sequences encoding the disclosed anti-α4β7 antibodies are provided herein, as are vectors comprising the polynucleotides. Additionally, prokaryotic cells transformed with vectors comprising nucleotide sequences encoding the disclosed anti-α4β7 antibodies, eukaryotic cells transfected with the vectors, and eukaryotic (e.g., mammalian) host cells engineered to express the nucleotide sequences are provided herein. Methods for producing the antibodies by culturing the host cells and recovering the antibodies are also provided.

[0009] The present disclosure provides methods of treating a subject, such as a human subject, diagnosed with HIV infection with an anti-α4β7 antibody. The methods generally include administering to the subject an amount of an anti-α4β7 antibody described herein effective to provide a therapeutic effect. The subject may be diagnosed with any clinical category of HIV infection.

[0010] Because the anti-α4β7 antibodies described herein target human α4β7 instead of viral proteins, they offer an advantageous therapeutic approach that does not induce HIV mutation-based resistance mechanisms, which often arise from treatments that target viral proteins due to the high mutation rate of HIV.

[0011] Based on the data presented herein, the anti-α4β7 antibodies described herein are expected to provide therapeutic benefit to subjects diagnosed with HIV infection. [Brief explanation of the drawings]

[0012] [Figure 1] Blocking of HuT78 cell adhesion to MAdCAM-1 by mouse antibody Ab-ml. [Figure 2] Figure 1 shows functional cynomolgus cross-reactivity of Ab-ml using a CHOK1-cα4β7 (cyno α4β7) cell adhesion assay to MAdCAM-1. [Figure 3] Ab-ml blocking of human MAdCAM-1 binding to primary human CD4+ memory T cells. [Figure 4] Binding of mouse-human chimeric Ab-c1 to human primary CD4+ memory T cells (hα4β7+CD4+CD45RO+) is shown. [Figure 5] Figure 1 shows binding of the liability-engineered humanized anti-α4β7 Ab-h1.9 scFv clone to the human α4β7 antigen displayed on yeast by flow cytometry. Clone Ab-h1.9(ae) had similar binding to the parental Ab-h1.9. [Figure 6A] Analysis of α4β7 expression in samples from 45 HIV+ individuals and 10 healthy (HIV-) donors is shown. α4β7 expression on CD4+ and CD8+ T cells (as percentages or as levels measured by MESF) was compared between HIV+ and HIV- individuals. Only comparisons significantly different by a two-tailed Mann-Whitney test are shown. T cell populations are abbreviated as follows: N+ = naive (CD28+CD45RO-), CM = central memory (CD28+CD45RO+CCR7+), TM = transient memory (CD28+CD45RO+CCR7-), EM = effector memory (CD28-CD45RO+), TE = terminal effector (CD28-CD45RO-); MESF = molecule of isosoluble fluorochrome. [Figure 6B]Analysis of α4β7 expression in samples from 45 HIV+ individuals and 10 healthy (HIV-) donors is shown. α4β7 expression on CD4+ and CD8+ T cells (as percentages or as levels measured by MESF) was compared between HIV+ and HIV- individuals. Only comparisons significantly different by a two-tailed Mann-Whitney test are shown. T cell populations are abbreviated as follows: N+ = naive (CD28+CD45RO-), CM = central memory (CD28+CD45RO+CCR7+), TM = transient memory (CD28+CD45RO+CCR7-), EM = effector memory (CD28-CD45RO+), TE = terminal effector (CD28-CD45RO-); MESF = molecule of isosoluble fluorochrome. [Figure 6C] Analysis of α4β7 expression in samples from 45 HIV+ individuals and 10 healthy (HIV-) donors is shown. α4β7 expression on CD4+ and CD8+ T cells (as percentages or as levels measured by MESF) was compared between HIV+ and HIV- individuals. Only comparisons significantly different by a two-tailed Mann-Whitney test are shown. T cell populations are abbreviated as follows: N+ = naive (CD28+CD45RO-), CM = central memory (CD28+CD45RO+CCR7+), TM = transient memory (CD28+CD45RO+CCR7-), EM = effector memory (CD28-CD45RO+), TE = terminal effector (CD28-CD45RO-); MESF = molecule of isosoluble fluorochrome. [Figure 6D]Analysis of α4β7 expression in samples from 45 HIV+ individuals and 10 healthy (HIV-) donors is shown. α4β7 expression on CD4+ and CD8+ T cells (as percentages or as levels measured by MESF) was compared between HIV+ and HIV- individuals. Only comparisons significantly different by a two-tailed Mann-Whitney test are shown. T cell populations are abbreviated as follows: N+ = naive (CD28+CD45RO-), CM = central memory (CD28+CD45RO+CCR7+), TM = transient memory (CD28+CD45RO+CCR7-), EM = effector memory (CD28-CD45RO+), TE = terminal effector (CD28-CD45RO-); MESF = molecule of isosoluble fluorochrome. [Figure 6E] Analysis of α4β7 expression in samples from 45 HIV+ individuals and 10 healthy (HIV-) donors is shown. α4β7 expression on CD4+ and CD8+ T cells (as percentages or as levels measured by MESF) was compared between HIV+ and HIV- individuals. Only comparisons significantly different by a two-tailed Mann-Whitney test are shown. T cell populations are abbreviated as follows: N+ = naive (CD28+CD45RO-), CM = central memory (CD28+CD45RO+CCR7+), TM = transient memory (CD28+CD45RO+CCR7-), EM = effector memory (CD28-CD45RO+), TE = terminal effector (CD28-CD45RO-); MESF = molecule of isosoluble fluorochrome. [Figure 6F]Analysis of α4β7 expression in samples from 45 HIV+ individuals and 10 healthy (HIV-) donors is shown. α4β7 expression on CD4+ and CD8+ T cells (as percentages or as levels measured by MESF) was compared between HIV+ and HIV- individuals. Only comparisons significantly different by a two-tailed Mann-Whitney test are shown. T cell populations are abbreviated as follows: N+ = naive (CD28+CD45RO-), CM = central memory (CD28+CD45RO+CCR7+), TM = transient memory (CD28+CD45RO+CCR7-), EM = effector memory (CD28-CD45RO+), TE = terminal effector (CD28-CD45RO-); MESF = molecule of isosoluble fluorochrome. [Figure 7A] HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 7B]HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 7C] HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 7D]HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 7E] HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 7F]HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 7G-1] HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 7G-2]HIV virion capture using Ab-h1.9d-WT is shown. All tests were performed using a virion capture assay in a bead format. Ab-h1.9d-WT was tested with six laboratory-grown HIV strains (Figures 7A-7F) at 5 nM and 15 nM, and a negative control antibody was tested only at 15 nM. The amount of HIV p24gag in the captured samples is shown in pg / mL in 10 μL assayed. Ab-h1.9d-WT was also tested with samples from two HIV-infected individuals using beads coated with 10 μg of antibody (Figures 7G-1 and 7G-2). The amount of HIV gag RNA in the input sample (Figure 7G-1) and the captured sample (Figure 7G-2), as detected by digital droplet PCR, is shown in copies / mL. [Figure 8A] Immune complexes (HIV virions with different antibodies) binding to FcγR are shown. Immune complexes were first formed by incubating antibodies (Ab-h1.9d-WT, Ab-h1.9d with the LALA mutation that significantly reduces FcγR binding, Ab-Vedo, and an isotype-negative control) with HIV NL4-3 and then captured on FcγR immobilized on a plate. FcγRI and FcγRIIIa (V158) were captured on nickel plates, while FcγRIIa (H131), FcγRIIa (R131), and FcγRIIIa (F158) were captured on neutravidin plates to increase detection sensitivity. The amount of HIV p24 gag detected is shown in pg / mL in the 10 μL assay. Results from a representative experiment are shown. [Figure 8B]Immune complexes (HIV virions with different antibodies) binding to FcγR are shown. Immune complexes were first formed by incubating antibodies (Ab-h1.9d-WT, Ab-h1.9d with the LALA mutation that significantly reduces FcγR binding, Ab-Vedo, and an isotype-negative control) with HIV NL4-3 and then captured on FcγR immobilized on a plate. FcγRI and FcγRIIIa (V158) were captured on nickel plates, while FcγRIIa (H131), FcγRIIa (R131), and FcγRIIIa (F158) were captured on neutravidin plates to increase detection sensitivity. The amount of HIV p24 gag detected is shown in pg / mL in the 10 μL assay. Results from a representative experiment are shown. [Figure 8C] Immune complexes (HIV virions with different antibodies) binding to FcγR are shown. Immune complexes were first formed by incubating antibodies (Ab-h1.9d-WT, Ab-h1.9d with the LALA mutation that significantly reduces FcγR binding, Ab-Vedo, and an isotype-negative control) with HIV NL4-3 and then captured on FcγR immobilized on a plate. FcγRI and FcγRIIIa (V158) were captured on nickel plates, while FcγRIIa (H131), FcγRIIa (R131), and FcγRIIIa (F158) were captured on neutravidin plates to increase detection sensitivity. The amount of HIV p24 gag detected is shown in pg / mL in the 10 μL assay. Results from a representative experiment are shown. [Figure 8D]Immune complexes (HIV virions with different antibodies) binding to FcγR are shown. Immune complexes were first formed by incubating antibodies (Ab-h1.9d-WT, Ab-h1.9d with the LALA mutation that significantly reduces FcγR binding, Ab-Vedo, and an isotype-negative control) with HIV NL4-3 and then captured on FcγR immobilized on a plate. FcγRI and FcγRIIIa (V158) were captured on nickel plates, while FcγRIIa (H131), FcγRIIa (R131), and FcγRIIIa (F158) were captured on neutravidin plates to increase detection sensitivity. The amount of HIV p24 gag detected is shown in pg / mL in the 10 μL assay. Results from a representative experiment are shown. [Figure 8E] Immune complexes (HIV virions with different antibodies) binding to FcγR are shown. Immune complexes were first formed by incubating antibodies (Ab-h1.9d-WT, Ab-h1.9d with the LALA mutation that significantly reduces FcγR binding, Ab-Vedo, and an isotype-negative control) with HIV NL4-3 and then captured on FcγR immobilized on a plate. FcγRI and FcγRIIIa (V158) were captured on nickel plates, while FcγRIIa (H131), FcγRIIa (R131), and FcγRIIIa (F158) were captured on neutravidin plates to increase detection sensitivity. The amount of HIV p24 gag detected is shown in pg / mL in the 10 μL assay. Results from a representative experiment are shown. [Figure 9A] Figure 9A shows the α4β7- and Fc-dependence of Ab-h1.9d-WT-mediated uptake of α4β7-coated beads in THP-1 cells. Phagocytosis scores of immune complexes (containing α4β7-coated beads and the indicated anti-α4β7 or control antibodies) in THP-1 cells treated with the complexes for 3 hours are plotted. Figure 9A shows data from one representative experiment. [Figure 9B]Figure 9B shows the α4β7- and Fc-dependence of Ab-h1.9d-WT-mediated uptake of α4β7-coated beads in THP-1 cells. Phagocytosis scores of immune complexes (α4β7-coated beads and the indicated anti-α4β7 or control antibodies) in THP-1 cells treated with the complexes for 3 hours are plotted. Figure 9B shows normalized data from three independent experiments. Significance was determined using one-way ANOVA coupled with Tukey's multiple comparison test. ****p<0.0001, ***p=0.0001-0.001, **p=0.001-0.01, *p=0.01-0.05, ns≧0.05. [Figure 9C] Figure 9C shows the α4β7- and Fc-dependence of Ab-h1.9d-WT-mediated uptake of α4β7-coated beads in THP-1 cells. Phagocytosis scores of immune complexes (α4β7-coated beads and the indicated anti-α4β7 or control antibodies) in THP-1 cells treated with the complexes for 3 hours are plotted. Figure 9D shows representative images of Ab-h1.9d-WT immune complex-treated cells with three internalized α4β7-coated beads obtained by image cytometry. [Figure 10] Figure 1 shows the binding of anti-α4β7 antibodies to α4β7+GFP+VLPs (virus-like particles). Ab binding to VLPs was determined using ELISA. Ab-h1.9d-WT, Ab-h1.9d-LALA, and Ab-Vedo bound to α4β7+GFP+VLP-coated plates with similar EC50 values. Representative data from two independent experiments are shown. [Figure 11A] Figure 11A shows the α4β7- and Fc-dependence of Ab-h1.9d-WT-mediated α4β7+GFP+VLP (virus-like particle) uptake by THP-1 cells. Figure 11A shows α4β7+GFP+VLP uptake by THP-1 cells as a percentage of GFP+ cells as measured by flow cytometry. The mean ± SD presented in Figures 10A-10B was collected from four and three independent experiments, respectively. Significance (****p=5.4×10-5) was calculated by two-tailed Student's t-test, with p<0.05 considered significant. [Figure 11B]Figure 11B shows the α4β7- and Fc-dependence of Ab-h1.9d-WT-mediated α4β7+GFP+VLP (virus-like particle) uptake by THP-1 cells. Figure 11B shows the inhibition of α4β7+GFP+VLP uptake by latrunculin A (LatA). In Figure 11B, THP-1 cells were pretreated with LatA for 2 hours and then incubated with VLPs and antibodies as in Figure 11A. [Figure 12A] Figure 12 shows the inhibition of HIV gp120 interaction with α4β7 by different antibodies. Figure 12A shows the binding of RPMI 8866 cells to the HIV gp120-V2 WT peptide but not to the control peptide. RPMI 8866 cells constitutively express α4β7 on the cell surface. The HIV gp120-V2 WT peptide and the HIV gp120-V2 control peptide were identical in sequence except for the four amino acids reported to mediate binding between α4β7 and gp120, which were mutated in the control peptide. [Figure 12B] Figure 12B shows the inhibition of HIV gp120 interaction with α4β7 by different antibodies. Figure 12B shows the inhibition of HIV gp120 peptide binding to RPMI 8866 cells expressing α4β7 by different antibodies. Ab-h1.9d-WT was more potent than Ab-Vedo at inhibiting the binding of HIV gp120-V2 WT peptide to RPMI 8866 cells expressing α4β7. [Figure 13] The percentages of human and cynomolgus monkey CD4+ and CD8+ T cell subsets bound by Ab-h1.9d-WT are shown. Binding of Ab-h1.9d-WT to human and cynomolgus monkey CD4+ and CD8+ T cells was assessed by flow cytometry analysis. The percentages of α4β7+ T cell subsets bound by Ab-h1.9d-WT were determined. Data were obtained from three human and five cynomolgus donors. [Figure 14A]Figure 1 shows the binding specificity of Ab-h1.9d-WT to various integrins. The binding specificity of Ab-h1.9d-WT was evaluated on recombinant cells expressing human (14A, 14C, and 14E) or cynomolgus monkey integrins (14B, 14D, and 14F). Binding to the target integrin α4β7 (Figures 14A / 14B) compared with α4β1 (Figures 14C / 14D) and αEβ7 (Figures 14E / 14F) integrins. Ab-Nata and etrolizumab-derived Ab-Etro were used as α4 and β7 integrin-specific positive controls, respectively, and Ab-Ctet was used as an isotype control. FACS binding results are expressed as MFI for the titrated mAbs. N=1. [Figure 14B] Figure 1 shows the binding specificity of Ab-h1.9d-WT to various integrins. The binding specificity of Ab-h1.9d-WT was evaluated on recombinant cells expressing human (14A, 14C, and 14E) or cynomolgus monkey integrins (14B, 14D, and 14F). Binding to the target integrin α4β7 (Figures 14A / 14B) compared with α4β1 (Figures 14C / 14D) and αEβ7 (Figures 14E / 14F) integrins. Ab-Nata and etrolizumab-derived Ab-Etro were used as α4 and β7 integrin-specific positive controls, respectively, and Ab-Ctet was used as an isotype control. FACS binding results are expressed as MFI for the titrated mAbs. N=1. [Figure 14C] Figure 1 shows the binding specificity of Ab-h1.9d-WT to various integrins. The binding specificity of Ab-h1.9d-WT was evaluated on recombinant cells expressing human (14A, 14C, and 14E) or cynomolgus monkey integrins (14B, 14D, and 14F). Binding to the target integrin α4β7 (Figures 14A / 14B) compared with α4β1 (Figures 14C / 14D) and αEβ7 (Figures 14E / 14F) integrins. Ab-Nata and etrolizumab-derived Ab-Etro were used as α4 and β7 integrin-specific positive controls, respectively, and Ab-Ctet was used as an isotype control. FACS binding results are expressed as MFI for the titrated mAbs. N=1. [Figure 14D] Figure 1 shows the binding specificity of Ab-h1.9d-WT to various integrins. The binding specificity of Ab-h1.9d-WT was evaluated on recombinant cells expressing human (14A, 14C, and 14E) or cynomolgus monkey integrins (14B, 14D, and 14F). Binding to the target integrin α4β7 (Figures 14A / 14B) compared with α4β1 (Figures 14C / 14D) and αEβ7 (Figures 14E / 14F) integrins. Ab-Nata and etrolizumab-derived Ab-Etro were used as α4 and β7 integrin-specific positive controls, respectively, and Ab-Ctet was used as an isotype control. FACS binding results are expressed as MFI for the titrated mAbs. N=1. [Figure 14E] Figure 1 shows the binding specificity of Ab-h1.9d-WT to various integrins. The binding specificity of Ab-h1.9d-WT was evaluated on recombinant cells expressing human (14A, 14C, and 14E) or cynomolgus monkey integrins (14B, 14D, and 14F). Binding to the target integrin α4β7 (Figures 14A / 14B) compared with α4β1 (Figures 14C / 14D) and αEβ7 (Figures 14E / 14F) integrins. Ab-Nata and etrolizumab-derived Ab-Etro were used as α4 and β7 integrin-specific positive controls, respectively, and Ab-Ctet was used as an isotype control. FACS binding results are expressed as MFI for the titrated mAbs. N=1. [Figure 14F]Figure 1 shows the binding specificity of Ab-h1.9d-WT to various integrins. The binding specificity of Ab-h1.9d-WT was evaluated on recombinant cells expressing human (14A, 14C, and 14E) or cynomolgus monkey integrins (14B, 14D, and 14F). Binding to the target integrin α4β7 (Figures 14A / 14B) compared with α4β1 (Figures 14C / 14D) and αEβ7 (Figures 14E / 14F) integrins. Ab-Nata and etrolizumab-derived Ab-Etro were used as α4 and β7 integrin-specific positive controls, respectively, and Ab-Ctet was used as an isotype control. FACS binding results are expressed as MFI for the titrated mAbs. N=1. [Figure 15] Nonspecific binding assessment of Ab-h1.9d-WT on HEK293 cells is shown. Nonspecific binding of Ab-h1.9d-WT to HEK293 cells was assessed by flow cytometry analysis. At the 100 μg / mL test concentration, the positive control showed high binding, and negligible binding was observed for both Ab-h1.9d-WT and the isotype control Ab-cTet. The percentage HEK293 cells bound by the test mAb (A) and the MFI of binding intensity by the test mAb (B) are presented. N=2. [Figure 16A] Figure 16A shows the internalization of α4β7 complexes with Ab-h1.9d-WT or Ab-Vedo on human primary cells. Figure 16B shows the internalization of Ab-h1.9d-WT on naive CD4+ T and CD8+ T cells from peripheral blood human donors. Dot plots from two donors show the percentage of α4β7 cells treated with Ab-h1.9d-WT at 4 or 37°C 18 hours after treatment. α4β7 remaining on the cell surface was detected using Alexa-647-labeled anti-β7 Ab-Etro. [Figure 16B]Figure 16B shows the internalization of α4β7 complexes with Ab-h1.9d-WT or Ab-Vedo on human primary cells. Figure 16B shows quantification of α4β7 internalization (reduced surface α4β7 expression relative to that observed after treatment at 4°C) in T cell subsets treated with Ab-h1.9d-WT (N = 2 donors). Naive cells were defined as CD45RA+. [Figure 17A] Figure 17A shows that Ab-h1.9d-WT blocks MAdCAM-1 costimulatory signals on human primary CD4+ T cells. In Figure 17A, activation of human primary CD4+ T cells by anti-CD3 and MAdCAM-1 in the presence of isotype control Ab and Ab-h1.9d-WT was measured by flow cytometry analysis as the percentage of Ki67+CD25+ cells (X-axis: Ki67+, Y-axis: CD25+). Data are from a representative donor. [Figure 17B] Figure 17B shows that Ab-h1.9d-WT blocks MAdCAM-1 costimulatory signals on human primary CD4+ T cells. In Figure 17B, activation of human primary CD4+ T cells by anti-CD3 and MAdCAM-1 in the presence of isotype control Ab, Ab-h1.9d-WT, and Ab-Vedo was measured as the percentage of Ki67+CD25+ cells (on the Y-axis). Data are from six individual healthy donors. Statistical analysis was performed using a two-tailed parametric paired t-test: **P<0.01, ****P<0.0001). [Figure 18] Figure 1 shows that Ab-h1.9d-WT does not block VCAM-1-mediated cell adhesion. Blockade of cell adhesion to VCAM-1 by Ab-h1.9d-WT was determined using the HuT78 cell adhesion assay. Ab-Nata, an anti-α4 mAb, served as a positive control, and Ab-cTet was used as a negative control. Representative data are shown, where a decrease in luminescence (RLU) indicates a decrease in cell binding due to ligand blockade. N=3. [Figure 19A]Flow cytometry shows the binding of Ab-h1.9d-WT to human FcγR-expressing cells compared to Ab-Vedo. Binding of Ab-h1.9d-WT (FIG. 19A) and Ab-Vedo (FIG. 19B) to human FcγR was analyzed using engineered CHO-K1 cells expressing various cell surface human FcγRs and is represented by the geometric mean of fluorescence. N=1. [Figure 19B] Flow cytometry shows the binding of Ab-h1.9d-WT to human FcγR-expressing cells compared to Ab-Vedo. Binding of Ab-h1.9d-WT (FIG. 19A) and Ab-Vedo (FIG. 19B) to human FcγR was analyzed using engineered CHO-K1 cells expressing various cell surface human FcγRs and is represented by the geometric mean of fluorescence. N=1. [Figure 20A] Figure 20 shows reporter-based ADCC and ADCP activity of Ab-h1.9d-WT. The ability of Ab-h1.9d-WT to induce Fc-mediated in vitro ADCC and ADCP activity was assessed by reporter assay. (Figure 20A) ADCC activity using engineered Jurkat human FcγRIIIa V158+ effector reporter cells and RPMI8866 target cells; N=2. [Figure 20B] Figure 20 shows reporter-based ADCC and ADCP activity of Ab-h1.9d-WT. The ability of Ab-h1.9d-WT to induce Fc-mediated in vitro ADCC and ADCP activity was assessed by reporter assay. (Figure 20B) ADCP activity using engineered Jurkat human FcγRIIa H131+ effector reporter cells and RPMI8866 target cells; N=2. Ab-Ritu was used as a positive control in the assay. Results are expressed as luminescence (RLU); a high signal indicates ADCC and ADCP activity. [Figure 21]Figure 1 shows the CDC activity of Ab-h1.9d-WT. The ability of Ab-h1.9d-WT to induce Fc-mediated in vitro CDC activity was assessed using RPMI8866 target cells and human serum as a source of complement factors; N = 2 donor sera tested. Ab-Ritu was used as a positive control. Results are expressed as percentage cell killing; higher cell killing indicates CDC activity. [Figure 22] Figure 1 shows the cytotoxicity-based ADCC activity of Ab-h1.9d-WT. The ability of Ab-h1.9d-WT to induce Fc-mediated in vitro ADCC was assessed in a FACS-based cytotoxicity assay using HuT78 as target cells and human primary NK cells from two donors as effector cells. Target cell killing (cytotoxicity) is expressed as the percentage ADCC for both donors. DETAILED DESCRIPTION OF THE INVENTION

[0013] Without being bound by theory, it is hypothesized that embodiments of the present invention exert viral control against HIV infection via two main mechanisms of action: 1) a Fab-dependent mechanism: blocking the interaction of α4β7 with its ligands, such as MAdCAM-1 and HIV gp120, thereby inhibiting the costimulation of CD4+ T cells mediated by the signaling of these ligands, suppressing HIV replication in these stimulated cells (Nawaz et al., Mucosal Immunol. 2018; Livia et al., PNAS. 2020), HIV infection of gastrointestinal tissues (Guzzo et al., Sci Immunol. 2017), and cell-to-cell viral spread (Arthos et al., Nat. Immunol. 2008), respectively; and 2) an Fc-dependent mechanism: anti-α4β7 mAbs bind to α4β7+ HIV virions, forming immune complexes that bind to mAbs. Through interaction of the Fc domain with FcγR on antigen-presenting cells (APCs), the viral peptides are internalized and processed, and the resulting viral peptides are subsequently presented on the surface of APCs to induce a new and durable HIV-specific immune response and suppress viral replication, inducing a "vaccination effect" (Parsons et al., Retrovirology 2018; Naranjo-Gomez et al., Curr. Opin. HIV AIDS 2019).

[0014] The α4β7 integrin is normally in a resting (inactive) state with low affinity for its ligands. Upon activation, it can bind to its ligands (e.g., MAdCAM-1 and gp120) with high affinity (Ye et al., Blood, 2012; Lertjuthaporn et al., PloS One, 2018). During HIV infection, a motif in the V2 region of HIV gp120 mimics MAdCAM-1 and can bind to α4β7 (Peachman et al., PloS One, 2015). The interaction between α4β7 and gp120 induces activation of lymphocyte function-associated antigen-1 (LFA-1), potentially inducing the formation of a virological synapse and thereby enhancing cell-to-cell transmission of HIV (Arthos et al., Nat Immunol, 2008). Cell-to-cell transmission is essential for promoting viral spread within tissues and is more important than cell-free virus for viral transmission. Upon binding to α4β7, embodiments of the present invention can reduce α4β7-mediated cell-to-cell spread of HIV by disrupting the interaction of α4β7 with gp120, induce internalization of α4β7-antibody binding complexes into cells, or inactivate α4β7. Thus, embodiments of the present invention demonstrate the ability to inhibit HIV replication and viral spread within tissues.

[0015] By targeting the α4β7 human protein instead of the viral protein, embodiments of the present invention do not induce the emergence of viral resistance mutations that are typically associated with treatments that target viral proteins due to the high mutation frequency of HIV.

[0016] 6.1. Abbreviations The antibodies described herein are, in many embodiments, described by their respective polypeptide sequences, which, unless otherwise indicated, are provided in N→C orientation.

[0017] The polynucleotides described herein are, in many embodiments, described by their respective polynucleotide sequences, unless otherwise indicated, in the 5' to 3' direction.

[0018] For polypeptide sequences, conventional three-letter or one-letter abbreviations for the genetically encoded amino acids may be used, as shown in Table 1 below.

[0019] [Table 1]

[0020] A particular sequence is defined by a structural formula that identifies amino acid residues that belong to a particular class (e.g., aliphatic, hydrophobic, etc.). As used herein, the various classes to which genetically encoded amino acids belong are shown in Table 2 below. Some amino acids may belong to more than one class. Cysteine, which contains a sulfhydryl group, and proline, which is conformationally restricted, have not been assigned a class.

[0021] [Table 2]

[0022] Abbreviations used throughout the various exemplary embodiments include those provided in Table 3 below:

[0023] [Table 3] TIFF0007765447000004.tif235166TIFF0007765447000005.tif97166

[0024] 6.2.Definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the meanings that are commonly understood by those of ordinary skill in the art.

[0025] As used herein, numbering of antibody amino acid residues is done according to the EU numbering scheme unless otherwise indicated.

[0026] 6.3. Anti-α4β7 antibody In one aspect, the present disclosure relates to an antibody that specifically binds to the α4β7 heterodimeric integrin receptor (also known as a4b7, LPAM-1, lymphocyte Peyer's patch adhesion molecule 1, and a dimer of integrin alpha-4 and integrin beta-7).

[0027] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to a particular antigen, e.g., α4β7. In some embodiments, the anti-α4β7 antibodies of the present disclosure bind to human α4β7, thereby modulating the immune system. The anti-α4β7 antibodies of the present disclosure contain complementarity-determining regions (CDRs), also known as hypervariable regions, in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As is known in the art, the amino acid positions / boundaries that define the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art. Some positions within a variable domain can be considered hybrid hypervariable positions, in that they can be determined to be within a hypervariable region under one set of criteria, while being outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in an extended hypervariable region. The present disclosure provides antibodies containing modifications at these hybrid hypervariable positions. Each naturally occurring heavy and light chain variable domain contains four FR regions connected by three CDRs, primarily in a β-sheet conformation, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antibody target binding site. See Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987).

[0028] Antibodies of the present disclosure may be polyclonal, monoclonal, genetically engineered, and / or otherwise modified in nature, including, but not limited to, chimeric antibodies, humanized antibodies, human antibodies, single-chain antibodies, etc. In various embodiments, the antibody comprises all or a portion of an antibody constant region. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. In specific embodiments, the anti-α4β7 antibodies described herein comprise IgG1. In other embodiments, the anti-α4β7 antibodies comprise IgG2. In yet other embodiments, the anti-α4β7 antibodies comprise IgG4. As used herein, the "constant region" of an antibody includes naturally occurring constant regions, allotypes or variants, such as any of T250Q, L234A, L235A, D356E, L358M, M428L and / or A431G in human IgG1.

[0029] The light constant region of an anti-α4β7 antibody can be a kappa (κ) light chain region or a lambda (λ) region. The λ light chain region can be any one of the known subtypes, such as λ1, λ2, λ3, or λ4. In some embodiments, the anti-α4β7 antibody comprises a kappa (κ) light chain region.

[0030] As used herein, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies are derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0031] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically chosen from a human immunoglobulin template.

[0032] "Humanized" forms of non-human (e.g., murine) antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.

[0033] A "human antibody" includes antibodies having the amino acid sequence of a human immunoglobulin, including antibodies isolated from a human immunoglobulin library or from animals transgenic for one or more human immunoglobulins and that do not express endogenous functional immunoglobulins. Human antibodies can be made by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0034] The anti-α4β7 antibodies of the present disclosure include full-length (intact) antibody molecules.

[0035] The anti-α4β7 antibody may be an antibody whose sequence has been modified to alter at least one constant region-mediated biological effector function. For example, in some embodiments, the anti-α4β7 antibody may be modified to reduce at least one constant region-mediated biological effector function compared to an unmodified antibody, e.g., to reduce binding to one or more Fc receptors (FcγR), such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and / or FcγRIIIb. FcγR binding may be reduced by mutating the antibody's immunoglobulin constant region segment in specific regions required for FcγR interaction (see, e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662). Reducing the FcγR binding ability of an antibody can also reduce other effector functions that depend on FcγR interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity ("ADCC").

[0036] The anti-α4β7 antibodies described herein include antibodies that have been modified to acquire or improve at least one constant region-mediated biological effector function compared to an unmodified antibody, e.g., to enhance FcγR interactions (see, e.g., U.S. Patent Application Publication No. 2006 / 0134709). For example, an anti-α4β7 antibody of the present disclosure can have a constant region that binds FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and / or FcγRIIIb with higher affinity than the corresponding unmodified constant region.

[0037] Additional substitutions that can modify FcγR binding and / or ADCC effector function of anti-α4β7 antibodies include a K322A substitution or a L234A and L235A double substitution in the Fc region. See, e.g., Hezareh et al., J. Virol., 75(24):12161-12168 (2001).

[0038] Anti-α4β7 antibodies of the present disclosure can comprise a modified (or variant) CH2 domain or entire Fc domain containing amino acid substitutions that increase binding to FcγRIIb and / or decrease binding to FcγRIIIa compared to the binding of the corresponding wild-type CH2 or Fc region. The variant CH2 or variant Fc domain can comprise one or more substitutions at positions 263, 266, 273, and 305. In some embodiments, the anti-α4β7 antibody comprises one or more substitutions relative to the wild-type CH2 domain selected from V263L, V266L, V273C, V273E, V273F, V273L, V273M, V273S, V273Y, V305K, and V305W.

[0039] Other examples of variant CH2 or variant Fc domains that can confer increased binding to FcγRIIb and / or reduced binding to FcγRIIIa compared to the binding of the corresponding wild-type CH2 or Fc region include those found in Vonderheide et al., Clin. Cancer Res., 19(5), 1035-1043 (2013), such as S267E or S267E / L328F in human IgG1.

[0040] Anti-α4β7 antibodies containing a human IgG4 constant region can contain the S228P mutation, which has been reported to prevent Fab arm exchange. See, e.g., Silva, JP et al., Journal of Biological Chemistry, 290(9), 5462-5469 (2015).

[0041] In some embodiments, anti-α4β7 antibodies include modifications that increase or decrease their binding affinity to the fetal Fc receptor, FcRn, for example, by mutating the immunoglobulin constant region segment in specific regions involved in FcRn interaction. In certain embodiments, anti-α4β7 antibodies of the IgG class are mutated so that at least one of amino acid residues 250, 314, and 428 in the heavy chain constant region is substituted, alone or in any combination thereof. For position 250, the substituted amino acid residue can be any amino acid residue other than threonine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. For position 314, the substituted amino acid residue can be any amino acid residue other than leucine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. For position 428, the substituted amino acid residue can be any amino acid residue other than methionine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. An exemplary substitution known to modify Fc effector function is the Fc substitution M428L, which may occur in combination with the Fc substitution T250Q. Additional specific combinations of suitable amino acid substitutions are identified in Table 1 of U.S. Patent No. 7,217,797. Such mutations increase binding to FcRn, protect the antibody from degradation, and extend its half-life.

[0042] Anti-α4β7 antibodies with high affinity for human α4β7 may be desirable for therapeutic and diagnostic applications. Accordingly, the present disclosure contemplates antibodies with high binding affinity for human α4β7. In specific embodiments, the anti-α4β7 antibodies bind to human α4β7 with an affinity of at least about 100 nM, but may exhibit higher affinities, e.g., at least about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or even higher. In some embodiments, the antibody binds to human α4β7 with an affinity ranging from about 1 pM to about 10 nM, from about 100 pM to about 10 nM, from about 100 pM to about 1 nM, or an affinity ranging between any of the aforementioned values.

[0043] The affinity of an anti-α4β7 antibody to human α4β7 can be determined using techniques well known in the art or described herein, such as, but not limited to, ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance, or fluorescence polarization assays.

[0044] Anti-α4β7 antibodies are generally referred to herein as (in N→C order): V H CDR#1, V H CDR#2 and V H A variable region (V) having three complementarity determining regions ("CDRs") designated CDR#3 H ), and heavy chains comprising (in N→C order) V L CDR#1, V L CDR#2 and V L A variable region (V) with three complementarity determining regions (CDRs) designated CDR#3 L ) and light chains comprising the amino acid sequences of exemplary CDRs and exemplary anti-α4β7 heavy and light chain Vs. H and V L The amino acid sequences of these exemplary CDRs and / or V regions are provided herein. Specific embodiments of anti-α4β7 antibodies include: H and / or V LSequences, as well as antibodies that compete with such antibodies for binding to human α4β7 are included.

[0045] In some embodiments, the anti-α4β7 antibody is suitable for administration to humans. In a specific embodiment, the anti-α4β7 antibody is humanized.

[0046] In some embodiments, the amino acid sequences of the CDRs of the anti-α4β7 antibody are selected from the sequences in Table 4.

[0047] [Table 4]

[0048] Specific exemplary embodiments of anti-α4β7 antibodies having the above CDRs are described herein. In some embodiments, the anti-α4β7 antibody has the CDRs of SEQ ID NOs: 12, 13, 14, 15, 16, and 17. In some embodiments, the anti-α4β7 antibody has the CDRs of SEQ ID NOs: 32, 33, 34, 35, 36, and 37. In some embodiments, the anti-α4β7 antibody has the CDRs of SEQ ID NOs: 42, 43, 44, 45, 46, and 47. In some embodiments, the anti-α4β7 antibody has the CDRs of SEQ ID NOs: 52, 53, 54, 55, 56, and 57. In some embodiments, the anti-α4β7 antibody has the CDRs of SEQ ID NOs: 62, 63, 64, 65, 66, and 67. In some embodiments, the anti-α4β7 antibody has the CDRs of SEQ ID NOs: 72, 73, 74, 75, 76, and 77. In some embodiments, the anti-α4β7 antibody has the CDRs of SEQ ID NOs: 82, 83, 84, 85, 86 and 87.

[0049] In some embodiments, the anti-α4β7 antibody is a V H Chain and V L Chains included:

[0050] [Table 5]

[0051] In some embodiments, the anti-α4β7 antibody comprises a V H chain, and V whose sequence corresponds to SEQ ID NO: 11 L In some embodiments, the anti-α4β7 antibody comprises a V chain whose sequence corresponds to any one of SEQ ID NOs: 20 or 22-23. H A V chain whose sequence corresponds to any one of SEQ ID NOs: 25 or 27 to 28 L In some embodiments, the anti-α4β7 antibody comprises a V chain whose sequence corresponds to a variant of SEQ ID NO: 21. H V, the sequence of which corresponds to the variant of SEQ ID NO: 26 L In some embodiments, the anti-α4β7 antibody comprises a V chain, the sequence of which corresponds to SEQ ID NO: 40. H chain, and V whose sequence corresponds to SEQ ID NO: 41 L In some embodiments, the anti-α4β7 antibody comprises a V chain whose sequence corresponds to SEQ ID NO: 50. H chain, and V whose sequence corresponds to SEQ ID NO: 51 L In some embodiments, the anti-α4β7 antibody comprises a V chain, the sequence of which corresponds to SEQ ID NO: 60. H chain, and V whose sequence corresponds to SEQ ID NO: 61 L In some embodiments, the anti-α4β7 antibody comprises a V chain, the sequence of which corresponds to SEQ ID NO: 70. H chain, and V whose sequence corresponds to SEQ ID NO: 71 L In some embodiments, the anti-α4β7 antibody comprises a V chain, the sequence of which corresponds to SEQ ID NO: 80. H chain, and V whose sequence corresponds to SEQ ID NO: 81 L Contains chains.

[0052] Those skilled in the art will recognize that the V H or V L It is understood that certain mutations in the sequence result in anti-α4β7 antibodies within the scope of the present disclosure. Mutations can be made to achieve the V sequence disclosed herein while retaining significant anti-α4β7 activity. H or V L The anti-α4β7 antibody may contain amino acid substitutions, additions, or deletions from the sequence. Thus, in some embodiments, the anti-α4β7 antibody has the V of any one of the antibodies shown in Table 5.H V having at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence H The anti-α4β7 antibody comprises the V sequence of any one of the antibodies shown in Table 5. H V having up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, or up to 2 mutations compared to the sequence H In some embodiments, the anti-α4β7 antibody can comprise the V sequence of any one of the antibodies shown in Table 5. H V having 5 or less, 4 or less, 3 or less, or 2 or less mutations compared to the sequence H In some embodiments, the anti-α4β7 antibody can comprise a V H In some embodiments, the V H The mutation in the sequence is V H CDR#1, V H CDR#2 or V H Located in CDR#3. In some embodiments, the anti-α4β7 antibody comprises any one of the V L V having at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence L The anti-α4β7 antibody comprises the V sequence of any one of the antibodies shown in Table 5. L V having up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, or up to 2 mutations compared to the sequence L In some embodiments, the anti-α4β7 antibody can comprise the V sequence of any one of the antibodies shown in Table 5. L V having 5 or less, 4 or less, 3 or less, or 2 or less mutations compared to the sequence LIn some embodiments, the anti-α4β7 antibody comprises a VL sequence with a single amino acid substitution. In some embodiments, the mutation in the VL sequence is located in VL CDR#1, VL CDR#2, or VL CDR#3.

[0053] In some embodiments, the anti-α4β7 antibody comprises a heavy chain amino acid sequence and / or a light chain amino acid sequence selected from the sequences in Table 6.

[0054] [Table 6]

[0055] In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO: 90 and a light chain whose sequence corresponds to SEQ ID NO: 100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO: 92 and a light chain whose sequence corresponds to SEQ ID NO: 100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO: 94 and a light chain whose sequence corresponds to SEQ ID NO: 100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO: 96 and a light chain whose sequence corresponds to SEQ ID NO: 100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO: 98 and a light chain whose sequence corresponds to SEQ ID NO: 100.

[0056] Post-translational modifications to the sequence of an anti-α4β7 antibody can be made to generate a truncated form, such as truncating one or more (eg, 1, 2, 3, or more) amino acid residues on the C-terminus of the antibody heavy chain.

[0057] In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO:91 and a light chain whose sequence corresponds to SEQ ID NO:100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO:93 and a light chain whose sequence corresponds to SEQ ID NO:100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO:95 and a light chain whose sequence corresponds to SEQ ID NO:100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO:97 and a light chain whose sequence corresponds to SEQ ID NO:100. In some embodiments, the anti-α4β7 antibody comprises a heavy chain whose sequence corresponds to SEQ ID NO:99 and a light chain whose sequence corresponds to SEQ ID NO:100.

[0058] In some embodiments, the anti-α4β7 antibody competes with a reference antibody for binding to human α4β7 in an in vitro assay. In some embodiments, the anti-α4β7 antibody competes for binding to human α4β7 on cells expressing human α4β7. The reference antibody can be any of the anti-α4β7 antibodies described herein. In some embodiments, the reference antibody is an antibody provided in Tables 4-6. In some embodiments, the reference antibody is an antibody provided in Table 8. In specific embodiments, the reference antibody is selected from a research-grade antibody generated using an amino acid sequence derived from an anti-human α4β7 antibody, or an antibody having an equivalent amino acid sequence thereto, e.g., vedolizumab.

[0059] In some embodiments, the anti-α4β7 antibody antagonizes, e.g., inhibits, the human α4β7 heterodimer of one α4 (SEQ ID NOs: 1-2) and one β7 (SEQ ID NOs: 3-4). α4β7 receptor antagonism can occur by a number of mechanisms, for example, by inhibiting the binding of α4β7 by at least one of its ligands, such as human MAdCAM-1 (SEQ ID NO: 5) or human VCAM-1 (SEQ ID NO: 6).

[0060] The anti-α4β7 antibodies described herein bind to human α4β7. The cross-reactivity of antibodies to α4β7 from other species, e.g., monkeys, e.g., cynomolgus monkeys, can provide advantages, such as the ability to test for biological activity in monkey animal models. Such animal model testing can be used to screen anti-α4β7 antibodies to select for properties related to efficacy, e.g., favorable pharmacokinetics, or properties related to safety, e.g., reduced hepatotoxicity. In some embodiments, the anti-α4β7 antibody binds to cynomolgus monkey α4β7 and human α4β7.

[0061] Assays for competition include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), sandwich ELISA, fluorescence-activated cell sorting (FACS) assays, and surface plasmon resonance assays.

[0062] When performing an antibody competition assay between a reference antibody and a test antibody (regardless of species or isotype), the reference can first be labeled with a detectable label, such as a fluorophore, biotin, or enzyme (or even radioactive) label, to enable subsequent identification. In this case, cells expressing human α4β7 are incubated with unlabeled test antibody, labeled reference antibody is added, and the intensity of bound label is measured. If the test antibody competes with the labeled reference antibody by binding to an overlapping epitope, the intensity will be reduced compared to a control reaction performed without the test antibody.

[0063] In a specific embodiment of this assay, 80% of maximal binding ("conc") under the assay conditions (e.g., a particular cell density) is achieved. 80% The concentration of labeled reference antibody that results in a 10× concentration of 10 ... 80% of unlabeled test antibody and conc 80% This is done using a labeled reference antibody.

[0064] Inhibition is calculated according to the following formula: inhibition constant or K ican be expressed as: K i =IC 50 / (1+[reference Ab concentration] / K d ), In the formula, IC 50 is the concentration of test antibody at which binding of the reference antibody is reduced by 50%, and K d is the dissociation constant of the reference antibody and is a measure of its affinity for human α4β7. Antibodies that compete with the anti-α4β7 antibodies disclosed herein have a K of 10 pM to 10 nM under the assay conditions described herein. i can have:

[0065] In various embodiments, a test antibody is considered to compete with a reference antibody if it reduces binding of the reference antibody by at least about 20% or more, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, or a range of percentages between any of the aforementioned values, at a reference antibody concentration that is 80% of maximal binding under the particular assay conditions used, and at a test antibody concentration that is 10-fold higher than the reference antibody concentration.

[0066] Another aspect of the present disclosure includes anti-α4β7 antibody binding fragments capable of specifically binding to human α4β7. In some embodiments, these anti-α4β7 binding fragments comprise at least one and up to all CDRs of an anti-α4β7 antibody disclosed herein. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, single-chain Fv fragments, and single-domain fragments.

[0067] An anti-α4β7 antibody or binding fragment thereof can have one or more amino acids inserted into one or more of its CDRs, as described, for example, in Jung and Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Patent Application Publication No. 2007 / 0280931.

[0068] 6.4. Polynucleotides Encoding Anti-α4β7 Antibodies, Expression Systems, and Methods for Producing Antibodies The present disclosure encompasses polynucleotide molecules encoding immunoglobulin light and heavy chain genes for anti-α4β7 antibodies, vectors containing such polynucleotides, and host cells capable of producing the anti-α4β7 antibodies of the present disclosure.

[0069] The anti-α4β7 antibodies of the present disclosure can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To express the antibody recombinantly, one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody are transfected into the host cells, whereby the light and heavy chains are expressed within the host cells and, optionally, secreted into the medium in which the host cells are cultured, from which the antibody can be recovered.

[0070] To generate polynucleotides encoding such anti-α4β7 antibodies, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained, for example, by using the polymerase chain reaction (PCR) to amplify and modify germline DNA or cDNAs encoding the light and heavy chain variable sequences.

[0071] V associated with anti-α4β7 antibodies H and V L Once DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V HA DNA fragment encoding the is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0072] V H The isolated DNA encoding the region is V H A full-length heavy chain gene can be obtained by operably linking the DNA encoding the heavy chain constant region (CH1, CH2, CH3, and optionally CH4) to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3, and optionally CH4). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but in certain embodiments, is IgG1 or IgG4. In the case of a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain CH1 constant region can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0073] V L The isolated DNA encoding the region is V LThe DNA encoding the light chain constant region CL can be converted to a full-length light chain gene (and a Fab light chain gene) by operably linking the DNA encoding the light chain constant region CL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but in certain embodiments is a kappa constant region.

[0074] To express the anti-α4β7 antibody of the present disclosure, DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0075] The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Prior to insertion of the light or heavy chain sequences associated with the anti-α4β7 antibody, the expression vector can already carry antibody constant region sequences. For example, the V H and V LOne approach to converting the sequences into full-length antibody genes is to insert them into expression vectors that already encode the heavy and light chain constant regions, respectively, thereby H The segment is operably linked to a C-H segment in a vector, L The segment is operably linked to a CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0076] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.

[0077] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0078] The antibodies of the present disclosure can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of the antibodies is carried out in eukaryotic cells, e.g., mammalian host cells, which optimally secrete properly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells (e.g., the DHFR selectable marker described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621, used with the DHFR selectable marker described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220). - Examples of cells that can be used to produce antibodies include CHO cells, NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods. The host cells can also be used to produce portions of an intact antibody, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures are within the scope of the present disclosure. For example, it may be desirable to transfect host cells with DNA encoding either the light chain or the heavy chain (but not both) of the anti-α4β7 antibody of the present disclosure.

[0079] Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to human α4β7. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

[0080] For recombinant expression of the anti-α4β7 antibodies of the present disclosure, two expression vectors of the present disclosure, a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide, can be co-transfected into a host cell. The two vectors can contain the same selectable marker, or they can each contain a distinct selectable marker. Alternatively, a single vector can be used that encodes both the heavy and light chain polypeptides.

[0081] Once a polynucleotide encoding one or more portions of an anti-α4β7 antibody is obtained, further modifications or mutations can be introduced into the coding sequence to generate polynucleotides encoding, for example, antibodies with different CDR sequences, antibodies with reduced affinity for Fc receptors, or antibodies of a different subclass.

[0082] The anti-α4β7 antibodies of the present disclosure can also be produced by chemical synthesis or by using a cell-free platform.

[0083] 6.5. Purification of anti-α4β7 antibodies Once a polypeptide of the present disclosure is produced by recombinant expression, it may be purified by any method known in the art for protein purification.

[0084] The polypeptides can be purified as monomers or dimers, for example, as an anti-α4β7 antibody comprising two polypeptides.

[0085] Once isolated, the anti-α4β7 antibodies can be further purified.

[0086] 6.6.How to use 6.6.1. Treatment benefits The data provided herein demonstrate that the disclosed anti-human α4β7 antibodies exhibit favorable binding affinity, specificity, and potency for α4β7, as well as a favorable binding profile to primary immune cells, FcγR binding, and a lack of ADCC and ADCP activity against human α4β7+ cells. These anti-human α4β7 antibodies were shown to bind to α4β7 in virions of different HIV strains grown in the laboratory and to α4β7 from patient HIV samples, subsequently forming immune complexes. These immune complexes can bind to different FcγRs and are phagocytosed by the human monocytic cell line THP-1. These antibodies also block the interaction of α4β7 with its ligands, such as MAdCAM-1 and the HIV gp120 protein. Therefore, anti-α4β7 antibodies, binding fragments, and / or pharmaceutical compositions containing them can be used therapeutically to induce viral suppression of HIV infection or reduce viral load in HIV-infected subjects via Fc- and Fab-dependent mechanisms. In some embodiments, viral suppression of an HIV infection comprises reducing the function of the HIV virus and / or reducing the replication of the HIV virus.

[0087] The disclosed anti-human α4β7 antibodies can be used in a method for treating HIV infection in a subject in need thereof. In some embodiments, the method includes reducing the viral load in the subject. In some embodiments, the viral load in the subject is reduced to an undetectable level. In some embodiments, the subject is a human subject infected with HIV.

[0088] In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that antagonizes α4β7 to provide a therapeutic benefit. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that binds to HIV virions. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that binds to α4β7 on HIV virions to form immune complexes. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that forms immune complexes with HIV virions. In some embodiments, these immune complexes are taken up by APCs by phagocytosis. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that blocks the interaction of α4β7 with its ligand, such as MAdCAM-1, and HIV gp120. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that blocks the interaction between α4β7 and HIV gp120 and inhibits cell-to-cell HIV spread. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that blocks CD4 T cell stimulation mediated by MAdCAM-1 or HIV gp120. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that suppresses HIV replication in CD4 T cells costimulated by MAdCAM-1 or HIV gp120. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that induces viral suppression of HIV replication. In some embodiments, the method comprises administering to a human subject with HIV infection an anti-α4β7 antibody that induces immune-mediated suppression of HIV.

[0089] 6.7. Sequence Listing Description A correlation of the sequences disclosed in the incorporated sequence listing and their brief descriptions is shown in Table 7.

[0090] [Table 7] [Example]

[0091] The following examples highlight certain features and characteristics of exemplary embodiments of the antibodies and binding fragments described herein and are provided for purposes of illustration and not limitation.

[0092] antibody Antibodies used throughout the examples, including Ab-h1.9d-WT, positive control and isotype control, are listed in Table 8.

[0093] [Table 8]

[0094] statistics Half maximal inhibitory concentration (IC 50 ) and median effective concentration (EC 50 ) Values ​​were determined by nonlinear regression analysis of concentration-response curves using GraphPad Prism. The significance of comparisons was determined by a two-tailed Mann-Whitney test. Except where noted, all values ​​are the mean or standard deviation of at least three independent experiments.

[0095] [Example 1] 7.1. Production of anti-human α4β7 antibodies 7.1.1. Hybridoma Screening Hybridoma-based technology was used to generate an initial panel of mouse anti-human α4β7 antibodies. Mice were immunized with HEK293, CHO-K1, or BaF3 recombinant cells expressing human α4β7 in addition to adjuvant. Selection of candidate hybridoma-derived antibodies was based on the criteria in Table 9.

[0096] [Table 9]

[0097] A panel of functional hybridoma mAbs was identified from this screen, and all candidates showed favorable profiles. However, none had rodent cross-reactivity, which can be explained by 1) human α4 / β7 shares 96% / 97% amino acid sequence homology with cynomolgus monkey α4 / β7 but only 84% / 85% sequence homology with rat and mouse α4 / β7; and 2) all selected functional hybridoma mAbs were highly selective for α4β7 and could bind to conformational epitopes on the α4β7 heterodimer.

[0098] 7.1.2. Antibody Characterization Small-scale antibody production by 200-300 ml roller bottle culture and Protein A affinity purification was performed on subcloned stable hybridoma cell lines. mAb purity was confirmed by SDS-PAGE and mass spectrometry. Purified antibodies were characterized through binding and functional assays to determine isoform and species cross-reactivity.

[0099] Binding Screening Purified antibodies were characterized for isoform and species cross-reactivity by FACS using BaF3-hα4β7, BaF3-hαEβ7, BaF3-cαEβ7, BaF3, CHOK1-cα4β7, CHOK1-mα4β7, and CHOK1-hα4β1 cell lines at a concentration of 1 μg / ml. FACS profiles, including the FACS profile of exemplary mAb Ab-m1, are summarized in Table 10.

[0100] [Table 10]

[0101] Functional Verification The purified antibody was characterized by an adhesion assay to MadCAM-1 in HuT78 cells. Ab-m1 showed potential blocking efficacy in the MadCAM-1 assay and was therefore functionally validated. Data from three independent experiments for Ab-m1 are summarized in Table 11. Representative data are shown in Figure 1.

[0102] [Table 11]

[0103] Functional characterization Further functional characterization of Ab-m1 was performed using an adhesion assay to MAdCAM-1 in CHOK1-cα4β7 to examine cynomolgus monkey cross-reactivity. Adhesion assay data for CHOK1-cα4β7 are summarized in Table 11 above. Representative data are shown in Figure 2.

[0104] To determine the affinity of Ab-m1 binding to human α4β7, FACS-based titrations were performed on BaF3-hα4β7. EC 50 were determined and are summarized in Table 12.

[0105] [Table 12]

[0106] Epitope binning was performed by competitive FACS using comparator antibodies conjugated to Alexa488 fluorophores in the presence of 50x excess unlabeled Abs shown in Table 13. Ab-m1 was characterized as belonging to the vedolizumab (Ab-Vedo)-like group based on the percentage of binding inhibition of Alexa488-labeled Abs by unlabeled Abs (Table 13).

[0107] [Table 13]

[0108] Candidate Profiling The affinity of mAb binding to human α4β7 was confirmed in primary human CD4+ memory T cells. Ab-ml inhibited human MACAM-1 binding to primary human CD4+ memory T cells with high potency (IC 50 = 63.7 pM, maximum inhibition 100%, Figure 3).

[0109] 7.1.3. VH / VL Sequencing, Chimeric Antibody Production and Characterization Hybridoma clones were recovered and expanded in complete hybridoma culture medium (DMEM with 10% FBS). Cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature and washed twice with PBS, pH 7.4. RNA was extracted from the cell pellet (approximately 1 x 10 cells) using Trizol. 7 cells).

[0110] Antibody VH and VL fragments were amplified separately from the hybridoma total RNA by RT-PCR using a mouse Ig primer set (Novagen, 69831-3). Positive PCR products of the appropriate size were inserted into a T vector for sequencing and identification of the VH and VL regions (Table 14). Full-length antibody sequences were constructed using a combination of the VH / VL sequences and theoretical constant region sequences.

[0111] After primer design, VH / VL were amplified individually and cloned into expression vectors to generate chimeric antibody constructs via homologous recombination. The amino acid sequences of the CDRs in VH and VL are shown in Table 14.

[0112] [Table 14]

[0113] The chimeric antibody was produced by transient transfection in HEK293 cells. After expression, the protein was purified. The high percentage of monomeric chimeric antibody was confirmed by SEC-HPLC.

[0114] Ab-c1 is a chimeric antibody with the variable region of Ab-m1 and a human IgG1 / κ constant region. The affinity of Ab-c1 binding to human primary CD4+CD45RO+ T cells was determined by FACS and showed a binding EC of 3.9 pM from one representative human whole blood sample. 50 The values ​​were as shown in FIG. 4 and Table 15. The ability of Ab-c1 to block α4β7 integrin binding to MadCAM-1 was also evaluated in HuT78 cells and CHOK1-cα4β7 cells (Table 15).

[0115] [Table 15]

[0116] The purified chimeric antibody Ab-c1 was further characterized by FACS using BaF3-hα4β7, BaF3-hαEβ7, BaF3-cαEβ7, BaF3, CHOK1-cα4β7, and CHOK1-hα4β1 cell lines at a concentration (1 μg / ml) to confirm its binding specificity and cynomolgus cross-reactivity. The FACS profiles of the tested chimeras are summarized in Figure 16.

[0117] [Table 16]

[0118] HuT78 was used to examine whether chimeric Ab-c1 could block MAdCAM-1-mediated adhesion to hα4β7 integrin. As shown in Table 17, Ab-c1 demonstrated the ability to inhibit HuT78 cell adhesion to MAdCAM-1.

[0119] CHOK1-cα4β7 was used to examine whether the chimeric antibody could inhibit MAdCAM-1-mediated adhesion to cα4β7. As shown in Table 17, Ab-c1 demonstrates the ability to inhibit CHOK1-cα4β7 cell adhesion to MAdCAM-1.

[0120] [Table 17]

[0121] Humanization Antibody Ab-m1 was selected based on binding and potency on both HuT78 cells and human primary CD4+ T memory cells, cynomolgus monkey cross-reactivity, CDR sequence diversity, binding selectivity, sequence liability (i.e., glycosylation), and epitope constellation. See Table 18 for designated characteristics.

[0122] [Table 18]

[0123] Humanized antibodies were designed by assembling VH and VL fragments according to Table 19 and incorporating human IgG1 and kappa constant regions.

[0124] [Table 19]

[0125] These humanized anti-α4β7 mAbs were produced and showed sufficient expression to be >95% monomeric by SEC. The identity was confirmed by MS, and the stability of the humanized anti-α4β7 was measured by DSC. Four of the humanized Ab-h1s were evaluated for their ability to block MACAM-1-mediated HuT78 cell adhesion, and all exhibited IC values ​​in the single-digit or low double-digit pM range. 50 The results showed strong efficacy with values ​​(Table 20).

[0126] [Table 20]

[0127] 7.1.5.Ab-h1.9 Liability Operation Antibody Ab-h1.9 was selected to generate variant VH and VL chains with reduced chemical liability for deamidation, isomerization, oxidation, glycosylation, hydrolysis, and cleavage.

[0128] The sequence of Ab-h1.9VH is: EVQLVQSGAEVKKPGSSVKVSCKASGFNIKNTYMHWVRQAPGQGLEWIGRIDPA NG HTEYAPKF QG RVTITADESTNTAYMELSSLRSEDTAVYYCYYV DS WGQGTTVTVSS (SEQ ID NO: 23), The sequence of Ab-h1.9VL is: DIQMTQSPSSLSASVGDRVTITCHAS QG ISDNIGWLQQKPGKSFKLLIYHGTNLEDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTFGGGTKVEIKR (SEQ ID NO: 28); Selected liability mutation sites are underlined and CDRs are in bold. Liabilities are present in VH-CDR2, VH-CDR3, and VL-CDR1.

[0129] Two rounds of liability-free anti-α4β7 clone selection using biotinylated human α4β7 extracellular domain protein were performed. Colonies from each library were sequenced, and colonies with additional liability in either CDR were removed. Clones from each library were screened by FACS for binding to the surface α4β7 antigen on yeast compared to the parent Ab-h1.9.

[0130] Liability-engineered clones identified that bound similarly to the parent (Figure 5) are presented in Table 21. These liability-engineered clones were converted along with their variable regions into an IgG format.

[0131] [Table 21]

[0132] Liability-engineered mAb Ab-h1.9a through Ab-h1.9e was tested for binding to α4β7-expressing HuT78 cells by FACS. These antibodies retained binding activity, with EC values ​​ranging from 253 pM to 702 pM. 50 This retention of α4β7 binding was surprising, considering that most of the VH-CDR3 (e.g., one-third of the amino acid residues in the CDRs) was mutated in the liability-engineered mAbs. Ab-h1.9d exhibited the strongest binding affinity (253 pM) and displayed good drug-like properties.

[0133] The protein production characteristics of antibodies Ab-h1.9(a)-(e) in the human IgG1 Fc LALA format were also tested, with Ab-h1.9d showing superior properties.

[0134] 7.1.6. Antibodies Exemplary Ab-h1.9d-derived IgG1 antibody conversions are shown in Table 22. Ab-h1.9d-HuIgG1 is a human IgG1 / kappa antibody with an HC of SEQ ID NO: 90, characterized by a canonical human heavy chain constant region, and an LC of SEQ ID NO: 100, characterized by a canonical human kappa light chain constant region. Ab-h1.9d-HuIgG1 may also have a C-terminal lysine-truncated HC of SEQ ID NO: 91. Ab-h1.9d-WT is an IgG1 / kappa antibody with an HC of SEQ ID NO: 92, characterized by variant CH3 substitutions D356E and L358M, and an LC of SEQ ID NO: 100. Ab-h1.9d-WT may also have a C-terminal lysine-truncated HC of SEQ ID NO: 93. Ab-h1.9d-LALA is an IgG1 / kappa antibody having an HC of SEQ ID NO: 94, characterized by variant CH2 substitutions L234A and L235A and variant CH3 substitutions D356E and L358M, and an LC of SEQ ID NO: 100. Ab-h1.9d-LALA may also have a C-terminal lysine-truncated HC of SEQ ID NO: 95. Ab-h1.9d-QL is an IgG1 / kappa antibody having an HC of SEQ ID NO: 96, characterized by variant CH2 substitution T250Q, and variant CH3 substitutions D356E, L358M, and M428L, and an LC of SEQ ID NO: 100. Ab-h1.9d-QL may also have a C-terminal lysine-truncated HC of SEQ ID NO: 97. Ab-h1.9d-LALA / QL is an IgG1 / kappa antibody having an HC of SEQ ID NO: 98 characterized by variant CH2 substitution T250Q, and variant CH3 substitutions L234A, L235A, D356E, L358M, and M428L, and an LC of SEQ ID NO: 100. Ab-h1.9d-LALA / QL may also have a C-terminal lysine-truncated HC of SEQ ID NO: 99.

[0135] [Table 22] TIFF0007765447000025.tif77162

[0136] After production, Ab-h1.9d-WT showed excellent properties as an anti-human α4β7 antibody, demonstrating high specificity (low binding to α4β7) and favorable PK / PD (low ADA) titers and high plasma exposure).

[0137] [Example 2] 7.2. α4β7 Expression on CD4+ and CD8+ T Cell Subsets from Healthy Donors or HIV+ Individuals 7.2.1 Materials and Methods During acute HIV infection, α4β7-expressing CD4+ T cells are preferentially infected and therefore depleted (Sivro et al., Sci Transl Med 2018). To investigate whether peripheral α4β7 expression recovers over time in HIV+ individuals receiving cART, we compared the percentage and expression levels of α4β7 on CD4+ and CD8+ T cell subsets derived from PBMCs of 10 healthy (HIV-) donors and 45 HIV+ individuals receiving cART (age: 26-66 years, median 42 years; HIV infection duration: 1-36 years, median 12 years) by flow cytometry analysis using a cocktail of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, and α4β7. Both the percentage (%) and expression levels (MESF) of α4β7 in CD4+ and CD8+ T cell populations were analyzed. CD4+ and CD8+ T cell subsets were defined as CD28+CD45RO- naive cells, CD28-CD45RO- terminal effector cells, CD28-CD45RO+ effector memory cells, CD28+CD45RO+CCR7+ central memory cells, and CD28+CD45RO+CCR7- transient memory cells.

[0138] 7.2.2.Results In both HIV- and HIV+ individuals receiving cART, the percentage of cells expressing α4β7 was higher in naive CD4+ or CD8+ T cells compared with memory T cell subsets, and α4β7 expression levels (as measured by MESF) were higher on central memory, transient memory, and effector memory cells than on naive cells (Figures 6A–6F). Both peripheral CD4+ and CD8+ T cells expressed similar levels of α4β7 (as measured by MESF) in HIV- and HIV+ individuals. This analysis showed that α4β7 expression on peripheral CD4+ or CD8+ T cells among HIV+ individuals receiving cART was highly variable but not different from α4β7 expression on peripheral CD4+ or CD8+ T cells from uninfected individuals. These results suggested that α4β7 expression on peripheral CD4+ T cells among HIV+ individuals could support the incorporation of α4β7 into budding HIV virions.

[0139] [Example 3] 7.3. HIV virion capture with Ab-h1.9d-WT 7.3.1 Materials and Methods To confirm that α4β7 is present in the HIV virion envelope and to test whether Ab-h1.9d-WT can bind to α4β7 incorporated into the HIV virion envelope and form immune complexes, Ab-h1.9d-WT was first tested in a virion capture assay (bead format) using laboratory-grown HIV-1 strains or samples from viremic HIV+ individuals according to published methods (Guzzo et al., Sci Immunol 2017). Six laboratory-grown HIV-1 strains (BCF06, CMU08, NL4-3, RU507, YBF30, and IIIB) representing different groups, genetic subtypes, and coreceptor usage (Table 23) were generated in activated primary human PBMCs (e.g., activated with OKT3 antibody or phytohemagglutinin (PHA)) in the presence of retinoic acid (RA), which induces α4β expression in cells. Protein G-conjugated immunomagnetic beads (Dynabeads, Thermo Fisher Scientific) were loaded with the appropriate antibody and then incubated with a viral stock (approximately 2 ng p24gag / reaction) of each HIV strain. After incubation, the loaded beads were washed to remove unbound viral particles and subsequently treated with Triton X-100 to lyse the captured virions for p24gag quantification. For HIV patient samples, the viral titer (copies / mL) of the sample was determined using the COBAS Taqman 2.0 assay. Ten micrograms of protein G-conjugated immunomagnetic beads loaded with the appropriate antibody were incubated with 400 μL of patient serum for 2 hours. The beads were then washed to remove unbound viral particles, and the RNA of the bound virions was subsequently extracted using a Qiagen viral RNA extraction kit according to the manufacturer's instructions. The copy number of virions captured from patient samples was subsequently quantified by digital droplet PCR using primers and probes targeting a conserved region in the LTR-gag in HIV subtype B.

[0140] EC of antibodies to capture HIV virions 50To determine the value, Ab-h1.9d-WT was tested in a virion capture assay adapted to a 96-well plate format. Serially diluted antibodies were added to prewashed Pierce™ Protein G-coated plates (Thermo Fisher). After incubation, the plates were washed to remove unbound antibody. Viral stocks (approximately 2 ng p24 gag) of each HIV strain were added to each well and incubated. The plates were washed to remove unbound viral particles and then treated with Triton X-100 to lyse the captured virions for p24 gag quantification. HIV p24 gag was detected using the High Sensitivity AlphaLISA p24 gag Detection Kit (Perkin Elmer).

[0141] 7.3.2.Results Ab-h1.9d-WT was able to capture virions from all six laboratory-grown HIV strains tested, whether clinical or laboratory-adapted, as indicated by the viral p24 gag protein in the captured virions (Figures 7A–7F). The amount of p24 gag protein from captured virions demonstrated a dose response to the antibody concentrations (5 and 15 nM) used in the assay. Ab-h1.9d-WT was also able to capture virions from HIV-1 patient samples (viral input shown in Figure 7G-1), as indicated by the higher number of HIV RNA copies in samples reacted with Ab-h1.9d-WT compared to samples reacted with the negative control antibody (Figure 7G-2).

[0142] Ab-h1.9d-WT inhibits its EC 50 To determine the EC value, the virions were then tested in a virion capture assay adapted to a 96-well plate format. 50The values ​​were similar for all viruses tested, ranging from 0.12 nM (0.019 μg / mL) to 0.25 nM (0.038 μg / mL) (Table 23). When tested against the same panel of viruses, Ab-Vedo was not as potent as Ab-h1.9d-WT in capturing HIV virions, with an EC 50 Values ​​are the EC of Ab-h1.9d-WT for each virus. 50 The EC values ​​were approximately 2-3 times higher (Table 23). HIV IIIB strains could be complemented by Ab-h1.9d-WT (Fig. 7F) and Ab-Vedo (data not shown) using the bead format assay, but their EC values ​​were significantly higher. 50 Values ​​could not be determined by plate format assay due to the low titer of the virus stock.

[0143] [Table 23]

[0144] Collectively, these data confirm the presence of α4β7 in virions from all laboratory-grown HIV strains and HIV-1 patient samples tested and demonstrate that Ab-h1.9d-WT is more potent than Ab-Vedo at binding to α4β7 on immune-complexed HIV virions, the first step in the series of steps required to induce the proposed "vaccination effect" for durable HIV viral control.

[0145] Ab-h1.9d-WT is a potent anti-α4β7 antibody that can bind to α4β7 on the virion envelope of all HIV strains tested and HIV-1 patient samples.

[0146] [Example 4] 7.4. Binding of immune complexes of Ab-h1.9d-WT and HIV virions to FcγR 7.4.1 Materials and Methods To test whether the immune complexes formed between Ab-h1.9d-WT and HIV virions can bind to FcγR in vitro, the appropriate antibodies were first mixed with HIV NL4-3 virus (prepared in human PBMCs activated in the presence of RA to induce α4β7 expression) to form immune complexes, which were subsequently incubated with His-tagged FcγR immobilized on nickel-coated plates (FcγRI and FcγRIIIa (V158) have relatively high affinity for Ab-h1.9d-WT, as shown in Table 32) or, to enhance detection sensitivity, biotinylated FcγR immobilized on neutravidin-coated plates (FcγRIIa (H131 or R131) and FcγRIIIa (F158) have relatively low affinity for Ab-h1.9d-WT, as shown in Table 32). After incubation, plates were washed to remove immune complexes that did not bind to FcγR and then treated with lysis buffer to dissolve captured immune complexes and release viral proteins for p24 gag quantification.

[0147] 7.4.2.Results Immune complexes formed between HIV virions and Ab-h1.9d-WT, which has a WT Fc domain, were able to bind to different FcγRs, as indicated by the presence of the HIV p24 capsid protein in the protein complex bound to the FcγRs (Fig. 8A–8E). HIV virions alone did not bind to FcγRs (data not shown), nor did immune complexes formed with Ab-h1.9d-LALA, an antibody identical to Ab-h1.9d-WT except that it has an engineered LALA mutation in its Fc domain that significantly reduces its binding to HIV virions and FcγRs (Fig. 8A–8E). Furthermore, immune complexes formed between HIV virions and Ab-Vedo showed minimal binding to FcγRs (Fig. 8A–8E). This is consistent with the significantly lower binding affinity of Ab-Vedo to different FcγRs than Ab-h1.9d-WT due to the engineered mutation in the Fc domain of Ab-Vedo. Because the binding affinity of Ab-h1.9d-WT to human FcγRIIa (H131 or R131) and FcγRIIIa (F158, also known as F176) is lower than that to FcγRI and FcγRIIIa (V158, also known as V176) (Figure 19A), instead of using the corresponding His-tagged FcγRs immobilized on nickel plates, biotinylated FcγRIIa (H131 or R131) and FcγRIIIa (F158) were immobilized on neutravidin-coated plates to enhance the capture of immune complexes of Ab-h1.9d-WT by these FcγRs.

[0148] The immune complex formed by Ab-h1.9d-WT and HIV virions could bind to different FcγRs, including FcγRIIa (responsible for ADCP), a step that may enable the complex to be internalized by APCs and induce the proposed "vaccination effect" for HIV control.

[0149] [Example 5] 7.5. Ab-h1.9d-WT mediates Fc-dependent uptake of α4β7-coated beads in THP-1 cells 7.5.1 Materials and Methods cell culture THP-1 cells were cultured in RPMI medium (Gibco) supplemented with 10% FBS (Sigma) at 37°C and 5% CO. Cells were passaged every 2 or 3 days and maintained at a density of 500,000–1,000,000 cells / ml.

[0150] Bead preparation Recombinant human α4β7 protein (R&D systems) was dialyzed overnight at 4°C in 1x PBS using a 3.5 kDa MWCO dialysis device (Thermo Fisher Scientific Inc.). The resulting α4β7 was then biotinylated with 100x molar excess NHS-biotin (Thermo Fisher Scientific Inc.) for 2 hours at 4°C. Excess biotin was removed by dialysis overnight at 4°C. For α4β7-coated beads, neutravidin-labeled fluorescent beads (Thermo Fisher Scientific Inc.) were incubated with biotinylated α4β7 for 1-24 hours at 4°C. The bead-protein conjugation reaction occurred at a ratio of 2 mg of α4β7 protein per ml of stock beads unless otherwise stated. The protein-conjugated beads were washed twice with 1x PBS (Sigma) containing 1% BSA and then diluted 100x prior to use. Successful conjugation of proteins to beads was confirmed in a phagocytosis assay comparing anti-α4β7 to an isotype control condition.

[0151] Phagocytosis assay The phagocytosis assay using protein-coated beads and THP-1 cells was adapted from a previously described study (Ackerman et al., 2011). The assay was performed in a 96-well plate. Immune complexes were formed by combining 10 μl of prepared α4β7-coated beads with 10 μl of the indicated antibody (10 μg / ml). These were incubated for 1–2 hours at 37°C and 5% CO2. THP-1 cells (100,000 / well) were then incubated with the immune complexes in a final volume of 200 μl for the indicated times. Following incubation, cells were stained with a fixable live / dead stain (ThermoFisher) and subsequently washed and fixed with FACS buffer. Data on the resulting cells was collected using a BD LSR Fortessa X20. Fluorescent bead and live / dead stain fluorescence were detected using the PE-CF594 and BV510 settings, respectively. Data were then analyzed using FlowJo software. The phagocytosis score was calculated as follows: phagocytosis score = (MFI x percent bead-positive cells) / 1000. Normalization: Data from three independent experiments were normalized and plotted on one graph. To normalize, one replicate in the "beads only" condition was set to 1 by dividing this value by the same value (performed for each experiment). All other values ​​were divided by this normalized value to display the normalized phagocytosis score, which represents the fold change from the "beads only" condition.

[0152] Imaging flow cytometry Bead internalization was confirmed using imaging flow cytometry. Fixed cells were analyzed on an ImagestreamX Mark II imaging flow cytometer (Luminex Corp.) at 40x magnification and medium sensitivity and medium speed settings. Fluorescent beads were imaged using 488 nm (5 mW) / 560-595 nm (excitation / emission). Live / dead staining was imaged using 405 nm (5 mW) / 430-480 nm (excitation / emission). Brightfield images were collected on channel 2 (camera 1). Data were analyzed using IDEAS analysis software v6.1 (Luminex Corp.). A standard gating strategy was used to find the appropriate cell population. Briefly, focused cells were identified using a high (>40) gradient root mean square (RMS) for brightfield image clarity. Single cells were identified by their high aspect ratio and low object area in the brightfield image. Cell object areas were identified in bright-field images, and four pixels were lost from the cell boundary to define an "intracellular mask." Cells with positive fluorescent signals in the intracellular mask were identified as true internalization events. The puncta count feature was used to count the number of internalized beads in cells with true internalization events. At least 2,000 cells with true internalization events were analyzed per sample.

[0153] statistical analysis Data were plotted using GraphPad Prism. Significance was determined using one-way analysis of variance coupled with Tukey's multiple comparison test. **** p<0.0001, *** p=0.0001~0.001, ** p=0.001~0.01, * p=0.01~0.05.

[0154] 7.5.2.Results The uptake of immune complexes by antigen-presenting cells is crucial for initiating downstream cellular and humoral immunity. It has been reported that THP-1 cells phagocytose antibody-fluorescent bead immune complexes in an Fc / FcγR-dependent manner (Ackerman et al., 2011). Therefore, we used THP-1 cells to examine whether Ab-h1.9d-WT mediates phagocytosis of α4β7-coated fluorescent beads. Cells treated with α4β7-bead / Ab-h1.9d-WT antibody immune complexes for 3 hours showed significant uptake of fluorescent beads compared with Ab-h1.9d-LALA and Ab-Vedo (featuring LALA) by flow cytometry (Figures 9A-9B). The LALA mutation has been shown to significantly reduce IgG Fc binding to FcγR. These data suggest that Ab-h1.9d-WT mediates robust Fc / FcγR-dependent phagocytosis of immune complexes containing α4β7-coated fluorescent beads.

[0155] Imaging flow cytometry using Amnis Imagestream was performed to confirm that the Ab-h1.9d-WT / α4β7 bead immune complexes were internalized by THP-1 cells. As expected, fluorescent beads were localized within THP-1 cells after Ab-h1.9d-WT immune complex treatment (Figure 9C).

[0156] [Example 6] 7.6. Ab-h1.9d-WT-induced uptake of α4β7+GFP+VLP / Ab immune complexes in THP-1 cells is α4β7- and Fc-dependent 7.6.1 Materials and Methods Antibody binding to α4β7-expressing GFP+ virus-like particles (VLPs) by ELISA α4β7+GFP+VLPs were generated from HEK293 cells sequentially transfected with an α4β7 cDNA construct and an HIV gag-GFP DNA construct, followed by purification of the VLPs from the cell culture supernatant. ELISA was performed to confirm the expression of α4β7 on the GFP+VLP surface and the binding specificity of various test Abs. Briefly, each well of a high-binding flat-bottom 96-well plate was filled with 7.5 × 10 α4β7 antibodies in PBS.7 Plates were coated with 50 μL of α4β7+GFP+VLP at a concentration of 100 μL of α4β7+GFP+VLPs and incubated overnight at 4°C. The wells were washed with PBS + 1% FBS and blocked with 100 μL of Superblock solution for 30 minutes at room temperature (RT). After three washes, each well was incubated with 50 μL of a 4-fold serially diluted primary antibody in PBS + 1% FBS for 1 hour at RT, followed by washing and incubation with 50 μL of HRP-conjugated donkey anti-human IgG-Fcγ-specific secondary antibody in PBS + 1% FBS for 1 hour at RT. After the final wash, TMB substrate was added to each well for color development, and the reaction was stopped by adding 2N H2SO4. The optical density (OD) for each well was measured at 450 nm using a plate reader.

[0157] Internalization of α4β7+GFP+VLP / anti-α4β7 immune complexes in THP-1 cells 5 × 10 for α4β7+GFP+VLP uptake experiments 4 THP-1 cells were mixed in flat-bottom 96-well plates without or with α4β7+GFP+VLPs at a final concentration of 1 μg / ml and a cell-to-particle ratio of 1:100 in 100 μL of RPMI, 10% FBS. Plates were incubated for 16 hours at 37°C in a CO2 incubator. Cells were then resuspended and transferred to V-bottom 96-well plates and washed once with 200 μL of PBS, 2% FBS via centrifugation. Cell pellets were resuspended in 200 μL of PBS, 0.5% paraformaldehyde and subjected to determination of the percent GFP+ cells by flow cytometry.

[0158] For inhibition of VLP uptake, THP-1 cells (5 × 10 6 / mL) were pretreated with or without latrunculin A (LatA) at a final concentration of 240 nM or with 0.1% DMSO (control) for 2 hours at 37°C in a CO incubator, followed by incubation with or without antibodies and α4β7+GFP+VLPs as indicated above.

[0159] 7.6.2.Results Ab-h1.9d-WT-induced uptake of α4β7+GFP+VLP / Ab immune complexes in THP-1 cells is α4β7- and Fc-dependent Recombinant virus-like particles (VLPs) produced from mammalian cells have a dynamic size ranging from 0.1 to 0.2 microns. Therefore, VLPs are more similar in size to virions than the fluorescently labeled beads previously used in experiments and were utilized as a tool to model the internalization / uptake of α4β7+ virion / Ab immune complexes by THP-1 cells. Purified α4β7-expressing GFP+ VLPs were assessed for their ability to bind anti-α4β7 Ab by ELISA. As shown in Figure 10, Ab-h1.9d-WT, Ab-h1.9d-LALA, and Ab-Vedo exhibited binding ECs comparable to those of α4β7+GFP+ VLPs. 50 The binding of these Abs to α4β7+GFP+VLPs was significantly higher than that of the control Ab (anti-CMV IgG) (0.050 nM, 0.048 nM, and 0.058 nM, respectively), whereas the isotype control Ab (anti-CMV IgG) did not bind to α4β7+GFP+VLPs at all. This data suggests that various anti-α4β7 Abs can bind to α4β7+GFP+VLPs with good binding affinity and specificity.

[0160] Next, uptake of α4β7+GFP+ VLPs by THP-1 cells was assessed in the presence of various anti-α4β7 Abs with or without functional Fc. As shown in Figure 11A, Ab-h1.9d-WT treatment induced detection of over 60% GFP+ THP-1 cells compared with 1.2% GFP+ cells with isotype Ab control treatment, representing a 50-fold induction of GFP+ cells. This suggests that α4β7+GFP+ VLP uptake by THP-1 cells is specific for Ab-h1.9d-WT and dependent on α4β7 expression. However, Ab-h1.9d-LALA or Ab-Vedo(LALA) showed minimal levels of VLP uptake in THP-1 cells, suggesting that anti-α4β7 Ab-induced α4β7+GFP+ VLP uptake by these cells requires, in addition to its α4β7-binding Fab domain, a functional Fc domain of the antibody that enables Fc / FcγR-mediated internalization of immune complexes.

[0161] To confirm that Ab-h1.9d-WT-mediated uptake of α4β7+GFP+VLPs was indeed due to internalization and not simply binding to cell surface FcγRs, THP-1 cells were pretreated with latrunculin A (LatA), a known internalization inhibitor, prior to their incubation with Ab / VLP immune complexes. As shown in Figure 11B, there was a 40% reduction in GFP+ cells in LatA-treated cells compared with untreated cells. This suggests that at least 40% of the total GFP signal observed in untreated (mock) or DMSO-treated controls arose from Ab-h1.9d-WT-dependent VLP internalization in THP-1 cells.

[0162] [Example 7] 7.7.Ab-h1.9d-WT did not exhibit neutralizing activity against HIV 7.7.1 Materials and Methods Some Abs that can bind to HIV virions, such as broadly neutralizing HIV antibodies, can block viral infection. To test whether Ab-h1.9d-WT binding to α4β7 on HIV virions can block HIV infection of host cells, we evaluated this in a virus neutralization assay using the TZM-bl indicator cell line (Arrildt et al., J Virol 2015).

[0163] HIV neutralization assay HIV virus (prepared with PHA and RA) corresponding to approximately 150,000 RLU (previously determined by virus titration on TZM-bl cells) was preincubated with serially diluted antibodies. TZM-bl cells containing DEAE-dextran were added to the mixture containing the preincubated HIV and antibodies and then incubated at 37°C for 48 hours. The cells were treated with Bright-Glo (Promega), and the luciferase signal was measured.

[0164] 7.7.2.Results When tested against the panel of HIV strains shown in Table 23, Ab-h1.9d-WT did not exhibit any neutralizing activity at concentrations up to 50 μg / mL, and the HIV broadly neutralizing antibodies targeting the CD4+ binding site exhibited a neutralization IC of approximately 0.1 μg / mL against all of the group M HIV strains tested (i.e., CMU08, NL4-3, and RU570). 50 had a value (data not shown).

[0165] Ab-h1.9d-WT binds to HIV virions but does not neutralize HIV infection, consistent with the notion that α4β7 is not a viral receptor on host cells, supporting the hypothesis that targeting α4β7 rather than virally encoded glycoproteins may circumvent viral resistance mechanisms.

[0166] [Example 8] 7.8. Inhibition of the interaction of α4β7 with HIV gp120 by different antibodies 7.8.1 Materials and Methods The interaction between α4β7 and HIV gp120 has been reported to activate LFA-1, potentially promoting cell-to-cell spread of HIV (Arthos et al., Nat Immunol 2008). To test whether Ab-h1.9d-WT could disrupt this interaction, an α4β7 / gp120 binding assay was set up using α4β7-expressing RPMI8866 cells that bound to HIV gp120-V2 peptide immobilized on the plate according to published methods (Peachman et al., PloS One). NeutrAvidin-coated high-capacity 96-well plates (Thermo Fisher) were coated with biotinylated HIV gp120-V2 WT or gp120-V2 control peptide (Table 24). The sequences of the two biotinylated peptides were identical except for the four amino acids reported to mediate binding between α4β7 and gp120 mutated in the control peptide.

[0167] [Table 24]

[0168] Before adding the antibody-cell mixture, the peptide-coated plates were washed to remove unbound peptide. To make the antibody-cell mixture stock, RPMI8866 cells (8 × 10 6 The antibody-cell mixture (2 × 10 cells / mL in 50 μL per well) was resuspended in cold blocking buffer supplemented with MnCl2 at a final concentration of 2 mM to activate the α4β7 conformation. Serially diluted Ab-h1.9d-WT, Ab-Vedo, or an isotype negative control antibody was mixed with the MnCl2-treated cells, and the mixture was incubated. 5 Cells) were added to each well of a plate coated with gp120 peptide and incubated. The plate was washed, and the viable cells attached to the peptide-coated plate were determined using CellTiter-Glo 2.0 reagent (Promega).

[0169] 7.8.2.Results The specificity of this assay is demonstrated by the binding of α4β7-expressing RPMI8866 cells to the HIV gp120-V2 WT peptide immobilized on the plate (Table 24), but not to an immobilized gp120-V2 control peptide harboring mutations in the four amino acids reported to mediate gp120 binding to α4β7 (Figure 12A). When tested in this assay format, Ab-h1.9d-WT inhibited binding of RPMI8866 cells to the HIV gp120-V2 WT peptide with an IC of 0.022 ± 0.016 μg / mL. 50 Ab-Vedo was blocked at an IC value lower than that of Ab-h1.9d-WT when tested in the same assay (Figure 12B). 50 IC value higher than 50 The Ab-h1.9d-WT had a β-blocking value (0.042 ± 0.023 μg / mL), indicating that it was less potent than Ab-h1.9d-WT in blocking this interaction (Figure 12B). Collectively, these results indicate that Ab-h1.9d-WT can effectively disrupt the interaction between α4β7 and gp120, potentially inhibiting cell-to-cell viral spread.

[0170] When tested in this assay format, Ab-h1.9d-WT inhibited binding of RPMI8866 cells to the HIV gp120-V2 WT peptide with an IC of 0.022 ± 0.016 μg / mL. 50 These results indicate that Ab-h1.9d-WT effectively disrupts the interaction between α4β7 and gp120, potentially inhibiting cell-to-cell viral spread.

[0171] [Example 9] 7.9. Binding of Ab-h1.9d-WT to human and cynomolgus monkey cells expressing α4β7 integrin 7.9.1 Materials and Methods Binding of Ab-h1.9d-WT was assessed on HuT78 cells (human T lymphoma cells expressing endogenous α4β7) using an ECL binding assay and on human and cynomolgus monkey peripheral lymphocytes using flow cytometry.

[0172] Furthermore, the binding EC of Ab-h1.9d-WT 50 Values ​​were determined and compared for both human and cynomolgus monkey blood-derived total, naive, and memory CD4+ and CD8+ T cells.

[0173] ECL cell binding assay HuT78 cells expressing endogenous α4β7 were cultured in IMDM medium containing 20% ​​FBS, penicillin (50 units / mL) and streptomycin (50 μg / mL). HuT78 cells were harvested, washed once, and then cultured at 1.5 × 10 6 Cells (7.5 x 10 in 50 µL) were resuspended in DPBS at 100 cells / mL. 4) was added to each well of an MSD high-binding plate. 6.7% fetal bovine serum (diluted in DPBS) was added, and the plate was incubated at 37°C for 1 hour. The supernatant was removed, and 25 μL of titrated Ab-h1.9d-WT antibody or isotype control, prepared through a 1:4 8-point dilution ranging from 1.5 μg / mL to 0.000091 μg / mL (in DPBS buffer containing 5% FBS and 1 mM MnCl2), was added to each well. The plate was then incubated at 37°C for 1 hour. The plate was washed twice with DPBS, and 25 μL of goat anti-human Ab sulfo-tag at a 1:500 dilution in 5% FBS / DPBS / 1 mM MnCl2 was added to each well, followed by incubation at 37°C for 30 minutes. Cells were washed twice with DPBS, then 150 μL of 2× MSD Read Buffer T was added to each well. Plates were read on a Sector Imager 6000 reader to generate binding curves and binding EC 50 Values ​​were generated using GraphPad Prism 7.0 software.

[0174] Human and cynomolgus monkey peripheral T cell binding assay Frozen human or cynomolgus monkey PBMCs (isolated from blood donors using standard Ficoll-Paque separation methods) were thawed in RPMI 1640 / 10% FBS medium and placed in FACS buffer (DPBS, w / o Ca). +2 / Mg +2 The cells were washed once with 1% BSA and resuspended in FACS buffer containing 5% goat serum. Approximately 1-2 × 10 5(in 100 μL) was added to a 96-well U-bottom plate and incubated on ice for 30 minutes. The plate was centrifuged, and the supernatant was removed. Titrated Ab-h1.9d-WT or isotype control (25 μL), prepared through a 1:5 dilution in FACS buffer containing 5% goat serum and a fluorochrome-conjugated antibody cocktail, with final concentrations ranging from 5 to 0.000016 μg / mL, was added to each well, and the plate was then incubated on ice for 1 hour. Compensation and FMO controls were also prepared simultaneously. Following incubation, the cells were centrifuged and washed twice with FACS buffer. The secondary antibody (PE conjugate) was diluted 1:2,000 in FACS buffer containing 5% goat serum, and 50 μL was added to each well. The plate was then incubated on ice for 1 hour. Following incubation, cells were washed twice with FACS buffer and resuspended in 200 μL of 0.5% PFA in PBS. Plates were read on a FACS (Canto II, BD) and live cells were gated based on forward and side scatter. Flow data (FCS 3.0 files) were analyzed using FlowJo Version 10 software to generate binding curves and binding EC 50 Values ​​were generated using GraphPad Prism 7.0 software.

[0175] 7.9.2.Results ECL cell binding As summarized in Table 25, Ab-h1.9d-WT had binding EC of 26 pM, 130 pM, and 62 pM for HuT78 cells, human, and cynomolgus monkey blood-derived lymphocytes, respectively. 50 The values ​​were shown.

[0176] [Table 25]

[0177] Human and cynomolgus monkey peripheral T cell binding assay Binding EC of Ab-h1.9d-WT to both total, naive, and memory CD4+ and CD8+ T cells derived from human and cynomolgus monkey blood50 The values ​​are shown in Table 26.

[0178] [Table 26]

[0179] average combined EC 50 The values ​​range from 10 to 165 pM on all T cell subsets evaluated. Ab-h1.9d-WT binds very similarly to human and cynomolgus monkey CD4+ and CD8+ T cells or their subsets because the mean EC for each corresponding cell type was 50 values ​​vary only 2-3 fold between these two species.

[0180] Binding of Ab-h1.9d-WT to human and cynomolgus monkey CD4+ and CD8+ T subsets was also analyzed to compare the percentage of Ab-h1.9d-WT-binding T cell subsets, as shown in Figure 13. Overall, the percentage of Ab-h1.9d-WT-binding cells in each T cell subset was comparable between humans and cynomolgus monkeys. These findings were not surprising given the fact that both α4 and β7 from humans and cynomolgus monkeys are highly homologous (97% amino acid identity, see Table 27).

[0181] [Table 27]

[0182] Furthermore, the functional cross-reactivity of Ab-h1.9d-WT in cynomolgus monkeys was confirmed in in vivo studies. Repeated administration of Ab-h1.9d-WT in cynomolgus monkeys increased peripheral blood CD4+ T cell counts when α4β7 receptors were fully occupied. These data demonstrated the on-target functional pharmacodynamic effects of Ab-h1.9d-WT and supported cynomolgus monkeys as a pharmacologically relevant species for the toxicological evaluation of Ab-h1.9d-WT.

[0183] In summary, Ab-h1.9d-WT binds strongly to both human and cynomolgus CD4+ and CD8+ T subsets, demonstrating excellent cynomolgus binding cross-reactivity.

[0184] [Example 10] Integrin binding specificity of 7.10.Ab-h1.9d-WT 7.10.1 Materials and Methods Recombinant cells expressing the individual human and cynomolgus monkey heterodimeric integrins α4β7, α4β1, and αEβ7 allowed for assessment of the binding specificity of Ab-h1.9d-WT.

[0185] Ab-h1.9d-WT was also assessed for nonspecific binding to human epithelial HEK293 cells.

[0186] Integrin binding specificity assay CHO-K1 or BAF3 cells expressing various human and cynomolgus integrins (α4β7, α4β1, or αEβ7), except for cynomolgus α4β1, were harvested, counted, and cultured at 1.5 × 10 per milliliter. 6 Cell density at 1000 saturates in FACS buffer (DPBS, w / o Ca). +2 / Mg +2 1.5 × 10 5100 μL of the cell-containing medium was added to each well of a 96-well U-bottom plate and centrifuged to remove the supernatant. Titrated Ab-h1.9d-WT, assay control, or isotype control (100 μL each) in FACS buffer was added to reconstitute the cell pellet. The well contents were mixed and then incubated on ice for 1 hour. The cells were washed twice with FACS buffer, and then 100 μL of a 1:600 ​​dilution of secondary antibody (goat anti-hu IgG Fcγ-specific Alexa Fluor 488) in FACS buffer was added to each well and mixed. The plate was incubated on ice for 45 minutes. The cells were then washed twice with FACS buffer and resuspended in 200 μL of 0.5% PFA in PBS. The plate was read on a FACS (Canto II, BD), and viable cells were measured based on forward and side scatter. Median fluorescence intensity values ​​for cell-bound antibodies were generated using GraphPad Prism 7.0 software.

[0187] For CHO-K1 cells expressing cynomolgus α4β1, an antibody staining cocktail containing 25 μL of a 1:25 dilution of CD29-APC and CD49d-BV421 mixture and 25 μL of Ab-h1.9d-WT, assay control, or isotype control, each prepared by 1:5 serial dilutions in FACS buffer, was added to each well of a 96-well U-bottom plate, followed by reconstitution of the cynomolgus α4β1 cell pellet. The well contents were mixed and then incubated on ice for 45 minutes. A staining control was also prepared. Cells were washed twice with FACS buffer, and then 50 μL of a 1:600 ​​dilution of secondary antibody (goat anti-hu IgG Fcγ-specific PE) in FACS buffer was added to each well and mixed. The plate was incubated on ice for an additional 45 minutes. Cells were then washed twice with FACS buffer and resuspended in 200 μL of 0.5% PFA in PBS. Plates were read on a FACS (Canto II, BD), and live cells were gated based on forward and side scatter. CD29+CD49d+ cells were then gated to determine median fluorescence intensity for cell-binding test antibodies using FlowJo version 10 software. Data were plotted using GraphPad Prism 7.0 software.

[0188] Nonspecific HEK293 cell binding assay HEK293 cells were cultured in complete DMEM (DMEM + 10% FBS + 1% Na-pyruvate). Cells were harvested using non-enzymatic dissociation buffer (Gibco, Cat. 13151-014), counted, and then dissociated into FACS buffer (2% BSA / PBS) at 1.5 × 10 6 The cells were resuspended at 7.5 × 10 cells / mL in each well of a 96-well U-bottom plate. 4The cells were dispersed. Ab-h1.9d-WT, positive control antibody, or isotype control antibody was added to each well at 100 μg / mL and incubated on ice for 1 hour. Following incubation, the cells were washed twice with FACS buffer and further incubated with 100 μL of goat anti-huIgG Fc-PE (Jackson, Cat109-116-098) diluted 1:100 in FACS buffer for 30 minutes on ice. The cells were then washed twice with FACS buffer and resuspended in 200 μL of FACS buffer. The plate was read by FACS (Canto II, BD). Binding data were analyzed using GraphPad Prism 7.0 software.

[0189] 7.10.2.Results Integrin binding specificity As shown in Figures 14A-14C, Ab-h1.9d-WT specifically bound to human α4β7-expressing cells but not to human α4β1-expressing cells, whereas the anti-α4 mAb, Ab-nata, bound to both integrins. Ab-h1.9d-WT bound minimally to human αEβ7-expressing cells, compared with the much stronger binding observed for the anti-β7 mAb, research-grade etrolizumab. Ab-h1.9d-WT displayed a similar binding specificity profile for cynomolgus integrins. These data demonstrate that Ab-h1.9d-WT has binding specificity for both human and cynomolgus α4β7.

[0190] Non-specific HEK293 cell binding Ab-h1.9d-WT was also evaluated for nonspecific binding to human epithelial HEK293 cells. As shown in Figure 15, Ab-h1.9d-WT showed no nonspecific binding to HEK293 cells at high concentrations (100 μg / mL), comparable to the control antibody, and the positive control mAb showed strong nonspecific binding to these cells.

[0191] [Example 11] 7.11.Ab-h1.9d-WT rabbit and rodent binding cross-reactivity 7.11.1 Materials and Methods The binding of Ab-h1.9d-WT to PBMCs isolated from rabbits, rats, and mice was assessed by flow cytometry.

[0192] Rabbit and rodent cross-reactive binding assays Thaw rabbit PBMCs and place them in RPMI1640 / 10% FBS medium and in FACS buffer (DPBS, w / o Ca). +2 / Mg +2 The cells (1 × 10 in 100 μL) were washed once with 1% BSA and resuspended in FACS buffer containing 5% goat serum. 5 ) was dispersed into a 96-well U-bottom plate and incubated on ice for 30 minutes. The plate was centrifuged to remove the supernatant, and 25 μL of titrated Ab-h1.9d-WT or isotype control plus 25 μL of a 1:10 diluted CD4-FITC antibody (all Abs diluted in FACS buffer) was added to each well, followed by reconstitution of the cell pellet. The well contents were mixed and then incubated on ice for 45 minutes. Appropriate staining controls were also prepared. Next, the cells were washed twice with FACS buffer, and 50 μL of secondary antibody-PE was added to the cell pellet at a 1:2000 dilution in FACS buffer containing 5% goat serum. The plate was further incubated on ice for 45 minutes. The cells were washed twice with FACS buffer and resuspended in 200 μL of 0.5% PFA in PBS. The plate was read on a FACS (Canto II, BD), and live cells were gated based on forward and side scatter. Percentage antibody-bound cells were determined using FlowJo version 10 software, and binding curves and binding EC 50 Values ​​were generated using GraphPad Prism 7.0 software.

[0193] Female C57B / 6N mice and female Lewis rats were received from Taconic Laboratories and Charles River Laboratories, respectively. PBMCs were isolated from pooled mice, and rat blood was isolated from rat animals. Erythrocytes were lysed using RBC lysis buffer (eBioscience). Cells were washed once with PBS and then resuspended in FACS buffer (DPBS, w / o Ca) containing 5% goat serum. +2 / Mg +2 Approximately 2.5 x 10 5 Cells (100 μL) were added to a 96-well U-bottom plate and then incubated on ice for 30 minutes. The appropriate fluorochrome-conjugated antibody was added to mouse cells (CD3-APC, α4β7-PE, or IgG2a-FITC control) or rat cells (CD3-APC, α4-FITC, or IgG2a-PE control) at a 1:50 dilution, along with Ab-h1.9d-WT or an isotype control. Well contents were mixed thoroughly, and the plate was incubated on ice for 1 hour. Final concentrations were 10 and 1 μg / mL for the isotype control and Ab-h1.9d-WT, respectively. Subsequently, cells were washed twice with FACS buffer, and 100 μL of secondary antibody-PE or secondary antibody-AF488 was added to the wells at a 1:800 dilution, and the wells were incubated on ice for 45 minutes. Following incubation, cells were washed twice with FACS buffer and resuspended in 200 μL of 0.5% PFA in PBS. Plates were read on a FACS (Canto II, BD), and live cells were gated based on forward and side scatter. The percentage of CD3+ cells that bound test antibody or isotype control was determined using FlowJo version 10 software and graphed using GraphPad Prism 7.0 software.

[0194] 7.11.2.Results As summarized in Table 28, Ab-h1.9d-WT exhibited similar binding EC2 activity for rabbit lymphocytes and CD4+ T cells compared to its human and cynomolgus counterparts. 50Values ​​are shown and Ab-h1.9d-WT did not show any measurable binding to rat or mouse PBMCs (lymphocytes and CD4+ T cells).

[0195] [Table 28]

[0196] [Example 12] 7.12.Ab-h1.9d-WT-induced α4β7 internalization 7.12.1 Materials and Methods Ab-h1.9d-WT was examined for its ability to induce internalization of cell surface α4β7 on human primary CD4+ and CD8+ naive T cells from two PBMC donors.

[0197] Internalization assay Ab-h1.9d-WT internalization was examined and quantified using a FACS protocol similar to that used to determine the cellular mechanisms of etrolizumab (Lichnog et al., Front Pharmacol). Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy blood donors (RBCs, Donors KP58219 and KP58239) using Ficoll-Paque (GE 17-1440-03) and SepMate tubes (StemCell 85450), resuspended in FBS containing 5% DMSO (Gibco 10438-026), and cryopreserved in liquid nitrogen. Frozen PBMCs were thawed, counted, and collected at 1 x 10 in RPMI medium + 10% FBS. 6 Reconstitute 100 μL of cells at 1 x 10 cells / mL per well. 5Human PBMCs were preincubated with 100 μL of 2x concentrated, unlabeled Ab-h1.9d-WT antibody at 1.25 μg / mL (final concentration: 0.625 μg / mL) for 1 hour at 4°C. Cells were centrifuged, washed, resuspended in 200 μL of RPMI + 10% FBS, and incubated at 4°C or 37°C, 5% CO2 for 18 hours. Following incubation, cells were washed twice with FACS buffer (PBS + 1% FBS) and then stained for 30 minutes with CD4+ (Biolegend 317410), CD8+ (Biolegend 344710), and CD45RA (Biolegend 304130) antibodies, with or without AF647-conjugated non-competitive anti-β7 antibodies. Cell fluorescence was acquired by flow cytometry (LSR-Fortessa). Data were analyzed using FlowJo 10 software, and the percentage internalization was calculated as 100 × [total cell-surface α4β7 expression before internalization (MFI at 4°C) - remaining cell-surface α4β7 expression after internalization (MFI at 37°C) / total cell-surface α4β7 expression before internalization (MFI at 4°C)].

[0198] 7.12.2.Results As shown in Figure 16A, Ab-h1.9d-WT bound to human PBMCs at 4°C for 18 hours, and CD4+ naive T cells from donor 1 (CD4+CD45RA) were assessed 18 hours after binding to human PBMCs to minimize internalization. + ) and 73% of CD8+ naive T cells (CD8+CD45RA +) remained β7-positive. However, after incubation of Ab-h1.9d-WT under the same protocol to promote internalization but at 37°C, only 17% of CD4+ naive T cells and 7% of CD8+ naive T cells were found to be β7-positive. These data indicate that significant internalization of surface α4β7 bound to Ab-h1.9d-WT occurred at 37°C. Similar results were observed with PBMCs from donor 2. Based on the quantification of α4β7 internalization at 37°C in Figure 16B (reduced surface α4β7 expression compared to that observed after treatment at 4°C), Ab-h1.9d-WT suppressed α4β7 expression. + We were able to induce α4β7 internalization by approximately 80% in both naive CD4+ and CD8+ T cells.

[0199] Ab-h1.9d-WT is more potent at inducing α4β7 internalization compared to Ab-Vedo.

[0200] [Example 13] MAdCAM-1 ligand blockade by 7.13.Ab-h1.9d-WT 7.13.1 Materials and Methods MAdCAM-1 ligand blocking assay For the FACS-based assay, HuT78 cells or human PBMCs were collected, washed once with DPBS, and 1.5 x 10 6 Adjust the density to cells / mL and add FACS buffer (DPBS, w / o Ca +2 / Mg +2 , 1% BSA / 1 mM MnCl2) and resuspended at 1 × 10 5Cells were dispersed in a 96-well U-bottom plate at 100 μL / well and centrifuged to remove the supernatant. A 50 μL mixture containing titrated Ab-h1.9d-WT or isotype control (50 μL) plus 0.3 μg / mL MAdCAM-1-mFc, 2 mM MnCl2, and a 1:50 dilution (30 μg / mL) of Alexa488-conjugated detection Ab in FACS buffer was added to each well. The plate was incubated on ice for 1 hour and then centrifuged. The plate was gently washed once with 200 μL of FACS buffer, and the cells were reconstituted in the same buffer. The plate was read by FACS (Canto II), and viable cells were determined by forward and side scatter gating. Flow data (FCS 3.0 files) were analyzed using FlowJo version 10 software to generate binding curves and inhibitory IC values. 50 Values ​​were generated using GraphPad Prism 7.0 software.

[0201] For plate-based assays, 96-well flat-bottom plates (Greiner, Cat. 655077) were filled with coating buffer (PBS w / o Ca +2 / Mg +2 Plates were coated with 100 μL of 20 μg / mL MAdCAM-1 hFc or isotype control (final concentration 2 μg / well) using PBS (0.1% BSA) overnight at 4°C. The following day, plates were washed with 200 μL of wash buffer (PBS w / Ca +2 / Mg +2 Wash twice with blocking buffer (PBS w / Ca, 0.1% BSA) and then +2 / Mg +2 The cells were blocked with Ab-h1.9d-WT and isotype control (1% BSA) for 1 hour at 37°C. During the blocking incubation, dilutions of Ab-h1.9d-WT and isotype control were prepared and HuT78 cells were harvested. A 2x starting concentration was prepared at 5 μg / mL, followed by serial 1:4.5 7-point dilutions (final concentrations ranging from 2.5 to 0.0003 μg / mL) performed in duplicate or triplicate. Cells were counted, washed, and resuspended in assay medium (IMDM, 1% BSA) at 2 x 10 6The cells were resuspended at 100,000 cells / mL, and MnCl2 was added to a final concentration of 2 mM. 100,000 cells were dispersed into each well of a 96-well plate. The diluted antibody was then added to the cells, and the mixture was incubated at 37°C and 5% CO2 for 30 minutes. The blocking solution from the MAdCAM-1 hFc-coated plate was decanted, and 100 μL / well of the pre-incubated HuT78 cell and mAb mixture was dispensed into each well of the MAdCAM-1 hFc-coated plate. The plate was spun at 1,000 rpm for 1 minute and incubated at 37°C and 5% CO2 for 30 minutes. The adherent plate was decanted and gently washed four times with 150 μL of wash buffer. After washing, 100 μL / well of the mixture (containing 50 μL of CellTiter-Glo reagent and 50 μL of assay medium) was added to each well. The plate was placed on an orbital shaker for 2 minutes and then incubated at RT for 10 minutes. Plate luminescence was read on a luminescence plate reader (Topcount, Perkin Elmer). Luminescence signals were plotted and IC 50 Values ​​were determined using a four-parameter curve fit analysis in GraphPad Prism 7.0.

[0202] 7.13.2.Results Ab-h1.9d-WT was tested for its ability to block binding of the recombinant extracellular domain of MAdCAM-1 protein to α4β7-expressing HuT78 cells and human lymphocytes (PBMCs) using both plate-based and FACS-based assays.

[0203] Using FACS- and plate-based assays, IC values ​​of 50 pM and 25 pM, respectively 50 values ​​were obtained.

[0204] Furthermore, Ab-h1.9d-WT blocked MAdCAM-1 binding to human blood-derived lymphocytes with an IC of 223 pM. 50 values ​​were observed (Table 29).

[0205] [Table 29]

[0206] These data demonstrated the strong inhibitory effect of Ab-h1.9d-WT on the MAdCAM-1 / α4β7 interaction.

[0207] [Example 14] 7.14.Ab-h1.9d-WT blocks MAdCAM-1 costimulation on human primary CD4+ T cells 7.14.1 Materials and Methods Human PBMC and CD4+ T cell isolation Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood collected from healthy donors, and human CD4+ T cells were then isolated from the PBMCs using a CD4 negative selection kit (Stem Cell Technologies).

[0208] CD4+ T cell activation and proliferation assays 96-well flat-bottom tissue culture plates were coated overnight at 4°C with 200 ng / well anti-CD3 antibody (Biolegend) in HBSS. The next day, the anti-CD3-coated plates were washed once with HBSS and incubated with 200 ng / well MAdCAM-1 (R&D systems) for 1 hour at 37°C. Following incubation, the plates were washed once with 200 μl of HBSS, and 50,000 CD4+ T cells were added to each well in the presence or absence of 1 μg / ml test antibody. After culturing the cells for 96 hours at 37°C and 5% CO2, the cells were washed and stained with Live-Dead Aqua viability dye (Thermofisher), followed by staining with the cell activation markers anti-CD25 FITC (clone MA0251 BD bioscience) and anti-Ki67 APC (Biolegend). Cells were analyzed on a flow cytometer, and data were analyzed using FlowJo software. Data from multiple donors were plotted and statistical analysis was performed using GraphPad Prism. Significance was determined using one-way ANOVA coupled with Tukey's multiple comparison test. ****p<0.0001, ** p=0.001~0.01.

[0209] 7.14.2.Results Ab-h1.9d-WT blocks MAdCAM-1 costimulation on human primary CD4+ T cells MAdCAM-1-mediated gut homing of α4β7+ CD4+ T cells plays a central role in HIV infection of GALT (gut-associated lymphoid tissue). In addition to this role, MAdCAM-1 has also been reported to deliver costimulatory signals to human primary CD4+ T cells and promote HIV replication (Nawaz et al., Mucosal Immunology 2018). HIV infection and replication require metabolic activation of these cells, and Ab-h1.9d-WT inhibited MAdCAM-1 binding to human lymphocytes with an IC of 223 pM. 50 Because Ab-h1.9d-WT can block MAdCAM-1 costimulatory signals on human primary CD4+ T cells (Table 35), we evaluated whether Ab-h1.9d-WT can block MAdCAM-1 costimulatory signals on human primary CD4+ T cells, which in turn inhibits MAdCAM-1-mediated viral replication in these cells.

[0210] When human primary CD4+ T cells from one representative healthy donor were incubated with plate-bound anti-CD3 alone for 96 hours, 22.6% of the cells were activated and exhibited a Ki67+CD25+ phenotype (Figure 17A). When cells were incubated with plate-bound anti-CD3 and MAdCAM-1, 56.6% of the CD4+ T cells were activated. This indicates that MAdCAM-1 delivered a costimulatory signal to CD4+ T cells mediated by interacting with cell surface α4β7. Addition of Ab-h1.9d-WT to the cells during the 96-hour incubation reduced cell activation to 20.8%, a level comparable to that of anti-CD3 alone, and an isotype control Ab had no inhibitory effect on cell activation. The data suggest that Ab-h1.9d-WT effectively and completely blocked the costimulatory signal exerted by MAdCAM-1. The assay was repeated with primary CD4 T cells from five additional individual donors, and the data are summarized in Figure 17B. Consistent with the data obtained from one representative donor in Figure 17A, Ab-h1.9d-WT almost completely blocked MAdCAM-1 / α4β7-mediated costimulation of CD4 T cells, while the isotype control Ab had no inhibitory effect.

[0211] [Example 15] VCAM-1 ligand blocking specificity of 7.15.Ab-h1.9d-WT 7.15.1 Materials and Methods The effect of Ab-h1.9d-WT on α4β7 / VCAM-1 interaction in a VCAM-1-mediated cell adhesion assay was evaluated.

[0212] VCAM-1 ligand blocking assay Plates (96-well flat-bottom, Greiner, Cat. 655077) were coated with coating buffer (PBS with Ca +2 / Mg +2On day 1, plates were coated with 100 μL of 20 μg / mL VCAM-1 hFc or isotype control (final concentration 2 μg / well) using PBS (0.1% BSA) overnight at 4°C. On day 2, plates were coated with 200 μL of wash buffer (PBS w / Ca). +2 / Mg +2 Wash three times with blocking buffer (PBS w / Ca, 0.1% BSA) and then +2 / Mg +2 The cells were blocked with 1% BSA (100 mM MgCl 2, 1% BSA) for 1 hour or longer at 37°C. During the blocking incubation, dilutions of Ab-h1.9d-WT, Ab-nata, and isotype control were prepared and HuT78 cells were harvested. A 2x starting concentration of antibody was prepared at 4 μg / mL, followed by 1:4 serial dilutions in assay medium (IMDM, 1% BSA). HuT78 cells were counted, washed, and diluted to 2x10 in assay medium. 6 The cells were resuspended at 100,000 cells / mL, and MnCl2 was added to a final concentration of 2 mM. 100,000 cells were dispersed into each well of a 96-well plate. The diluted antibody was then added to the cells, and the mixture was incubated at 37°C and 5% CO2 for 30 minutes. The blocking solution from the VCAM-1 hFc-coated plate was decanted, and 100 μL of the pre-incubated HuT78 cell and mAb mixture was dispensed into each well of the VCAM-1 hFc-coated plate. The plate was incubated at 37°C and 5% CO2 for 30 minutes and then gently washed three times with wash buffer. After washing, 100 μL / well of the mixture (containing 50 μL of CellTiter-Glo reagent and 50 μL of assay medium) was added to each well. The plate was placed on an orbital shaker for 2 minutes and then incubated at room temperature for 10 minutes. Plate luminescence was read on a luminescence plate reader (Topcount, Perkin Elmer). Plot the luminescence signal and IC using a four-parameter curve fit analysis in GraphPad Prism 7.0 50 value was determined.

[0213] 7.15.2.Results In addition to α4β7 binding to MAdCAM-1, which allows gut homing of blood lymphocytes, α4β7 can also bind to VCAM-1 expressed on endothelial cells (Table 30).

[0214] [Table 30]

[0215] Natalizumab, an anti-α4 mAb, Ab-nata, can block α4β7 and α4β1 binding to VCAM-1, but it also blocks trafficking of circulating lymphocytes to the brain, causing progressive multifocal leukoencephalopathy (PML). Therefore, evaluation of the effect of Ab-h1.9d-WT on the α4β7 / VCAM-1 interaction in a VCAM-1-mediated cell adhesion assay was a critical safety parameter to examine using Ab-nata as a positive control. As expected, Ab-nata inhibited VCAM-1-mediated HuT78 cell adhesion with a mean IC of 56 pM. 50 In contrast, Ab-h1.9d-WT did not show any detectable blockade of VCAM-1-mediated HuT78 cell adhesion (FIG. 18).

[0216] Ab-h1.9d-WT selectively blocks the α4β7 / MAdCAM-1 interaction with high potency, but shows no inhibition of the α4β7 / VCAM-1 interaction.

[0217] [Example 16] 7.16. Ab-h1.9d-WT binding affinity to human and cynomolgus monkey FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcRn 7.16.1 Materials and Methods Ab-h1.9d-WT was evaluated by BIAcore for its binding affinity to a panel of recombinant human and cynomolgus monkey FcγR extracellular domain (ECD) proteins compared to the antibody IgG1 control, trastuzumab.

[0218] Ab-h1.9d-WT binding to human FcγRs was also assessed via flow cytometry by using CHO-K1 cells engineered to express various cell surface human FcγRs.

[0219] The binding of Ab-h1.9d-WT to human and cynomolgus FcRn was assessed at pH 6.0 and pH 7.4 by BIAcore using recombinant human and cynomolgus FcRn ECD proteins (Table 31).

[0220] [Table 31]

[0221] Human and cynomolgus monkey FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa surface plasmon resonance (SPR) binding assays The binding kinetics of Ab-h1.9d-WT to His-tagged human FcγRs were determined by SPR measurements performed on a Biacore T200 instrument (GE Healthcare) at 25°C using an anti-His capture. Approximately 10,000 RU of mouse anti-His antibody (R&D) diluted to 25 μg / mL in 10 mM sodium acetate (pH 4.5) was immobilized onto a CM5 biosensor chip using a standard amine coupling kit according to the manufacturer's instructions. Unreacted sites on the biosensor surface were blocked with 1 M ethanolamine. The activated and unactivated surfaces on flow cell 1 served as references. Chip preparation and binding kinetic measurements were performed in assay running buffer, HBS-EP+ (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20). Next, human and cynomolgus monkey FcγR were captured on flow cell 2 until a capture level of 250–500 RU was reached. Ab-h1.9d-WT samples were injected over all flow cells at a flow rate of 50 μL / min for 1–5 min (1 min for human and cynomolgus monkey FcγRIIb and FcγRIIa, 2 min for human FcγRIIIa, and 5 min for human and cynomolgus monkey FcγRI and cynomolgus monkey FcγR). Analyte concentrations ranged from 0.78–200 nM for human and cynomolgus monkey FcγRI and cynomolgus monkey FcγRIII, 46.9–12,000 nM for human and cynomolgus monkey FcγRII, and 7.8–4,000 nM for FcγRII (2-fold serial dilutions). A buffer-only injection was included for double reference. Bound FcγR dissociation was monitored for 1 to 5 minutes (1 minute for FcγRIIb and FcγRIIa, 3 minutes for FcγRIIIa, and 5 minutes for FcγRI). The chip surface was regenerated with 100 mM HCl injected for 2 seconds at a flow rate of 100 μL / min across all eight channels. Three experiments using the same CM5 chip were performed for each sample. The results of these three experiments were averaged.

[0222] Human FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa cell binding assay CHO-K1-expressing hFcγR cells were 150 cm 2Cells grown in culture flasks are conjugated with a specific amount of fluorophore, CellTrace CFSE. 商標 and CellTrace Violet 商標 The cells were loaded with Ab-h1.9d-WT (Molecular Probes) according to the manufacturer's instructions, establishing a unique fluorescent footprint (barcode method) for each line. Lines were mixed and incubated with monomeric Ab-h1.9d-WT at different concentrations (0, 0.01, 0.1, 1, 10, 50, 100, and 250 μg / mL) in RPMI 1640 / 2 mM L-glutamine / 10% ultra-low IgG heat-inactivated FBS (binding medium) for 1 hour at 4°C. Following incubation, the cells were resuspended in PBS, pH 7.4 (without Ca). +2 / Mg +2 ), and then incubated with the secondary antibody (F(ab')2 goat anti-human IgG (H+L) linked to AF647 in binding medium for 15 min at 4 °C to detect cell-bound Ab-h1.9d-WT. Following incubation, the cells were resuspended in PBS, pH 7.4 (without Ca). +2 / Mg +2 The cells were washed twice more with AF647. Cell surface fluorescence was detected and recorded using a flow cytometry analyzer (LSR-Fortessa). The recorded fluorescence data was analyzed using FlowJo software version 10 (Tristar), and Ab-h1.9d-WT binding to CHO-K1 hFcγR cells was reported as the geometric mean of AF647 fluorescence (a binding curve of gMFI as a function of Ab-h1.9d-WT concentration was generated).

[0223] Human and cynomolgus monkey FcRn surface plasmon binding assay For the FcRn binding assay, Ab-h1.9d-WT was directly immobilized onto a CM5 chip amine-coupled to a density of 750 RU according to the manufacturer's protocol. Human and cynomolgus monkey FcRn recombinant proteins were injected over all flow cells at concentrations ranging from 5.5 to 12,000 nM (3-fold serial dilutions) at a flow rate of 50 μL / min for 1 min, followed by a 1-min dissociation period. The surface was regenerated with a 15-s injection of HBS-EP + pH 7.4. Samples were prepared and run in two running buffers: MES EP + pH 6.0 and HBS-EP + pH 7.4. Three experiments using different CM5 chips were performed for each sample (each in duplicate). The results of these three experiments were averaged. Data from human FcγRI, FcγRIIIa (F158), FcγRIIIa (V158), and cynomolgus monkey FcγRI and FcγRIII binding in all samples were consistent with a constant R max The data were fitted to a one-to-one global kinetic model with the following equation: Data from human FcγRIIb, FcγRIIa(H131), FcγRIIa(R131), cynomolgus monkey FcγRIIa, FcγRIIb, and FcRn binding in all samples were fitted to a steady-state affinity model. FcγR and FcRn data were fitted using Biacore T200 evaluation software version 2.0.

[0224] 7.16.2.Results Human and cynomolgus monkey FcγR1, FcγRIIa, FcγRIIb, and FcγRIIIa binding The binding kinetic parameters are summarized in Table 32 (for humans) and Table 33 (for cynomolgus monkeys).

[0225] [Table 32]

[0226] Ab-h1.9d-WT and trastuzumab had similar measurable binding affinity to both hFcγRI and hFcγRII(H131), but weaker binding to the hFcγRII(R131) and hFcγRIIb receptors. Ab-h1.9d-WT and trastuzumab had measurable binding affinity to hFcγRIIIa(F158 / V158), with higher affinity for the V158 polymorphic variant, as expected.

[0227] [Table 33]

[0228] Both Ab-h1.9d-WT and trastuzumab displayed binding to both cynomolgus FcγRI and cynomolgus FcγRIII, but no measurable binding parameters could be determined for the cynomolgus FcγRIIa and cynomolgus FcγRII receptors, likely due to their weak binding to these receptors. Human FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa cell binding assay Ab-h1.9d-WT showed the highest binding to human FcγR1 and the FcγRIIa (V176) polymorphic variant, but relatively low binding to other human FcγRs [FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa (F176), and FcγRIIIb] (Figures 19A-19B).

[0229] Human and cynomolgus monkey FcRn surface plasmon binding assay Ab-h1.9d-WT displayed measurable binding to human and cynomolgus monkey FcRn under acidic conditions (pH 6.0), but no measurable binding at neutral pH (pH 7.4) (Table 34). The FcRn-binding properties were comparable to those of the control (trastuzumab).

[0230] [Table 34]

[0231] [Example 17] 7.17.Ab-h1.9d-WT in vitro ADCC, ADCP and CDC activities 7.17.1 Materials and Methods Ab-h1.9d-WT was evaluated for in vitro ADCC and ADCP activity using native α4β7 / CD20-expressing RPMI8866 cells as target cells and reporter Jurkat cells expressing hFcγRIIIa (V158) or hFcγRIIa (H131) as effector cells. This Jurkat cell line contains an NFAT response element driving the expression of firefly luciferase as a reporter. The anti-CD20 mAb, Ab-Ritu, was used as a positive control in these assays.

[0232] Furthermore, Ab-h1.9d-WT was evaluated in a cytotoxicity-based ADCC assay using α4β7 / CD52-expressing HuT78 cells as target cells, human primary NK cells as effector cells, and Campath (anti-CD52) as a positive control antibody.

[0233] ADCC reporter assay Target α4β7-expressing RPMI8866 cells were grown and maintained in culture medium (RPMI1640, 2 mM L-glutamine, and 10% FBS). Log-phase cells were harvested, counted, washed, and diluted to 6 × 10 5 The cells / mL stocks were resuspended in assay medium (RPMI 1640 containing low IgG serum) and placed at 37°C, 5% CO2 until ready to use. Ab-h1.9d-WT and control mAbs were prepared at a 3x starting concentration of 30 μg / mL and then serially diluted 1:100 (2 pt.) in a Costar 3956 dilution plate. Antibodies (25 μL) were mixed in duplicate with an equal volume of target RPMI 8866 cells (25 μL) according to the plate layout. Effector Jurkat cells (human FcγRIIIa V158 variant and Promega 商標 Rapidly thaw 3 x 10 cells (stably expressing an NFAT response element driving the expression of firefly luciferase) in assay medium.6 The Jurkat effector cells (25 μL) were then added to the 96-well assay plate containing the RPMI8866 target and antibody. Control wells contained medium alone, effector (E) and target (T) alone, E + T, E + mAb (30 μg / mL), or T + mAb (30 μg / mL). All wells were adjusted to 75 μL per well according to the plate layout. The final cell numbers were 75,000 Jurkat effector cells / well and 15,000 RPMI8866 target cells / well, corresponding to a 5:1 E to T ratio. The final antibody concentrations per well were 67 nM (10 μg / mL), 0.67 nM (0.1 μg / mL), and 0.0067 nM (0.001 μg / mL). The plates were incubated at 37°C and 5% CO2 for 6 hours, during which time Bio-Glo 商標 The buffer and substrate (Cat7110) were equilibrated to RT prior to use. At the end of the incubation, Bio-Glo substrate was reconstituted with buffer to form the enzyme / substrate solution (Bio-Glo Reagent). An equal volume of 75 μL / well Bio-Glo was added to all wells. The plate was then incubated for 10 minutes at RT. Plate luminescence was read on a luminescence plate reader (Topcount, Perkin Elmer). Luminescence signals were plotted as RLU using GraphPad Prism 7.0 software.

[0234] ADCP reporter assay Target α4β7-expressing RPMI8866 cells were grown and maintained in culture medium (RPMI1640, 2 mM L-glutamine, and 10% FBS). Log-phase cells were harvested, counted, washed, and diluted to 2 × 10 5The cells / mL stocks were resuspended in assay medium (RPMI 1640 containing low IgG serum) and placed at 37°C, 5% CO2 until ready to use. Ab-h1.9d-WT and control mAbs were prepared at a 3x starting concentration of 30 μg / mL and then serially diluted 1:100 (2 pt.) in a Costar 3956 dilution plate. Antibodies (25 μL) were mixed in duplicate with an equal volume of target cells (25 μL) according to the plate layout. Effector Jurkat cells (human FcγRIIIa H131 variant and Promega 商標 Rapidly thaw 1 x 10 cells (stably expressing an NFAT response element driving the expression of firefly luciferase) from Invitrogen and place in assay medium. 6 The Jurkat effector cells (25 μL) were then added to the 96-well assay plate containing the RPMI8866 target and antibody. Control wells contained medium alone, effector (E) and target (T) alone, Effector (E) + Target (T), Effector (E) + mAb (30 μg / mL), or T + mAb (30 μg / mL). All wells were adjusted to 75 μL per well according to the plate layout. The final cell numbers were 25,000 Jurkat effector cells / well and 5,000 target cells / well, corresponding to a 5:1 Effector to Target ratio. The final antibody concentrations per well were 67 nM (10 μg / mL), 0.67 nM (0.1 μg / mL), and 0.0067 nM (0.001 μg / mL). The plates were incubated at 37°C and 5% CO2 for 6 hours, during which time Bio-Glo 商標 The buffer and substrate (Cat7110) were equilibrated to RT prior to use. At the end of the incubation, Bio-Glo substrate was reconstituted with buffer to form the enzyme / substrate solution (Bio-Glo Reagent). An equal volume of 75 μL / well Bio-Glo was added to all wells. The plate was then incubated for 10 minutes at RT. Plate luminescence was read on a luminescence plate reader (Topcount, Perkin Elmer). Luminescence signals were plotted as RLU using GraphPad Prism 7.0 software.

[0235] CDC assay RPMI8866 cells were grown and maintained in culture medium (RPMI1640, 2 mM L-glutamine, and 10% FBS). Log-phase cells were harvested, counted, washed, and diluted to 4 × 10 6 The cells / mL stocks were resuspended in assay medium (RPMI 1640 minus phenol red, Cat. 11835-030) and placed at 37°C, 5% CO2 until ready for use. Ab-h1.9d-WT and control mAbs were prepared at 3x starting concentrations of 45 μg / mL and 0.45 μg / mL in a Costar 3956 dilution plate. Human donor sera (HMN19169 and HMN19170) were thawed using cold running water and immediately placed on ice. Antibodies, controls (25 μL), and medium were added to an assay plate (Costar 3599). Donor serum (25 μL each), target cells (25 μL), and diluted mAb (25 μL) were diluted to 100% sera in 33% serum complement, 1x10 5 Cells and 15 μg / mL mAb were mixed in a final volume of 75 μL per well. The plates were then incubated at 37°C and 5% CO for 2 hours. After incubation, a cell-permeable dye (Sigma; Resazurin sodium salt) at 1.5 mg / mL 5x stock solution was diluted 1:5 in DPBS, and 25 μL of dye was added to each well. The plates were incubated for an additional 16 hours, and then absorbance (545 / 600) was read using a Clariostar plate reader. Percent target-specific cell lysis was calculated in Excel using the formula: 100-100 × [(absorbance with mAb incubation) / (control absorbance)], and results were plotted using GraphPad Prism 7.0 software.

[0236] ADCC cytotoxicity assay Collect the target α4β7-expressing HuT78 cells and resuspend them in PBS (w / o Ca). +2 / Mg +2 Wash twice with 1x10 PBS (1% BSA) and 7The cells were resuspended in PBS at 100 μg / mL and then labeled with CFSE at a final concentration of 2 μM for 8 minutes at room temperature. After incubation, FBS was added to a final concentration of 10% to quench the labeling. The cells were washed twice with RPMI + 10% FBS medium, and the CFSE-labeled HuT78 cells were then incubated with 100 μL of Ab-h1.9d-WT or control antibody at 2× concentration at 10 μg / mL in a 96-well V-bottom plate at 37°C and 5% CO for 30 minutes. Subsequently, 100 μL of 2.5×10 5 NK (FcγRIIIa V158) effector cells (preincubated with IL-2 at 200 U / mL) were added to target cells at a 5:1 ratio. The well contents were mixed thoroughly and then incubated for 5 hours at 37°C in a CO2 incubator. The NK and HuT78 cell mixture was washed twice with PBS and diluted to 1 × 10 with azide- and protein-free PBS containing 1 μL of FVD dye / mL. 6 The plates were resuspended at 1000 cells / mL and incubated at RT for 20 minutes. Cells were washed twice with FACS buffer, resuspended in 200 μL of 0.5% PFA in PBS, and the plates were read on a FACS (Canto II, BD). Flow data (FCS 3.0 files) were analyzed using FlowJo version 10 software. All HuT78 target cells (live and dead) were gated to determine the % dead targets within the total target cell population. The formula used to calculate % ADCC was % ADCC = 100 × [% dead targets in (E + T + Ab) mix - % dead targets in (E + T) mix] / [100 - % dead targets in (E + T) mix]. Percentage ADCC was graphed using GraphPad Prism 7.0 software.

[0237] 7.17.2.Results ADCC reporter activity, ADCP reporter activity, and CDC As expected, Ab-Ritu exhibited strong concentration-dependent in vitro ADCC and ADCP activities, whereas Ab-h1.9d-WT and the isotype control exhibited none of these activities at the three concentrations tested (10, 0.1, and 0.001 μg / mL) (Figures 20A and 20B). Only minimal signals were detected in the three negative control assay conditions: target and effector alone, target plus antibody, and effector plus antibody (data not shown). Ab-Ritu also exhibited strong in vitro CDC activity at 0.15 and 15 μg / mL (100 nM) on RPMI8866 cells, whereas Ab-h1.9d-WT and the isotype control exhibited no CDC signals at the corresponding concentrations using two human serum donors (Figure 21).

[0238] ADCC cytotoxicity In this in vitro assay, Campath (Ab-Alem) demonstrated potent cytotoxicity against target cells. In contrast, Ab-h1.9d-WT did not induce any in vitro cytotoxicity at a concentration of 10 μg / mL (67 nM) when the assay was performed using NK effector cells isolated from two different donors with the FcγRIIIa V158 genotype (Figure 22). Thus, although Ab-h1.9d-WT binds to human FcγRs as shown above (Example 12), it does not induce undesirable Fc-mediated ADCC, ADCP, and CDC activity against uninfected α4β7+ cells in vitro.

[0239] [Example 18] 7.18. Homology modeling of Ab-h1.9d-WT binding site on target 7.18.1 Materials and Methods The binding mode of Ab-h1.9d-WT to α4β7 was investigated by homology modeling. The crystal structure of recombinant human α4β7 extracellular domain protein in complex with another anti-α4β7 antibody, vedolizumab, has been published (Yu et al., J Cell Biol 2012). Based on that data and the unique sequence of the Fab region of Ab-h1.9d-WT, we set out to model the binding mode of the candidate antibody compared to the benchmark antibodies vedolizumab and AMG181.

[0240] 7.18.2.Results Consistent with the in vitro characterization data, Ab-h1.9, a humanized variant of hybridoma Ab-m1 and the parent of Ab-h1.9d-WT, and the two benchmark mAbs bind to α4β7 primarily through interactions with the β7 subunit, but also to a lesser extent with the α4 subunit. This model explains the lack of binding to α4β1 and the very low binding to αEβ7. Interestingly, this model suggests subtle differences between Ab-h1.9 and the two benchmark mAbs, in that Ab-h1.9 binds to slightly more residues on the α4 subunit. The overlapping epitopes evident in the model predict that Ab-h1.9 and vedolizumab should compete for binding to their targets. Indeed, FACS-based binding competition studies demonstrated that Ab-m1 could compete with Ab-Vedo binding to α4β7+ cells, indicating that Ab-m1 and Ab-Vedo bind to similar binding epitopes (Table 13).

[0241] 7.19. Discussion Expression levels of α4β7 on peripheral human CD4+ and CD8+ T cell subsets from HIV+ and HIV− individuals were comparable, suggesting that α4β7 expression on CD4+ T cells from HIV+ individuals can support the incorporation of α4β7 into budding HIV virions.

[0242] Ab-h1.9d-WT is a potent anti-α4β7 antibody that can bind to α4β7 on the virion envelope of all laboratory-grown HIV strains tested and HIV patient samples. The immune complex formed by Ab-h1.9d-WT and HIV virions can bind to different FcγRs through its Fc domain, which is the step by which Ab-h1.9d-WT can be phagocytosed into APCs and induce the proposed "vaccination effect" for HIV control.

[0243] Ab-h1.9d-WT binds to HIV virions but does not neutralize HIV infection, consistent with the idea that α4β7 is not a viral receptor on host cells. By targeting the α4β7 integrin, a host protein on the HIV viral envelope, Ab-h1.9d-WT may exhibit a higher barrier to resistance compared with other antibodies that target the HIV virally-encoded gp120 / 41 glycoprotein in the viral envelope, such as HIV broadly neutralizing antibodies.

[0244] Ab-h1.9d-WT can disrupt the interaction between α4β7 and its ligands, such as MadCAM-1 or HIV gp120, through a Fab-dependent mechanism, inhibiting MadCAM-1- or gp120-mediated CD4 T cell costimulation and potentially inhibiting HIV replication in these stimulated cells.

[0245] Ab-h1.9d-WT can potentially inhibit cell-to-cell HIV viral transmission by a Fab-mediated mechanism through its ability to disrupt the interaction between α4β7 and HIV gp120.

[0246] Compared with Ab-h1.9d-WT, Ab-Vedo showed reduced activity in capturing HIV virions and disrupting the interaction between α4β7 and HIV gp120. Furthermore, although Ab-Vedo was able to bind to HIV virions and form immune complexes, these immune complexes bound to FcγR with much lower affinity than complexes formed by Ab-h1.9d-WT due to engineered mutations in the Ab-Vedo Fc domain that reduced Fc function.

[0247] Vedolizumab has demonstrated moderate efficacy in two clinical trials for HIV research. This efficacy can be attributed to Fab dependence (e.g., antibody binding to α4β7, disrupting its interaction with its ligands, such as MAbCAM-1 and HIV gp120, thus inhibiting CD4 T cell costimulation, HIV replication in these stimulated cells, and cell-to-cell viral spread), but not to an Fc-dependent mechanism of action. Vedolizumab's reduced binding affinity for FcγRs renders it poorly able to mediate Fc-dependent mechanisms. In contrast, Ab-h1.9d-WT possesses intact Fc functionality and is distinct from vedolizumab in its ability to induce sustained HIV viral suppression through its Fc-dependent mechanism of action. Induction of a new, durable HIV-specific immune response (vaccination effect) requires the binding of immune complexes formed between HIV virions and anti-α4β7 antibodies (with intact Fc domains) to FcγRs on APCs. Indeed, Ab-h1.9d-WT can mediate the uptake of α4β7-coated beads or α4β7-expressing GFP+ VLPs (virus-like particles) in THP-1 cells in an α4β7- and Fc-dependent manner. Collectively, Ab-h1.9d-WT demonstrates activity across several proposed mechanisms for HIV control, including Fc- and Fab-dependent mechanisms. Therefore, Ab-h1.9d-WT is expected to be a more potent agent than vedolizumab for sustained reduction of HIV viral load due to its higher affinity for α4β7 and its intact Fc functionality, which induces a "vaccination effect."

[0248] The key attributes of Ab-h1.9d-WT from comprehensive in vitro characterization are summarized in Table 35.

[0249] [Table 35]

[0250] Ab-h1.9d-WT is an antagonistic anti-α4β7 human IgG1 / κ monoclonal antibody that binds to α4β7 but not to α4β1, but minimally to αEβ7. Ab-h1.9d-WT strongly binds to both human and cynomolgus monkey CD4+ and CD8+ T subsets, demonstrating excellent cynomolgus monkey binding cross-reactivity. However, Ab-h1.9d-WT does not bind to rodent PBMCs. Ab-h1.9d-WT selectively blocks the α4β7 / MAdCAM-1 interaction with high potency without inhibiting the α4β7 / VCAM-1 interaction. Ab-h1.9d-WT can block MAdCAM-1-mediated costimulation of human primary CD4+ T cells. As expected for human IgG1, Ab-h1.9d-WT binds to human FcγRs in vitro against uninfected α4β7+ cells without triggering ADCC, ADCP, or CDC activity (an essential requirement for Fc-mediated "vaccination effect" in vivo). The lack of in vitro Fc effector activity by Ab-h1.9d-WT may be partially explained by reduced cell surface α4β7 expression due to antibody-induced target internalization. Furthermore, Ab-h1.9d-WT can mediate the uptake of α4β7-coated beads or α4β7-expressing GFP+ VLPs (virus-like particles) in THP-1 cells in an α4β7- and Fc-dependent manner. Ab-h1.9d-WT exhibits the intended in vitro pharmacological properties required for a clinical candidate.

[0251] As provided in this disclosure, Ab-h1.9d-WT is a potent α4β7-selective antagonist that is distinct from and improved upon vedolizumab. Key properties of Ab-h1.9d-WT compared to Ab-Vedo are shown in Tables 36-40 and summarized in Table 41.

[0252] [Table 36]

[0253] [Table 37]

[0254] [Table 38]

[0255] Table 39 - Binding affinity of Ab-h1.9d-WT to human FcγRs compared to Ab-Vedo by BIAcore

[0256] [Table 39]

[0257] Table 40 - Binding affinity of Ab-h1.9d-WT to cynomolgus monkey FcγR compared to Ab-Vedo by BIAcore

[0258] [Table 40]

[0259] Table 41 - Key characteristics of Ab-h1.9d-WT compared to Ab-Vedo

[0260] [Table 41]

[0261] Key parameters of Ab-h1.9d-WT, such as its better binding affinity for α4β7 and its ability to bind human FcγRs without triggering Fc-mediated effector functions, are important differentiators from vedolizumab, and these properties are expected to drive improved potency over vedolizumab. Ab-h1.9d-WT also possesses the in vitro pharmacological properties required for an anti-α4β7 clinical candidate.

[0262] 8. Exemplary Embodiments While various specific embodiments have been illustrated and described, some of which are illustrated below, it will be recognized that various modifications can be made without departing from the spirit and scope of the invention. 1. An anti-human α4β7 antibody comprising: (i) a VH chain region comprising three CDRs; and (ii) a VL chain region comprising three CDRs, VH CDR#1 is GFNIKNTYMH (SEQ ID NO: 72); VH CDR#2 is RIDPAKGHTEYAPKFLG (SEQ ID NO: 73); VH CDR#3 is VDV (SEQ ID NO: 74); VL CDR#1 is HASQDISDNIG (SEQ ID NO: 75); VL CDR#2 is HGTNLED (SEQ ID NO: 76); and VL CDR#3 is VQYAQFPWT (SEQ ID NO: 77), Anti-human α4β7 antibody. 2. EVQLVQSGAEVKKPGSSVKVSCKASGFNIKNTYMHWVRQAPGQGLEWIGRIDPAKGHTEYAPKFLGRVTITADESTNTAYMELSSLRSEDTAVYYCYYVDVWGQGTTVTVSS (SEQ ID NO: 70) the VH chain region of DIQMTQSPSSLSASVGDRVTITCHASQDISDNIGWLQQKPGKSFKLLIYHGTNLEDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTFGGGTKVEIK (SEQ ID NO: 71) VL chain region of 2. The anti-human α4β7 antibody of embodiment 1, comprising: 3. The anti-human α4β7 antibody of embodiment 1 or 2, which is humanized. 4. The anti-human α4β7 antibody according to any one of embodiments 1 to 3, which is an IgG. 5. The anti-human α4β7 antibody of any of embodiments 1 to 4, comprising a kappa light constant region. 6. An anti-human α4β7 antibody according to any one of embodiments 1 to 5, which is an IgG1. 7. An anti-human α4β7 antibody according to any one of embodiments 1 to 6, comprising a variant CH3 domain having the amino acid substitutions D356E and L358M. 8. An anti-human α4β7 antibody according to any one of embodiments 1 to 7, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 93, and a light chain having the amino acid sequence of SEQ ID NO: 100. 9. The anti-human α4β7 antibody of any one of embodiments 1 to 8, comprising a variant CH2 domain having the amino acid substitutions L234A and / or L235A. 10. The anti-human α4β7 antibody of any one of embodiments 1 to 9, comprising a variant CH2 domain having the amino acid substitution T250Q and / or a variant CH3 domain having the amino acid substitution M428L. 11. A polynucleotide comprising a nucleotide sequence encoding an anti-human α4β7 antibody, wherein the antibody comprises: (i) a VH chain region comprising three CDRs; and (ii) a VL chain region comprising three CDRs; VH CDR#1 is GFNIKNTYMH (SEQ ID NO: 72); VH CDR#2 is RIDPAKGHTEYAPKFLG (SEQ ID NO: 73); VH CDR#3 is VDV (SEQ ID NO: 74); VL CDR#1 is HASQDISDNIG (SEQ ID NO: 75); VL CDR#2 is HGTNLED (SEQ ID NO: 76); and VL CDR#3 is VQYAQFPWT (SEQ ID NO: 77), Polynucleotide. 12. An expression vector comprising the polynucleotide of embodiment 11. 13. A eukaryotic host cell transfected with the vector of embodiment 12. 14. A eukaryotic host cell engineered to express the polynucleotide of embodiment 11. 15. The eukaryotic host cell of embodiment 13 or 14, which is a mammalian host cell. 16. A method for producing an anti-human α4β7 antibody, comprising the steps of (a) culturing the eukaryotic host cell of embodiment 15, and (b) recovering the anti-human α4β7 antibody. 17. A prokaryotic host cell transformed with a vector according to embodiment 12. 18. A prokaryotic host cell engineered to express the polynucleotide of embodiment 17. 19. The prokaryotic host cell of embodiment 18, which is a bacterial host cell. 20. A method for producing an anti-human α4β7 antibody, comprising the steps of (a) culturing the prokaryotic host cell of embodiment 19, and (b) recovering the anti-human α4β7 antibody. 21. A method for inducing viral suppression of HIV infection in an HIV-infected subject, comprising administering to the subject an amount of an anti-human α4β7 antibody described in any one of embodiments 1 to 10. 22. The method of embodiment 21, wherein viral suppression is immune-mediated. 23. A method for treating HIV infection in an HIV-infected subject, comprising administering to the subject an amount of an anti-human α4β7 antibody according to any one of embodiments 1 to 10. 24.Anti-human α4β7 antibody that suppresses HIV. 25. The anti-human α4β7 antibody of embodiment 24, which inhibits HIV replication and / or HIV infection. 26. An anti-human α4β7 antibody according to embodiment 24, which inactivates human α4β7 and / or reduces the activation of human α4β7 on cells expressing human α4β7. 27. An anti-human α4β7 antibody according to embodiment 24, which induces internalization of human α4β7 on cells expressing human α4β7. 28. The anti-human α4β7 antibody of embodiment 24, which inhibits CD4 T cell costimulation. 29. The anti-human α4β7 antibody of embodiment 28, wherein the CD4 T cell costimulation is mediated by MAdCAM-1 or HIV gp120. 30. The anti-human α4β7 antibody of embodiment 25, which inhibits HIV replication in CD4 T cells. 31. The anti-human α4β7 antibody of embodiment 30, wherein the CD4 T cells are MAdCAM-1-stimulated CD4 T cells or HIV gp120-stimulated CD4 T cells. 32. The anti-human α4β7 antibody of embodiment 24, which disrupts at least one interaction between α4β7 and its ligand. 33. An anti-human α4β7 antibody according to embodiment 32, wherein the α4β7 ligand is MAdCAM-1. 34. The anti-human α4β7 antibody of embodiment 32, wherein the α4β7 ligand is HIV gp120. 35. An anti-human α4β7 antibody according to embodiment 34, which inhibits gp120-mediated cell-to-cell spread of HIV. 36. The anti-human α4β7 antibody of embodiment 24, which binds to HIV virions. 37. The anti-human α4β7 antibody of embodiment 36, wherein the antibody binding to HIV virions forms an immune complex. 38. The anti-human α4β7 antibody of embodiment 37, wherein the immune complex binds to FcγR on a cell. 39. The anti-human α4β7 antibody of embodiment 37, wherein the immune complex binds to FcγR on antigen-presenting cells (APCs). 40. The anti-human α4β7 antibody of embodiment 39, wherein the immune complex is taken up by the APC by phagocytosis. 41. An anti-human α4β7 antibody according to embodiment 40, which induces an HIV-specific immune response. 42. An anti-human α4β7 antibody according to embodiment 40 or 41, which induces a vaccination effect against HIV. 43. The anti-human α4β7 antibody of embodiment 41, wherein the HIV-specific immune response results in viral control of HIV in HIV-infected individuals. 44. The anti-human α4β7 antibody of embodiment 43, wherein the viral control results in a reduction in viral load. 45. The anti-human α4β7 antibody of embodiment 43, wherein the viral control results in a lower viral set point. 46. ​​The anti-human α4β7 antibody of embodiment 43, wherein viral control results in delayed viral rebound after antiretroviral treatment interruption (ATI). 47. An anti-human α4β7 antibody according to any one of embodiments 24 to 46, having a set of six complementarity determining regions (CDRs) or a variable heavy chain region and a variable light chain region from an antibody selected from Ab-m1, Ab-c1, Ab-h1.1, Ab-h1.2, Ab-h1.3, Ab-h1.4, Ab-h1.5, Ab-h1.6, Ab-h1.7, Ab-h1.8, Ab-h1.9, Ab-h1.9a, Ab-h1.9b, Ab-h1.9c, Ab-h1.9d, or Ab-h1.9e. 48. An anti-human α4β7 antibody according to any one of embodiments 1 to 10, which inhibits HIV. 49. An anti-human α4β7 antibody according to embodiment 48, which inhibits HIV replication and / or HIV infection. 50. An anti-human α4β7 antibody according to embodiment 48, which inactivates human α4β7 and / or reduces the activation of human α4β7 on cells expressing human α4β7. 51. An anti-human α4β7 antibody according to embodiment 48, which induces internalization of human α4β7 on cells expressing human α4β7. 52. The anti-human α4β7 antibody of embodiment 48, which inhibits CD4 T cell costimulation. 53. The anti-human α4β7 antibody of embodiment 52, wherein the CD4 T cell costimulation is mediated by MAdCAM-1 or HIV gp120. 54. The anti-human α4β7 antibody of embodiment 49, which inhibits HIV replication in CD4 T cells. 55. The anti-human α4β7 antibody of embodiment 54, wherein the CD4 T cells are MAdCAM-1-stimulated CD4 T cells or HIV gp120-stimulated CD4 T cells. 56. The anti-human α4β7 antibody of embodiment 48, which disrupts at least one interaction between α4β7 and its ligand. 57. An anti-human α4β7 antibody according to embodiment 56, wherein the α4β7 ligand is MAdCAM-1. 58. The anti-human α4β7 antibody of embodiment 56, wherein the α4β7 ligand is HIV gp120. 59. An anti-human α4β7 antibody according to embodiment 58, which inhibits gp120-mediated cell-to-cell spread of HIV. 60. The anti-human α4β7 antibody of embodiment 48, which binds to HIV virions. 61. The anti-human α4β7 antibody of embodiment 60, wherein the antibody binding to HIV virions forms an immune complex. 62. The anti-human α4β7 antibody of embodiment 61, wherein the immune complex binds to FcγR on cells. 63. The anti-human α4β7 antibody of embodiment 61, wherein the immune complex binds to FcγR on antigen-presenting cells (APCs). 64. The anti-human α4β7 antibody of embodiment 63, wherein the immune complex is taken up by the APC by phagocytosis. 65. An anti-human α4β7 antibody according to embodiment 64, which induces an HIV-specific immune response. 66. An anti-human α4β7 antibody according to embodiment 64 or 65, which induces a vaccination effect against HIV. 67. An anti-human α4β7 antibody according to embodiment 64 or 65, wherein the HIV-specific immune response results in viral control of HIV in HIV-infected individuals. 68. The anti-human α4β7 antibody of embodiment 67, wherein the viral control results in a reduction in viral load. 69. The anti-human α4β7 antibody of embodiment 67, wherein viral control results in a lower viral set point. 70. The anti-human α4β7 antibody of embodiment 67, wherein viral control results in delayed viral rebound after antiretroviral treatment interruption (ATI). 71. A pharmaceutical composition comprising the antibody of any one of the preceding embodiments.

Claims

1. A polynucleotide comprising a nucleotide sequence encoding an anti-human α4β7 antibody, wherein the antibody comprises: (i) a VH chain region comprising three CDRs; and (ii) a VL chain region comprising three CDRs; VH CDR#1 is GFNIKNTYMH (SEQ ID NO:72); VH CDR#2 is RIDPAKGHTEYAPKFLG (SEQ ID NO: 73); VH CDR#3 is VDV (SEQ ID NO:74); VL CDR#1 is HASQDISDNIG (SEQ ID NO:75); VL CDR#2 is HGTNLED (SEQ ID NO:76); and VL CDR#3 is VQYAQFPWT (SEQ ID NO: 77); Polynucleotide.

2. An expression vector comprising the polynucleotide of claim 1.

3. A eukaryotic host cell transfected with the vector of claim 2.

4. The eukaryotic host cell of claim 3, which is a mammalian host cell.

5. A method for producing an anti-human α4β7 antibody, comprising the steps of (a) culturing the eukaryotic host cell of claim 4 and (b) recovering the anti-human α4β7 antibody.

6. 1. A pharmaceutical composition for use in a method of inducing viral suppression of HIV infection in an HIV-infected subject, comprising: the pharmaceutical composition comprises an amount of an anti-human α4β7 antibody; The method includes administering the pharmaceutical composition to a subject; The anti-human α4β7 antibody An anti-human α4β7 antibody comprising: (i) a VH chain region comprising three CDRs; and (ii) a VL chain region comprising three CDRs, VH CDR#1 is GFNIKNTYMH (SEQ ID NO:72); VH CDR#2 is RIDPAKGHTEYAPKFLG (SEQ ID NO: 73); VH CDR#3 is VDV (SEQ ID NO:74); VL CDR#1 is HASQDISDNIG (SEQ ID NO:75); VL CDR#2 is HGTNLED (SEQ ID NO:76); and VL CDR#3 is VQYAQFPWT (SEQ ID NO: 77); The pharmaceutical composition is an anti-human α4β7 antibody.

7. 1. A pharmaceutical composition for use in a method of treating an HIV infection in an HIV-infected subject, comprising: the pharmaceutical composition comprises an amount of an anti-human α4β7 antibody; The method includes administering the pharmaceutical composition to a subject; The anti-human α4β7 antibody An anti-human α4β7 antibody comprising: (i) a VH chain region comprising three CDRs; and (ii) a VL chain region comprising three CDRs, VH CDR#1 is GFNIKNTYMH (SEQ ID NO:72); VH CDR#2 is RIDPAKGHTEYAPKFLG (SEQ ID NO: 73); VH CDR#3 is VDV (SEQ ID NO:74); VL CDR#1 is HASQDISDNIG (SEQ ID NO:75); VL CDR#2 is HGTNLED (SEQ ID NO:76); and VL CDR#3 is VQYAQFPWT (SEQ ID NO: 77); The pharmaceutical composition is an anti-human α4β7 antibody.

8. A pharmaceutical composition comprising an antibody, The antibody An anti-human α4β7 antibody comprising: (i) a VH chain region comprising three CDRs; and (ii) a VL chain region comprising three CDRs, VH CDR#1 is GFNIKNTYMH (SEQ ID NO:72); VH CDR#2 is RIDPAKGHTEYAPKFLG (SEQ ID NO: 73); VH CDR#3 is VDV (SEQ ID NO:74); VL CDR#1 is HASQDISDNIG (SEQ ID NO:75); VL CDR#2 is HGTNLED (SEQ ID NO:76); and VL CDR#3 is VQYAQFPWT (SEQ ID NO: 77); The pharmaceutical composition is an anti-human α4β7 antibody.

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