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WO2023137161A1 - Triple blocage de tigit, cd112r et pd-l1 - Google Patents

Triple blocage de tigit, cd112r et pd-l1 Download PDF

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WO2023137161A1
WO2023137161A1 PCT/US2023/010775 US2023010775W WO2023137161A1 WO 2023137161 A1 WO2023137161 A1 WO 2023137161A1 US 2023010775 W US2023010775 W US 2023010775W WO 2023137161 A1 WO2023137161 A1 WO 2023137161A1
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amino acid
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Sue J. SOHN
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Amgen Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Nivolumab and pembrolizumab are two such inhibitors that target the PD-1 pathway, and each has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic melanoma.
  • FDA U.S. Food and Drug Administration
  • Studies of checkpoint inhibitors in combination with other agents are underway or recently have been completed. The combination of nivolumab and ipilimumab, a CTLA-4 receptor blocking antibody, for example, was tested in a Phase III clinical trial on patients with unresectable stage III or IV melanoma.
  • TIGIT and CD112R are data demonstrating the induction of TIGIT and CD112R on activated human T cells and TILs (tumor infiltrating leukocytes) in primary human tumor tissues as well as data supporting the high co-expression levels of the ligands of TIGIT and CD112R (CD155 and CD112) on tumor cells.
  • the data provided herein support that, while blocking the single interaction of TIGIT or CD112R with its ligand enhances primary human T cell activity, the simultaneous blockade of both receptors (TIGIT and CD112R) from binding to their respective ligands greatly enhances primary human T cell activity.
  • the data furthermore support that blockade of yet a third interaction involving PD-1 and its ligand, in addition to the blockade of TIGIT and CD112R interactions, significantly increases the overall primary human T cell activity.
  • the increase in activity achieved with the blockade of all three molecules (PD-1, TIGIT, CD112R) is beyond that achieved with single blockade (TIGIT only or CD112R only) and double blockade (blockade of both TIGIT and CD112R, both TIGIT and PD-1, or both CD112R and PD-1).
  • the present disclosure provides TIGIT antigen binding proteins (e.g., antibodies and antigen binding fragments thereof), CD112R antigen binding proteins (e.g., antibodies and antigen binding fragments thereof), and combinations thereof.
  • Compositions comprising TIGIT antigen binding proteins, CD112R antigen binding proteins and PD-1 antigen binding proteins are furthermore provided by the present disclosure.
  • the compositions comprise a TIGIT antibody or TIGIT-binding fragment thereof and/or a CD112R antibody or CD112R-binding fragment thereof, and/or a PD-1 antibody or PD-1 binding fragment thereof.
  • compositions comprise a TIGIT antibody or TIGIT-binding fragment thereof and/or a CD112R antibody or CD112R-binding fragment thereof, and/or a PD-L1 antibody or PD-L1 binding fragment thereof.
  • the composition comprises a TIGIT antibody and a CD112R antibody, and, optionally further comprising a PD-L1 antibody.
  • Related conjugates, fusion proteins, nucleic acids, vectors, host cells and kits are provided herein.
  • the present disclosure also provides pharmaceutical compositions comprising a TIGIT antigen binding protein, CD112R antigen binding protein, or combinations thereof, optionally, further comprising a PD-1 antigen binding protein or a PD-L1 antigen binding protein, or a conjugate, fusion protein, nucleic acid, vector, or host cell, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical composition comprises a 1:1 ratio of a TIGIT antibody and a CD112R antibody.
  • the pharmaceutical composition comprises a 1:1:1 ratio of a TIGIT antibody, a CD112R antibody, and a PD-1 antibody or a PD-L1 antibody.
  • Methods of making the antigen binding proteins are provided. Also provided herein are methods of treating a subject comprising administering to the subject a CD112R antigen-binding protein, a TIGIT antigen-binding protein, and a PD-L1 antigen-binding protein, optionally, an anti- CD112R antibody, an anti-TIGIT antibody, and an anti-PD-L1 antibody.
  • each antigen-binding protein is separately formulated and administered to the subject.
  • at least two of the antibodies are formulated together and thus administered together.
  • at least the anti-CD112R and anti-TIGIT antibodies are co-formulated and co- administered and the anti-PD-L1 antibody is administered separately.
  • the method comprises administering a composition comprising all three antigen-binding proteins.
  • Figure 1A is a series of plots depicting co-expression profiles of cells of the indicated tumor indication. In the top row, co-expression of TIGIT family members with each other and with PD-1 is shown. In the bottom row, co-expression of ligands of the TIGIT family members with each other and with PD-1 is shown.
  • Figure 1B is a series of plots depicting the expression of TIGIT, CD112R, CD226, or PD-1 (data based on single cell RNA seq data).
  • Figure 1C is a series of FACS plots depicting the co-expression of TIGIT, CD112R, and PD-1.
  • Figure 1D is a table listing the % of CD4 T-cells, CD8 T-cells, or Natural Killer (NK) cells positive for expression of CD112R, TIGIT, or PD-1 in tumor infiltrating T/NK cells.
  • Figure 1E is a series of plots depicting the expression of Epcam, CD45, CD112, CD155, CD11c, and CD11b in tumor vs. PBMC.
  • FIG. 2A is an illustration of a Jurkat reporter gene assay (RGA).
  • the assay system uses engineered CHO cells that stably express CD112 and CD3 engager and purified human pan T cells pre-activated with CD3/CD28 antibodies.
  • CD112R expressed on the surface of the T cells binds to CD112 expressed on the surface of the CHO cells, IL-2 release is expected to be suppressed.
  • Figure 2B are graphs depicting the increase of CD112R expression of activated T-cells (right) relative to not activated T-cells (left).
  • FIG. 2C is a graph demonstrating the binding between antibodies or ligand to CHO cells expressing human CD112R (represented by GeoMean fold) plotted as a function of concentration of tool antibodies (PL-52575, PL-52576, and PL-52577) or human or mouse IgG matched control antibodies (HuIgG isotype and MuIgG isotype, respectively). The binding of CD112 ligand is also shown.
  • Figure 2D is a graph demonstrating the calculated % inhibition of the binding between the ligand and human CD112R expressed on CHO cells plotted as a function of concentration of tool antibodies (PL-52575, PL-52576, and PL-52577) or human or mouse IgG matched control antibodies (HuIgG isotype and MuIgG isotype, respectively).
  • Figure 2E is a graph of IL-2 concentration (pg/mL) plotted as a function of concentration of tool antibodies (PL-52575, PL-52576, and PL-52577) or human or mouse IgG matched control antibodies (HuIgG isotype and MuIgG isotype, respectively) upon interaction with CHO cells transfected with empty vector (Vector) or vector encoding CD112 (CD112).
  • EC50 for each tool antibody is indicated in the table below the X-axis.
  • Figure 2F is a graph of IL-2 concentration (pg/mL) plotted as a function of concentration of CD226 antibody or isotype-matched control antibody in an assay where T cell are co-cultured with CHO cells transfected with empty vector (Vector-CHO) or vector encoding CD112 (CD112-CHO). In one instance, T-cells without any antibody is shown.
  • Figure 3 is a schematic of the screening cascade utilized to discover anti-CD112R antagonist antibodies.
  • Figure 4A is a schematic of the Jurkat reporter gene assay (RGA).
  • Figure 4B is a graph of the fold-induction of luciferase activity plotted for Harvests 6-9.
  • Figure 4C is a graph of the binding activity to primary cyno T cells plotted for a panel of 350 hits and for positive binders.
  • Figure 5A is a table listing characteristics of Harvests 1-3 and Figure 5B is a table listing characteristics of Harvests 6-9.
  • Figure 6 is a graph of the relative binding activity for CD112R antibodies with 100 pM Kd as an arbitrary cut off threshold.
  • Figure 7A is a graph of the Clading results showing the sequence diversity of antibodies.
  • Figure 7B is a table listing exemplary EC50 values of the indicated antibodies and germline and HC CDR3 sequence information.
  • Figure 8 is a graph of the % inhibition of the binding achieved by the indicated antibody or tool antibody (PL-52575, PL-52577). Irrelevant mouse and human antibodies are used as controls. The table below the X-axis lists EC50 values and the max % inhibition for each tool antibody.
  • Figure 9A is a graph of the signal during the different stages of the competition assay for three scenarios: A2, B2, and F2, wherein A2 is when two different antibodies are used to determine if the second antibody competes with the first antibody for binding to ligand. B2 is when the same antibody is used as throughout the assay. F2 is when an irrelevant control antibody is used.
  • Figure 9B is a table listing antibodies that compete with each other (Bin A) for binding to ligand, as determined by the competition assay.
  • Figures 10A and 10B is a graph of the fold over isotype control plotted as a function of concentration of the indicated antibody.
  • the results of the assay using cyno PBMCs and human T- cells are shown in Figures 10A and 10B, respectively.
  • the graphs of Figures 10A and 10B plot the fold over isotype control signal plotted as a function of the log concentration of the indicated antibody.
  • Figure 11A is a graph of the NFAT luciferase activity plotted as a function of concentration of the indicted antibody.
  • Figure 11B is a table listing the EC50 of the antibody as determined in the Jurkat RGA.
  • Figure 12A is a graph of the % inhibition of binding of TIGIT to CD155-Fc plotted as a function of tool TIGIT antibody concentration.
  • Figure 12B is a graph of the % inhibition of binding to CD112-Fc as a function of antibody concentration.
  • Figure 12C is an illustration of a cellular assay to test activity of tool TIGIT antibodies.
  • Figure 12D is a graph of the binding of CD226-Fc to the different cells of Figure 12C without any antibodies.
  • Figure 12E is a graph of the binding of CD226-Fc in the presence of the indicated concentration of tool antibody or control antibody.
  • Figure 12F is a graph of the concentration of IFN ⁇ made by T-cells in the presence of the indicated concentration of tool antibody or control antibody.
  • Figure 12G is a graph of the concentration of NFAT luciferase activity induced in the presence of the indicated concentration of tool antibody or control antibody.
  • Figure 12H is a graph of the binding in the presence of the indicated concentration of tool antibody or control antibody.
  • 1F4 is a tool antibody like 10A7 and MBSA43.
  • Figure 12I is a series of plots demonstrating the binding activity of the indicated antibody to primary pre-activated cyno T- cells relative to isotype control.
  • Figure 13 is a schematic of the screening assays utilized to discover anti-TIGIT antibodies.
  • FIG 14A is schematic of the Jurkat reporter gene assay (RGA). CHO cells expressing CD3 engager and CD155 are co-cultured with Jurkat T-cells expressing an NFAT-luciferase construct and TIGIT in the presence of antibodies or controls.
  • Figure 14B is a graph of the NFAT-luciferase activity induced in the presence of the indicated TIGIT antibody or tool antibody (MBSA43) or control antibody (human IgG4 isotype control, human IgG2 isotype control, murine IgG1 isotype control).
  • Figure 14C is a graph of the IFN ⁇ made by T-cells in the presence of the indicated TIGIT antibody or tool antibody (MBSA43) or control antibody (human IgG isotype control, murine IgG1 isotype control).
  • Figure 15 is a graph of the luciferase activity induced in the presence of the indicated TIGIT antibody (AB1 or AB2) or tool antibody (MBSA43) by Jurkat T cells transfected with the IL-2- Luciferase reported construct and TIGIT. One set of cells was engineered to knock out CD226 expression (CD226KO).
  • Figure 16A is a schematic of an IFN ⁇ release assay using T cells expressing TIGIT, CD226 and CD112R and CHO cells expressing CD155, CD112, and an scFV anti-CD3.
  • Figure 16B is a graph of the IFN ⁇ released in the presence of the indicated combinations of antibodies.
  • HuIgG1 and mIgG1 are isotype matched control antibodies.
  • Tool CD112R antibodies include PL-52577.
  • Figure 17D is a graph of the expected vs. observed results of the IFN ⁇ release assay for the indicated combination of antibodies or for the single antibody.
  • Figure 17E is a graph of the IFN ⁇ released in the presence of the indicated combinations of antibodies of 2 or three antibodies or single antibody in dissociated human tumor cell assay. IFN ⁇ concentration of supernatant collected on Day 3 vs Day 6 is shown.
  • Figure 17F is a graph of the % of cells positive for expression for indicated molecules in CD4 T cells in PBMC or tumor cells.
  • Figure 17G is a graph of the % of cells positive for expression for indicated molecules in CD8 T cells in PBMC or tumor cells.
  • Figure 17H is a graph of the % of cells positive for expression of PD-1 on Day 6 of dissociated tumor cell cultures wherein the cells were treated with anti-TIGIT antibody, anti-CD112R antibody or isotype control antibody.
  • Figure 17I is a graph of the % of cells positive for expression of TIGIT on Day 6 of dissociated tumor cell cultures wherein the cells were treated with anti-PD-1 antibody, anti-CD112R antibody or isotype control antibody.
  • Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein.
  • FIG. 19A and 19B are graph of the amount of IFN- ⁇ (pg/mL) produced by cytotoxic T lymphocytes upon stimulation with a formulation comprising anti-CD112R mAb (24F1), anti-TIGIT mAb (43B7.002.015) and anti-PD-1 mAb at varying ratios as indicated.
  • the total antibody concentration of the formulations in Figures 19A and 19B is 1.5 nM and 30 nM, respectively.
  • Figures 20A-20C are graphs relating to the expression of TIGIT, CD112R, and the ligands CD155, CD112, and PDL1 by ex vivo primary human tumor tissues and matching blood, as determined by FACS analysis.
  • TIGIT+ and CD112R+ cells are graphed in Figure 20A, and the percentages of TIGIT+ and CD112R+ cells expressed by combined T/NK among tumor infiltrating lymphocytes (TIL) or blood are shown in Figure 20B.
  • Ligand expression was analyzed on a subset of the samples, for which the percentages of CD155-, CD112-, and PD-L1- positive cells in EpcamHI tumor cells are shown in Figure 20C.
  • the connecting lines indicate values from individual donors. These data represent combined results from tumor tissues from four indications (PANC, CRC, GIST, and TNBC).
  • Figure 21 is a graph of the mean serum mAb concentration plotted as a function of time post IV administration to male cynomolgus monkeys.
  • the line with circles plots the mean serum concentration of anti-TIGIT mAb of Group 1 animals
  • the line with squares plots the mean serum concentration of anti-CD112R mAb of Group 1 animals
  • the line with triangles (pointing up) plots the mean serum concentration of anti-TIGIT mAb of Group 2 animals
  • the line with triangles (pointing down) plots the mean serum concentration of anti-CD112R mAb of Group 3 animals.
  • Figures 22A-22D demonstrate CD112R+TIGIT blockade additively enhances human primary NK cell activity against tumor cells.
  • Figure 22A is a graph of the % target positive NK cells where targets are CD226, TIGIT, CD112R, PD-1 or CD96, showing that purified human NK cells express high levels of CD226, TIGIT, and CD112R and low levels of PD-1 and CD96.
  • Figure 22B is a graph of the % ligand-positive cells expressing CD155, CD112, or PDL1 and shows target tumor cells used in the assay (SKBR3 tumor cell line) express high levels of CD155 and CD112 and a low level of PD-L1.
  • Figure 22C is a graph of the extent of tumor cell killing (relative to isotype control antibody) by the purified human NK cells stimulated by anti-PD-1 antibody, anti-TIGIT antibody, anti-CD112R antibody, or a combination of anti-TIGIT antibody and anti-CD112R antibody, or a combination of anti-PD-1 antibody, anti-TIGIT antibody, and anti-CD112R antibody (3X).
  • Figure 22D is a graph of the extent of IFN ⁇ production (relative to isotype control antibody) stimulated by the indicated antibody mixtures comprising anti-PD-1 antibody, anti-TIGIT antibody, anti-CD112R antibody, or a combination of anti-TIGIT antibody and anti-CD112R antibody, or a combination of anti-PD-1 antibody, anti-TIGIT antibody, and anti-CD112R antibody (3X).
  • Figures 22C and 22D show that individual blockade of TIGIT or CD112R enhanced NK cell activity compared to isotype (HuIgG1) or PD-1 antibody-treated cells, but the blockade of both TIGIT and CD112R additively enhanced both tumor cell killing and IFNg production at 16hrs.
  • NK cell activity is shown as fold change over isotype control. Each mAb was added at 10ug/ml.
  • a single cell suspension generated from dissociated ex vivo tumor tissue was cultured in the presence of the indicated antibody mixtures comprising anti-PD-1 antibody, anti-TIGIT antibody, anti-CD112R antibody, or a combination of anti-TIGIT antibody and anti-CD112R antibody, or a combination of anti-PD-1 antibody, anti-TIGIT antibody, and anti-CD112R antibody (10ug/ml each) and T/NK cell activity was measured by IFNg levels in the supernatant on day 3.
  • Figure 23 is a graph of the increase (relative to isotype control antibody) in primary TIL response in dissociated ex vivo tumor tissue, showing that the mixture comprising all three antibodies targeting CD112R+TIGIT+PD-1 at a ratio of 1:1:1 stimulates the highest TIL response. The average values from 5 different tissues and error bars indicating SEM are shown in this graph.
  • Figures 24A-24C represent graphs of the tumor growth response over time in several different mouse tumor models.
  • Figure 24A shows tumor growth in CT26 syngeneic tumor model, where tumor volume is plotted as a function of days post tumor implantation in wildtype (WT) mice, TIGITxCD112R KO (double KO) mice, CD112R KO mice, or TIGIT KO mice, that were treated with isotype control or anti-PD1 antibody.
  • the numbered groups correspond to (1) WT mice treated with an isotype control antibody, (2) TIGITxCD112R KO mice treated with an isotype control antibody, (3) WT mice treated with anti-PD-1 antibody, (4) CD112R KO mice treated with anti-PD-1 antibody, (5) TIGIT KO mice treated with anti-PD-1 antibody and (6) TIGITxCD112R KO mice treated with anti-PD-1 antibody.
  • Figure 24B is a graph of the tumor volume in B16F10 syngeneic tumor model where group numbers correspond to (1) WT mice treated with an isotype control antibody, (2) WT mice treated with an anti-PD-1 antibody, (3) TIGITxCD112R KO (dKO) mice treated with an isotype control antibody, and (4) TIGITxCD112R KO (dKO) mice treated with anti- PD-1 antibody.
  • Figure 24C is a graph of the tumor volume measured over time in a xenograft model treated with (1) isotype control antibody, (2) anti-PD-1 antibody, (3) a combination formulation comprising anti-TIGIT mAb and anti-CD112R mAb, and (4) a combination formulation comprising anti-TIGIT mAb and anti-CD112R mAb and an anti-PD-1 mAb.
  • FIG. 25A-25H is a graph of the % high molecular weight (HMW) species ( Figures 25A-25D) or % antibody main peak ( Figures 25E-25H) plotted as a function of time (weeks) measured in a formulation comprising (1) anti-CD112R mAb (CD112R), (2) anti-TIGIT mAb (TIGIT-10), (3) another anti-TIGIT mAb (TIGIT-12), (4) both anti-CD112R mAb and TIGIT-10 mAb and (5) both anti-CD112R mAb and TIGIT-12 after storage at -30 °C ( Figures 25A and 25E), 4 °C ( Figures 25B and 25F), 25 °C ( Figures 25C and 25G) and 40 °C ( Figures 25D and 25H), as measured by SEC.
  • HMW high molecular weight
  • Figures 25E-25H % antibody main peak plotted as a function of time (weeks) measured in a formulation comprising
  • Figure 26 is a table listing the % LMW+HMW peaks of formulations comprising 70 mg/mL or 140 mg/mL anti-CD112R mAb, TIGIT-10, or TIGIT-12, or a formulation comprising both anti- CD112R and TIGIT-10 at a 1:1 ratio or comprising both anti-CD112R and TIGIT-12 at a 1:1 ratio.
  • Figure 27 is a graph of the viscosity (cP) of formulations comprising 70 mg/mL or 140 mg/mL anti-CD112R mAb, TIGIT-10, or TIGIT-12, or a formulation comprising both anti-CD112R and TIGIT-10 at a 1:1 ratio or comprising both anti-CD112R and TIGIT-12 at a 1:1 ratio.
  • TIGIT and CD112R also known as PVRIG
  • TIGIT and CD112R belong to a family of receptors that contain immunoglobulin (Ig) domain(s) in the extracellular region. These receptors interact with ligands that also contain Ig domain(s).
  • TIGIT-CD155 and CD112R-CD112 represent the primary receptor-ligand pairings based on the rank order by affinity measurements.
  • Another family member, CD226, can interact with both CD112 and CD155, both with weaker affinity than that for TIGIT-CD155 and CD112R-CD112. Engagement of CD226 enhances T/NK cell activity specifically in the context of T/NK cell response to tumor cells.
  • TIGIT and CD112R are co-expressed at high levels, because 1) TIGIT and CD112R both bind to the ligands at higher affinities than does CD226 and effectively limit ligand accessibility and 2) TIGIT and CD112R intracellular domains contain ITIM or ITIM-like domains that are thought to generate inhibitory signals, although the nature of such signal has not been extensively characterized in T cells.
  • Transcripts of CD155 and CD112, the primary ligands for TIGIT and CD112R are present in a wide range of tissues and cell types. This contrasts with PD-L1, whose expression is more restricted, with preferential expression in antigen presenting cells and tumor cells.
  • ligands have been shown to be induced by different types of stimuli: while PD-L1 is upregulated by exposure to IFN ⁇ , CD155 and CD112 are not regulated by exposure to this cytokine and are instead upregulated in response to DNA damage, viral infection, and reactive oxygen species (ROS). Therefore, ligand induction response further differentiates the pathways engaged by TIGIT and CD112R from that by PD-1.
  • the present disclosure provides antigen binding proteins, e.g., antibodies and antigen- binding fragments thereof, that bind to TIGIT, e.g., TIGIT binding proteins, also referred herein as TIGIT antigen binding proteins.
  • the TIGIT antigen binding protein is an antibody that specifically binds TIGIT (e.g., a TIGIT antibody, an ⁇ -TIGIT antibody).
  • TIGIT e.g., a TIGIT antibody, an ⁇ -TIGIT antibody.
  • the present disclosure additionally provides antigen binding proteins which bind to CD112R, e.g., CD112R binding proteins, also referred herein as CD112R antigen binding proteins.
  • the CD112R antigen binding protein is an antibody that specifically binds CD112R ((e.g., a CD112R antibody, an ⁇ -CD112R antibody)).
  • binding strength of the presently disclosed TIGIT antigen binding protein for binding to TIGIT is described in terms of its affinity. In exemplary aspects, the binding strength of the presently disclosed TIGIT antigen binding protein for TIGIT is described in terms of K D . Likewise, the binding strength of the presently disclosed CD112R antigen binding protein for binding to CD112R is described in terms of its affinity, and, in exemplary aspects, the binding strength of the presently disclosed CD112R antigen binding protein for CD112R is described in terms of K D .
  • K D is the equilibrium dissociation constant, a ratio of k off /k on , between an antigen binding protein and its target or antigen.
  • K D is inversely proportional to the affinity.
  • the K D value relates to a concentration of the antigen binding protein, and thus the lower the K D value, the higher the affinity of the antigen binding protein.
  • the K D of the TIGIT antigen binding proteins and CD112R antigen binding proteins provided herein is micromolar, nanomolar, picomolar or femtomolar.
  • the K D of the TIGIT antigen binding proteins or the CD112R antigen binding proteins provided herein is within a range of about 10 -4 to 10 -6 M, or 10 -7 to 10 -9 M, or 10 -10 to 10 -12 M, or 10 -13 to 10 -15 M.
  • the K D of the TIGIT antigen binding proteins or the CD112R antigen binding proteins provided herein is within a range of about 10 -12 to 10 -8 M, optionally, 10 -11 to 10 -10 M.
  • the antigen binding protein e.g., antibody
  • the amino acid sequence of human TIGIT is provided herein as SEQ ID NO: 1.
  • amino acids 1-21 of SEQ ID NO: 1 represents the signal peptide
  • amino acids 22-244 of SEQ ID NO: 1 represents the mature human TIGIT amino acid sequence.
  • the antigen binding protein binds to the human TIGIT with a K D that is about 50 nM or less (e.g., about 40 nM or less, about 30 nM or less, about 20 nM or less, or about10 nM or less). In exemplary aspects, the antigen binding protein binds to the human TIGIT with a K D that is less than or about 5 nM, less than or about 4 nM, less than or about 3 nM, less than or about, 2 nM, or less than or about 1 nM.
  • the K D of the antigen binding protein for human TIGIT is less than 1 nM, e.g., less than 0.75 nM, less than 0.5 nM, or less than 0.25 nM.
  • the K D of the antigen binding protein for human TIGIT is greater than or about 0.001 nM or greater than or about 0.01 nM and less than 0.5 nM.
  • the K D of the antigen binding protein for human TIGIT is about 0.01 nM to about 0.5 nM, about 0.02 nM to about 0.5 nM, about 0.03 nM to about 0.5 nM, about 0.04 nM to about 0.5 nM, about 0.05 nM to about 0.5 nM, about 0.06 nM to about 0.5 nM, about 0.07 nM to about 0.5 nM, about 0.08 nM to about 0.5 nM, about 0.09 nM to about 0.5 nM, about 0.1 nM to about 0.5 nM, about 0.2 nM to about 0.5 nM, about 0.3 nM to about 0.5 nM, about 0.4 nM to about 0.5 nM, about 0.01 nM to about 0.4 nM, about 0.01 nM to about 0.3 nM, about 0.01 nM to about 0.2 nM, about 0.01 nM to about 0.1 nM, about
  • the antigen binding protein also binds to cynomolgus monkey (cyno) TIGIT.
  • the amino acid sequence of cyno TIGIT is provided herein as SEQ ID NO: 2024.
  • amino acids 1-21 of SEQ ID NO: 2024 represents a signal peptide and amino acids 22-245 of SEQ ID NO: 2024 represents the mature cyno TIGIT protein.
  • the antigen binding protein binds to cynomolgus monkey TIGIT with a K D that is about 1 nM to about 25 nM, e.g., about 5 nM to about 20 nM, or about 5 nM to about 15 nM.
  • the antigen binding protein binds to cynomolgus (cyno) monkey TIGIT with a K D of about 8 nM to about 14 nM.
  • the antigen binding protein binds with high affinity to both human TIGIT and cyno TIGIT.
  • the K D of the antigen binding protein for human TIGIT is about 0.01 nM and less than 0.5 nM and the K D of the antigen binding protein for cyno TIGIT is about 8 nM to about 14 nM.
  • the K D of the antigen binding protein for human TIGIT is within about 100- fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold, or less, of the K D of the antigen binding protein for cyno TIGIT.
  • the EC50 value of the TIGIT antigen binding protein for human T-cells expressing human TIGIT is within about 100-fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold, or less, of the EC50 value of the TIGIT antigen binding protein for cyno PBMCs expressing cyno TIGIT.
  • the antigen binding protein binds to human CD112R.
  • the amino acid sequence of human CD112R is provided herein as SEQ ID NO: 3.
  • amino acid 53 of SEQ ID NO: 3 represents the first amino acid of the extracellular domain.
  • the antigen binding protein binds with high affinity to both human CD112R and cyno CD112R.
  • the antigen binding protein binds to the human CD112R with a K D that is about 50 nM or less (e.g., about 40 nM or less, about 30 nM or less, about 20 nM or less, or about10 nM or less).
  • the antigen binding protein binds to the human CD112R with a K D that is less than or about 5 nM, less than or about 4 nM, less than or about 3 nM, less than or about, 2 nM, or less than or about 1 nM.
  • the K D of the antigen binding protein for human CD112R is less than 1 nM, e.g., less than 0.75 nM, less than 0.5 nM, or less than 0.25 nM.
  • the KD of the antigen binding protein for human CD112R is greater than or about 0.001 nM or greater than or about 0.01 nM and less than 3 nM.
  • the K D of the antigen binding protein for human CD112R is about 0.01 nM to about 5 nM, about 0.05 nM to about 5 nM, about 0.10 nM to about 5 Nm, about 0.5 nM to about 5 Nm, about 1 nM to about 5 Nm, about 2 nM to about 5 Nm, about 3 nM to about 5 Nm, about 4 nM to about 5 nm, about 0.01 nM to about 4 Nm, about 0.01 nM to about 3 Nm, about 0.01 nM to about 2 Nm, about 0.01 nM to about 1 Nm, about 0.01 nM to about 0.5 Nm, about 0.01 nM to about 0.1 Nm, or about 0.01 nM to about 0.05 Nm.
  • the antigen binding protein binds to cyno CD112R.
  • the amino acid sequence of cyno CD112R is provided herein as SEQ ID NO: 2022 of which amino acid 53 is the first amino acid of the extracellular domain.
  • the antigen binding protein binds to cynomolgus monkey (cyno) CD112R with a K D that is about 1 nM to about 25 Nm, e.g., about 5 nM to about 20 Nm, or about 5 nM to about 15 Nm.
  • the antigen binding protein binds to cyno CD112R with a KD of about 0.05 nM to about 0.15 Nm.
  • the KD of the antigen binding protein for human CD112R is about 0.01 nM and less than 5 nM and the K D of the antigen binding protein for cyno CD112R is about 0.05 nM to about 0.15 nM.
  • the K D of the antigen binding protein for human CD112R is within about 100-fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold, or less, of the K D of the antigen binding protein for cyno CD112R.
  • the EC50 value of the CD112R antigen binding protein for human T-cells expressing human CD112R is within about 100- fold, about 50-fold, about 25-fold, about 10-fold, about 5-fold, or about 2-fold, or less, of the EC50 value of the CD112R antigen binding protein for cyno PBMCs expressing cyno CD112R.
  • the antigen binding protein e.g., antibody, exhibits a binding affinity for its target (TIGIT or CD112R) which is increased relative to the binding affinity of the native interaction between TIGIT and CD155, TIGIT and CD112, or CD112R and CD112.
  • the increase in binding affinity may be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85% increase, at least or about a 90% increase, at least or about a 95% increase, relative to the binding affinity of human TIGIT for its ligand (CD155) or relative to the binding affinity of human CD112R for its ligand (CD112).
  • the antigen binding protein exhibits an increase which is about a 2-, 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100-, 105-, 110-, 115-, 120-, 125-, 130-, 135-, 140-, 145-, 150-, 175-, 200-, 225-, 250-, 275-, 300-, 325-, 350-, 375-, 400-, 425-, 450-, 475-, 500-, 525-, 550-, 575-, 600-, 625-, 650-, 675-, 700-, 725-, 750-, 775-, 800-, 825-, 850-, 875-, 900-, 925-, 950-, 975-fold, 1000-fold, or more increase in binding affinity for its target (TIGIT or CD
  • the antigen-binding protein e.g., antibody
  • the TIGIT-binding proteins of the present disclosure compete with the reference antibody for binding to human TIGIT and thereby reduce the amount of human TIGIT bound to the reference antibody as determined by an in vitro competitive binding assay.
  • the antigen-binding proteins of the present disclosure inhibit the binding interaction between human TIGIT and the reference antibody and the inhibition is characterized by an IC50.
  • the antigen-binding proteins exhibit an IC 50 of less than about 250 nM for inhibiting the binding interaction between human TIGIT and the reference antibody. In various aspects, the antigen-binding proteins exhibit an IC50 of less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm.
  • the antigen-binding proteins exhibit an IC 50 of less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than 0.5 nM or less than 0.1 nM.
  • the antigen-binding proteins of the present disclosure compete with the reference antibody for binding to human TIGIT and thereby reduce the amount of human TIGIT bound to the reference antibody as determined by a FACS-based assay in which the fluorescence of a fluorophore- conjugated secondary antibody which binds to the Fc of the reference antibody is measured in the absence or presence of a particular amount of the antigen-binding protein of the present disclosure.
  • the FACS-based assay is carried out with the reference antibody, fluorophore- conjugated secondary antibody and cells which express TIGIT.
  • the cells are genetically-engineered to overexpress TIGIT.
  • the cells are HEK293T cells transduced with a viral vector to express TIGIT.
  • the cells endogenously express TIGIT.
  • the cells which endogenously express TIGIT are pre-determined as low TIGIT-expressing cells or high TIGIT- expressing cells.
  • the antigen-binding protein e.g., antibody
  • the antigen-binding protein e.g., antibody
  • greater than 80% (e.g., greater than 85%, greater than 90%) of the binding interactions between human TIGIT and CD155 are inhibited in the presence of the presently disclosed antigen binding protein, e.g., antibody.
  • the binding interactions between human TIGIT and CD155 are inhibited in the presence of the presently disclosed antigen binding protein, e.g., antibody, as determined by a FACS-based receptor-ligand competition binding assay, such as that described herein at Example 5.
  • the antigen-binding protein e.g., antibody
  • the antigen-binding protein inhibits a binding interaction between human CD112R and a reference antibody, which reference antibody is known to bind to CD112R but is not an antigen-binding protein of the present disclosure.
  • the CD112R -binding proteins of the present disclosure compete with the reference antibody for binding to human CD112R and thereby reduce the amount of human CD112R bound to the reference antibody as determined by an in vitro competitive binding assay.
  • the antigen- binding proteins of the present disclosure inhibit the binding interaction between human CD112R and the reference antibody and the inhibition is characterized by an IC50.
  • the antigen- binding proteins exhibit an IC 50 of less than 250 nM for inhibiting the binding interaction between human CD112R and the reference antibody.
  • the antigen-binding proteins exhibit an IC 50 of less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 90 nm, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, or less than about 10 nM.
  • the antigen-binding proteins exhibit an IC 50 of less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than 0.5 nM or less than 0.1 nM.
  • the antigen-binding proteins exhibit an IC 50 of about 0.05 nM to about 0.5 nM (e.g., about 0.06 nM, about 0.07 nM, about 0.08 nM, about 0.09 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM).
  • the antigen-binding proteins of the present disclosure compete with the reference antibody for binding to human CD112R and thereby reduce the amount of human CD112R bound to the reference antibody as determined by a FACS-based assay in which the fluorescence of a fluorophore-conjugated secondary antibody which binds to the Fc of the reference antibody is measured in the absence or presence of a particular amount of the antigen- binding protein of the present disclosure.
  • the FACS-based assay is carried out with the reference antibody, fluorophore-conjugated secondary antibody and cells which express CD112R.
  • the cells are genetically-engineered to overexpress CD112R.
  • the cells are HEK293T cells transduced with a viral vector to express CD112R.
  • the cells endogenously express CD112R.
  • the cells which endogenously express CD112R are pre-determined as low CD112R - expressing cells or high CD112R -expressing cells.
  • the antigen-binding protein e.g., antibody, inhibits a binding interaction between human CD112R and its native ligand, e.g., CD112.
  • the antigen-binding protein e.g., antibody
  • greater than 90% of the binding interactions between human CD112R and CD112 are inhibited in the presence of the presently disclosed antigen binding protein, e.g., antibody.
  • greater than 95% e.g., greater than 96%, greater than 97%, greater than 98%, greater than 99% or nearly 100%
  • the presently disclosed antigen binding protein e.g., antibody
  • FACS-based receptor-ligand competition binding assay such as that described herein at Example 3.
  • Other binding assays e.g., competitive binding assays or competition assays, which test the ability of an antibody to compete with another antigen-binding protein for binding to an antigen, or to an epitope thereof, are known in the art.
  • the antigen binding protein e.g., antibody
  • the presently disclosed TIGIT binding protein binds to TIGIT and thereby inhibits the binding interaction between TIGIT and CD155.
  • the presently disclosed TIGIT binding protein inhibits the binding interaction between TIGIT and other ligands (e.g., CD112).
  • the presently disclosed CD112R binding protein binds to CD112R and thereby inhibits the binding interaction between CD112R and CD112.
  • the presently disclosed CD112R binding protein inhibits the binding interaction between CD112R and other ligands (e.g., CD96, CD226, TIGIT).
  • the antigen binding protein e.g., antibody, is an antagonist which inhibits the biological activity of the target or antigen.
  • the CD112R binding protein binds to CD112R and inhibits the signal transduction pathway(s) activated upon CD112 binding to CD112R.
  • the TIGIT binding protein binds to TIGIT and inhibits the signal transduction pathway(s) activated upon CD155 binding to TIGIT. Additional signal transduction pathway(s) may be inhibited upon binding of the TIGIT binding protein to TIGIT of upon binding of the CD112R binding protein to CD112R.
  • the reduction or inhibition provided by the antigen binding protein, e.g., antibody may not be a 100% or complete inhibition or abrogation or reduction.
  • the antigen binding protein may inhibit the TIGIT and/or CD112R protein(s) to any amount or level.
  • the reduction or inhibition provided by the antigen binding protein is at least or about 10% reduction or inhibition (e.g., at least or about 20% reduction or inhibition, at least or about 30% reduction or inhibition, at least or about 40% reduction or inhibition, at least or about 50% reduction or inhibition, at least or about 60% reduction or inhibition, at least or about 70% reduction or inhibition, at least or about 80% reduction or inhibition, at least or about 90% reduction or inhibition, at least or about 95% reduction or inhibition, at least or about 98% reduction or inhibition).
  • the antigen binding protein of the present disclosure inhibits binding CD112 to CD112R or TIGIT to CD155.
  • the inhibition may be characterized in terms of a half maximal inhibitory concentration (IC50) which is a measure of the effectiveness of the antigen binding protein in inhibiting a specific biological or biochemical function.
  • IC50 half maximal inhibitory concentration
  • Suitable methods for measuring the inhibitory or antagonist activity of the antigen binding proteins of the present disclosure are known in the art.
  • the antagonist or inhibitory activity of the antigen binding proteins may be assayed by measuring the level of TCR activation, given that simultaneous binding of CD112R to CD112 and/or TIGIT to CD155 and activation of the T-cell receptor (TCR) of a T-cell produces an inhibitory signal which inactivates or shuts off TCR-mediated responses, and, therefore, blocking the interaction between CD112R to CD112 and/or TIGIT to CD155 upon TCR ligation lead to TCR-mediated activities including one or more of phosphorylation of the TCR subunits, recruitment of Zap70 to the TCR, phosphorylation of LAT and/or SLP-76, calcium mobilization or calcium release from the endoplasmic reticulum (ER), activation of PLC-gamma, production of diacylglycerol (DAG) and inositol triphosphate (IP3), activation of Protein Kinase C, MARPK/Erk signaling, NF- ⁇ b activation
  • TCR
  • the inhibitory or antagonist activity of the antigen binding proteins of the present disclosure may be assayed by measuring for IL-2 production, IFN-gamma production and/or activation of NF- ⁇ b and/or NFAT, for instance.
  • a luciferase reporter gene assay is used with Jurkat T cells, wherein, upon TCR activation and in the presence of TIGIT binding proteins and/or CD112R binding proteins, luciferase activity is measured.
  • luciferase activity is expected to be higher than the luciferase activity observed in the absence of the TIGIT binding proteins and/or CD112R binding proteins.
  • Jurkat RGA are described herein in the Examples (See, e.g., Example 3 and Example 5).
  • the antagonist activity of the TIGIT binding proteins and/or CD112R binding proteins may be measured by a receptor-ligand binding assay or a Jurkat RGA.
  • the antagonist activity or inhibitory activity of the antigen binding proteins of the present disclosure may be measured by an Jurkat RGA and the activity is expressed as an EC50.
  • the EC50 of the CD112R antigen binding protein or the TIGIT antigen binding protein is within about 0.01 nM to about 10 nM, about 0.01 nM to about 9 nM, about 0.01 nM to about 8 nM, about 0.01 nM to about 7 nM, about 0.01 nM to about 6 nM, about 0.01 nM to about 5 nM, about 0.01 nM to about 4 nM, about 0.01 nM to about 3 nM, about 0.01 nM to about 2 nM, about 0.01 nM to about 1 nM, about 0.01 nM to about 0.5 nM, about 0.01 nM to about 0.1 nM, about 0.01 nM to about 0.05 nM, about 0.05 nM to about 10 nM, about 0.1 nM to about 10 nM, about 0.5 nM to about 10 nM, about 1 nM to about 10 nM, about 2
  • the CD112R antigen binding protein exhibits an EC 50 in a Jurkat RGA as described above and/or as described in Figures 7B or in Tables 2-5.
  • the TIGIT antigen binding protein exhibits an EC 50 in a Jurkat RGA as described above and/or as described in Tables 13- 15.
  • the IC 50 of the CD112R antigen binding protein is, in exemplary aspects, less than about 10 nM, optionally, less than 5 nM. In exemplary aspects, the IC50 of the CD112R antigen binding protein is less than 2 nM or less than 1 nM.
  • the IC 50 of the CD112R antigen binding protein is about 0.5 nM to about 2 nM. In various instances, the IC 50 of the CD112R antigen binding protein is within about 0.01 nM to about 10 nM, about 0.01 nM to about 9 nM, about 0.01 nM to about 8 nM, about 0.01 nM to about 7 nM, about 0.01 nM to about 6 nM, about 0.01 nM to about 5 nM, about 0.01 nM to about 4 nM, about 0.01 nM to about 3 nM, about 0.01 nM to about 2 nM, about 0.01 nM to about 1 nM, about 0.01 nM to about 0.5 nM, about 0.01 nM to about 0.1 nM, about 0.01 nM to about 0.05 nM, about 0.05 nM to about 10 nM, about 0.1 nM to about 10 nM, about 0.5 nM to about
  • the IC 50 of the CD112R antigen binding protein is a measure of the effectiveness of the CD112R antigen binding protein in inhibiting the binding interaction between CD112R and CD112 as determined by a receptor-ligand binding assay. See, e.g., Example 3.
  • the IC 50 of the TIGIT antigen binding protein is, in exemplary aspects, less than about 10 nM, optionally, less than 5 nM. In exemplary aspects, the IC 50 of the TIGIT antigen binding protein is less than 2 nM or less than 1 nM. In exemplary aspects, the IC 50 of the TIGIT antigen binding protein is about 0.5 nM to about 2 nM.
  • the IC50 of the TIGIT antigen binding protein is within about 0.01 nM to about 10 nM, about 0.01 nM to about 9 nM, about 0.01 nM to about 8 nM, about 0.01 nM to about 7 nM, about 0.01 nM to about 6 nM, about 0.01 nM to about 5 nM, about 0.01 nM to about 4 nM, about 0.01 nM to about 3 nM, about 0.01 nM to about 2 nM, about 0.01 nM to about 1 nM, about 0.01 nM to about 0.5 nM, about 0.01 nM to about 0.1 nM, about 0.01 nM to about 0.05 nM, about 0.05 nM to about 10 nM, about 0.1 nM to about 10 nM, about 0.5 nM to about 10 nM, about 1 nM to about 10 nM, about 2 nM to about 10 nM, about
  • the IC 50 of the TIGIT antigen binding protein is a measure of the effectiveness of the TIGIT antigen binding protein in inhibiting the binding interaction between TIGIT and CD155 or CD112 as determined by a receptor- ligand binding assay. See, e.g., Example 5.
  • the antigen-binding proteins of the present disclosure can take any one of many forms of antigen-binding proteins known in the art.
  • the antigen-binding protein is an antibody or immunoglobulin, or an antigen binding antibody fragment thereof, or an antibody protein product.
  • antibodies form a family of plasma proteins known as immunoglobulins and comprise of immunoglobulin domains.
  • an antibody refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions.
  • an antibody may be an IgG which is a “Y-shaped” structure of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • An antibody has a variable region and a constant region.
  • variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and substantially varies among other antibodies that bind to different antigens.
  • the constant region allows the antibody to recruit cells and molecules of the immune system.
  • the variable region is made of the N-terminal regions of each light chain and heavy chain, while the constant region is made of the C-terminal portions of each of the heavy and light chains.
  • CDRs of antibodies have been described in the art. Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition.
  • a variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol.
  • Antibodies can comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region.
  • the heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region.
  • the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.
  • the antibody can be a monoclonal antibody or a polyclonal antibody.
  • the antibody comprises a sequence that is substantially similar to a naturally-occurring antibody produced by a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, and the like.
  • the antibody can be considered as a mammalian antibody, e.g., a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, and the like.
  • the antibody is a human antibody.
  • the antibody is a chimeric antibody or a humanized antibody.
  • the term “chimeric antibody” refers to an antibody containing domains from two or more different antibodies.
  • a chimeric antibody can, for example, contain the constant domains from one species and the variable domains from a second, or more generally, can contain stretches of amino acid sequence from at least two species.
  • a chimeric antibody also can contain domains of two or more different antibodies within the same species.
  • the term “humanized” when used in relation to antibodies refers to antibodies having at least CDR regions from a non-human source which are engineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies.
  • humanizing can involve grafting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanizing also can involve select amino acid substitutions to make a non-human sequence more similar to a human sequence.
  • an antibody can be cleaved into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab') 2 fragment and a pFc' fragment.
  • the antigen binding protein of the present disclosure comprises an antigen binding antibody fragment.
  • the term “antigen binding antibody fragment” refers to a portion of an antibody molecule that is capable of binding to the antigen of the antibody and is also known as “antigen-binding fragment” or “antigen-binding portion”.
  • the antigen binding antibody fragment is a Fab fragment or a F(ab’) 2 fragment.
  • Antibody protein products include those based on the full antibody structure and those that mimic antibody fragments which retain full antigen-binding capacity, e.g., scFvs, Fabs and VHH/VH (discussed below).
  • the smallest antigen binding antibody fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions.
  • a soluble, flexible amino acid peptide linker is used to connect the V regions to a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment [fragment, antigen-binding].
  • Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells.
  • Other antibody protein products include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains.
  • minibodies minibodies that comprise different formats consisting of scFvs linked to oligomerization domains.
  • the smallest fragments are VHH/VH of camelid heavy chain Abs as well as single domain Abs (sdAb).
  • V-domain antibody fragment which comprises V domains from the heavy and light chain (VH and VL domain) linked by a peptide linker of ⁇ 15 amino acid residues.
  • VH and VL domain V domains from the heavy and light chain linked by a peptide linker of ⁇ 15 amino acid residues.
  • a peptibody or peptide-Fc fusion is yet another antibody protein product.
  • the structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain.
  • Peptibodies are well-described in the art. See, e.g., Shimamoto et al., mAbs 4(5): 586-591 (2012).
  • antibody protein products include a single chain antibody (SCA); a diabody; a triabody; a tetrabody; bispecific or trispecific antibodies, and the like.
  • SCA single chain antibody
  • Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, BsAb fragments, bispecific fusion proteins and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015).
  • the antigen binding protein of the present disclosure comprises any one of these antibody protein products.
  • the antigen binding protein of the present disclosure comprises any one of an scFv, Fab VHH/VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., a diabody, triabody, tetrabody), miniAb, peptibody VHH/VH of camelid heavy chain antibody, sdAb, diabody; a triabody; a tetrabody; a bispecific or trispecific antibody, BsIgG, appended IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.
  • the antigen binding protein of the present disclosure comprises an antibody protein product in monomeric form, or polymeric, oligomeric, or multimeric form.
  • the antibody comprises two or more distinct antigen binding regions fragments
  • the antibody is considered bispecific, trispecific, or multi-specific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that are recognized and bound by the antibody.
  • the antigen binding protein of the present disclosure is a bispecific antibody (bsAb) comprising two scFv, one which binds to TIGIT and one which binds to CD112R.
  • the scFv which binds to CD112R comprises the light chain variable region and heavy chain variable region of 29E10, 24F1 or 11E4.
  • the scFv which binds to TIGIT comprises the light chain variable region and heavy chain variable region of 43B7.002.015, 66H9.009, or 58A7.002.008.
  • each scFv is linked to a heavy chain and/or light chain, optionally, an IgG heavy chain and/or an IgG light chain.
  • the CD112R antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence set forth in Table A1 or a variant sequence thereof which differs by only 1-4 amino acids (e.g., 1, 2, 3, 4 amino acids) or which has at least or about 90% sequence identity; (b) an HC CDR2 amino acid sequence set forth in Table A1 or a variant sequence thereof which differs by only 1-4 amino acids or which has at least or about 90% sequence identity; (c) an HC CDR3 amino acid sequence set forth in Table A1 or a variant sequence thereof which differs by only 1-4 amino acids or which has at least or about 90% sequence identity; (d) a light chain (LC) CDR1 amino acid sequence set forth in Table A1 or a variant sequence thereof which differs by only 1-4 amino acids or which has at least or about 90% sequence identity; (e)
  • the CD112R antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence set forth in Table A1 and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table A1.
  • the CD112R antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, and a HC CDR3 amino acid sequence set forth in Table A1 and at least 1 or 2 of the LC CDR amino acid sequences set forth in Table A1.
  • the CD112R antigen binding protein comprises all three such CDRs.
  • the CD112R antigen binding protein comprises 3, 4, 5, or all 6 of the amino acid sequences designated by the SEQ ID NOs: in a single row of Table A1.
  • the CD112R antigen binding protein comprises each of the LC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table A1 and at least 1 or 2 of the HC CDR amino acid sequences designated by the SEQ ID NOs: in the same single row or another single row of Table A1.
  • the CD112R antigen binding protein comprises each of the HC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table A1 and at least 1 or 2 of the LC CDR amino acid sequences designated by the SEQ ID NOs: in the same single row or another single row of Table A1.
  • the CD112R antigen binding protein comprises six CDR amino acid sequences listed in a single row of Table A1 or comprising six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 13-18; (b) SEQ ID NOs: 23-28; (c) SEQ ID NOs: 33-38; (d) SEQ ID NOs: 43-48; I SEQ ID NOs: 53-58; (f) SEQ ID NOs: 63-68; (g) SEQ ID NOs: 73-78; (h) SEQ ID NOs: 83-88, (i) SEQ ID NOs: 93-98, (j) SEQ ID NOs: 103-108, (k) SEQ ID NOs: 233-238, (l) SEQ ID NOs: 1973- 1978, (m) SEQ ID NOs: 1983-1988, (n) SEQ ID NOs: 1993-1998, and (o) SEQ ID NOs: 2003-2008.
  • the CD112R antigen binding protein comprises the six CDR amino acid sequences as described above and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 2019 or SEQ ID NO: 2020 [0095]
  • the amino acid sequences of Table A1 are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s), e.g., framework residues.
  • the intervening amino acids comprise a framework region.
  • the TIGIT antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence set forth in Table A2 or a variant sequence thereof which differs by only 1-4 amino acids (e.g., 1, 2, 3, 4 amino acids) or which has at least or about 90% sequence identity; (b) an HC CDR2 amino acid sequence set forth in Table A2 or a variant sequence thereof which differs by only 1-4 amino acids or which has at least or about 90% sequence identity; (c) an HC CDR3 amino acid sequence set forth in Table A2 or a variant sequence thereof which differs by only 1-4 amino acids or which has at least or about 90% sequence identity; (d) a light chain (LC) CDR1 amino acid sequence set forth in Table A2 or a variant sequence thereof which differs by only 1-4 amino acids or which has at least or about 90% sequence identity; (e) an LC CDR2 amino acid sequence set forth in Table A
  • the TIGIT antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises a LC CDR1 amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence set forth in Table A2 and at least 1 or 2 of the HC CDR amino acid sequences set forth in Table A2.
  • the TIGIT antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, and a HC CDR3 amino acid sequence set forth in Table A2 and at least 1 or 2 of the LC CDR amino acid sequences set forth in Table A2.
  • the TIGIT antigen binding protein comprises all three such CDRs.
  • the TIGIT antigen binding protein comprises 3, 4, 5, or all 6 of the amino acid sequences designated by the SEQ ID NOs: in a single row of Table A2.
  • the TIGIT antigen binding protein comprises each of the LC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table A2 and at least 1 or 2 of the HC CDR amino acid sequences designated by the SEQ ID NOs: in the same single row or another single row of Table A2.
  • the TIGIT antigen binding protein comprises each of the HC CDR amino acid sequences designated by the SEQ ID NOs: of a single row of Table A2 and at least 1 or 2 of the LC CDR amino acid sequences designated by the SEQ ID NOs: in the same single row or another single row of Table A2.
  • the TIGIT antigen binding protein comprises six CDR amino acid sequences listed in a single row of Table A2 or comprising six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 113-118; (b) SEQ ID NOs: 123-128; (c) SEQ ID NOs: 133-138; (d) SEQ ID NOs: 143-148; (e) SEQ ID NOs: 153-158; (f) SEQ ID NOs: 163-168; (g) SEQ ID NOs: 173-178; (h) SEQ ID NOs: 183-188, (i) SEQ ID NOs: 193-198, (j) SEQ ID NOs: 203-208, (k) SEQ ID NOs: 213-218, (l) SEQ ID NOs: 223-228, and (m) SEQ ID NOs: 2013-2018.
  • the TIGIT antigen binding protein comprises the six CDR amino acid sequences as described above and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 2019 or SEQ ID NO: 2020.
  • the amino acid sequences of Table A2 are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acid(s), e.g., framework residues.
  • the intervening amino acids comprise a framework region.
  • the CD112R antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises a pair of HC variable region and LC variable region amino acid sequences listed in a single row of Table B1 or comprising one of the following pairs of amino acid sequences: (a) SEQ ID NOs: 11-12; (b) SEQ ID NOs: 21-22; (c) SEQ ID NOs: 31-32; (d) SEQ ID NOs: 41-42; (e) SEQ ID NOs: 51-52; (f) SEQ ID NOs: 61-62; (g) SEQ ID NOs: 71-72; (h) SEQ ID NOs: 81-82, (i) SEQ ID NOs: 91-92, (j) SEQ ID NOs: 101-102, (k) SEQ ID NOs: 231-232, (l) SEQ ID NOs: 1971-1972, (m) SEQ ID NOs: 1981-1982, (n) SEQ ID NOs
  • the CD112R antigen binding protein comprises the pair of HC variable region and LC variable region amino acid sequences as described above and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 2019 or SEQ ID NO: 2020.
  • the CD112R antigen binding protein comprises a pair of full- length (FL) HC and FL LC amino acid sequences listed in a single row of Table B1 or comprising one of the following pairs of amino acid sequences: (a) SEQ ID NOs: 9-10; (b) SEQ ID NOs: 19-20; (c) SEQ ID NOs: 29-30; (d) SEQ ID NOs: 39-40; (e) SEQ ID NOs: 49-50; (f) SEQ ID NOs: 59-60; (g) SEQ ID NOs: 69-70; (h) SEQ ID NOs: 79-80, (i) SEQ ID NOs: 89-90, (j) SEQ ID NOs: 99-100, (k) SEQ
  • the TIGIT antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises a pair of HC variable region and LC variable region amino acid sequences listed in a single row of Table B2 or comprising one of the following pairs of amino acid sequences: (a) SEQ ID NOs: 111-112, (b) SEQ ID NOs: 121-122, (c) SEQ ID NOs: 131- 132, (d) SEQ ID NOs: 141-142, (e) SEQ ID NOs: 151-152, (f) SEQ ID NOs: 161-162, (g) SEQ ID NOs: 171-172, (h) SEQ ID NOs: 181-182, (i) SEQ ID NOs: 191-192, (j) SEQ ID NOs: 201-202, (k) SEQ ID NOs: 111-112, (b) SEQ ID NOs: 121-122, (c) SEQ ID NOs: 131- 132, (d) SEQ ID NOs:
  • the TIGIT antigen binding protein comprises the pair of HC variable region and LC variable region amino acid sequences as described above and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 2019 or SEQ ID NO: 2020.
  • the TIGIT antigen binding protein comprises a pair of full-length (FL) HC and FL LC amino acid sequences listed in a single row of Table B2 or comprising one of the following pairs of amino acid sequences: (a) SEQ ID NOs: 109-110, (b) SEQ ID NOs: 119-120, (c) SEQ ID NOs: 129-130, (d) SEQ ID NOs: 139-140, (e) SEQ ID NOs: 149-150, (f) SEQ ID NOs: 159-160, (g) SEQ ID NOs: 169- 170, (h) SEQ ID NOs: 179-180, (i) SEQ ID NOs: 189-190, (j) SEQ ID NOs
  • the CD112R antigen binding protein or the TIGIT antigen binding protein comprises an amino acid sequence which is similar to an above-referenced amino acid sequence, yet the antigen-binding protein substantially retains its biological function, e.g., its ability to bind to its target or antigen, e.g., human TIGIT, human CD112R, or to decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of TIGIT with its binding partner, CD155, or from the interaction of CD112R with its binding partner, CD112.
  • its target or antigen e.g., human TIGIT, human CD112R
  • the CD112R antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises an amino acid sequence which differs by only 1, 2, 3, 4, 5, 6, or more amino acids, relative to a parent amino acid sequence having an amino acid sequence referenced in Table A1 or Table B1.
  • the TIGIT antigen binding protein comprises an amino acid sequence which differs by only 1, 2, 3, 4, 5, 6, or more amino acids, relative to a parent amino acid sequence having an amino acid sequence referenced in Table A2 or Table B2.
  • the antigen binding protein (e.g., the CD112R antigen binding protein or the TIGIT antigen binding protein) comprises a variant sequence of the parent sequence, which variant sequence differs by only one or two amino acids, relative to the parent sequence.
  • the antigen-binding protein comprises one or more amino acid substitutions that occur outside of the CDRs, e.g., the one or more amino acid substitutions occur within the framework region(s) of the heavy or light chain.
  • the antigen binding protein comprises one or more amino acid substitutions, yet the antigen-binding protein retains the amino acid sequences of the six CDRs.
  • the antigen binding protein comprises an amino acid sequence having only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions, relative to the parent sequence(s).
  • conservative amino acid substitution refers to the substitution of one amino acid with another amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and/or aromaticity, and includes exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III.
  • the antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises an amino acid sequence comprising at least one amino acid substitution relative to the parent sequence, and the amino acid substitution(s) is/are non-conservative amino acid substitution(s).
  • non-conservative amino acid substitution is defined herein as the substitution of one amino acid with another amino acid having different properties, e.g., size, charge, hydrophobicity, hydrophilicity, and/or aromaticity, and includes exchanges outside the above five groups.
  • the antigen binding protein e.g., an antibody or antigen binding fragment thereof
  • the substitute amino acid is a naturally-occurring amino acid.
  • the antigen binding protein comprises an amino acid sequence comprising at least one amino acid substitution relative to the parent sequence, and the substitute amino acid is a non-standard amino acid, or an amino acid which is not incorporated into proteins during translation.
  • Non-standard amino acids include, but are not limited to: selenocysteine, pyrrolysine, ornithine, norleucine, ⁇ -amino acids (e.g., ⁇ -alanine, ⁇ -aminoisobutyric acid, ⁇ - phenlyalanine, ⁇ -homophenylalanine, ⁇ -glutamic acid, ⁇ -glutamine, ⁇ -homotryptophan, ⁇ -leucine, ⁇ - lysine), homo-amino acids (e.g., homophenylalanine, homoserine, homoarginine, monocysteine, homocysteine), N-methyl amino acids (e.g., L-abrine, N-methyl-alanine, N-methyl-isoleucine, N- methyl-leucine), 2-aminocaprylic acid, 7-aminocephalosporanic acid, 4-aminocinnamic acid, alpha- aminocyclohex
  • the antigen binding protein comprises an amino acid sequence which has greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity to the parent amino acid sequence(s).
  • the antigen-binding protein comprises an amino acid sequence which has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity to the parent amino acid sequence.
  • the antigen-binding protein comprises an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity along the full-length of the parent amino acid sequence.
  • the antigen-binding protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity along the full-length of the parent amino acid sequence.
  • the CD112R antigen binding protein e.g., an antibody or antigen binding fragment thereof
  • the CD112R antigen binding protein comprises a variant sequence of a HC variable region amino acid sequence or a variant sequence of a LC variable region amino acid sequence listed in Table B1 which variant sequence differs from the sequence of Table B1 by only 1 to 12 amino (e.g., 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) acids or which has at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity).
  • the CD112R antigen binding protein comprises a variant sequence of a FL HC amino acid sequence or a variant sequence of a FL LC amino acid sequence listed in Table B1 which variant sequence differs from the sequence of Table B1 by only 1 to 46 amino acids or which has at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity).
  • the CD112R antigen binding protein comprises a pair of HC variable region and LC variable region amino acid sequences listed in a single row of Table C1 or comprising one of the following pairs of amino acid sequences: (a) the first 100 amino acids of each of SEQ ID NOs: 241 and 242; (b) the first 100 amino acids of each of SEQ ID NOs: 247 and 248; (c) the first 100 amino acids of each of SEQ ID NOs: 249 and 250; (d) the first 100 amino acids of each of SEQ ID NOs: 251 and 252; or (e) the first 100 amino acids of each of SEQ ID NOs: 263 and 264.
  • the CD112R antigen binding protein comprises the first 105, first 106, first 107, first 108, first 109, first 110, first 111, first 112, first 113, first 114, or first 115 amino acids of SEQ ID NO: 242, SEQ ID NO: 248, SEQ ID NIO: 250, SEQ ID NO: 252, or SEQ ID NO: 264, and/or the first 115, first 116, first 117, first 118, first 119, first 120, first 121, first 122, first 123, first 124, first 125, first 126, or first 127 amino acids of SEQ ID NO: 241, SEQ ID NO: 247, SEQ ID NIO: 249, SEQ ID NO: 251, or SEQ ID NO: 263.
  • the CD112R antigen binding protein comprises a pair of full-length (FL) HC and FL LC amino acid sequences listed in a single row of Table C1 or comprising one of the following pairs of amino acid sequences: (a) SEQ ID NOs: 241-242; (b) SEQ ID NOs: 247-248; (c) SEQ ID NOs: 249-250; (d) SEQ ID NOs: 251-252; or (e) SEQ ID NOs: 263-264.
  • the CD112R antigen binding protein comprises a FL LC amino acid sequence having an odd numbered SEQ ID NO. listed in the “Engineered*” column of Table C1 and a FL HC amino acid sequence having a SEQ ID NO.
  • the CD112R antigen binding protein comprises at least a portion of a sequence of any one of the SEQ ID NOs: of Table C1, wherein the portion comprises the first 100 amino acids of the amino acid sequence, the first 105 amino acids of the amino acid sequence, the first 110 amino acids of the amino acid sequence, the first 115 amino acids of the amino acid sequence, or the first 120 amino acids of the amino acid sequence.
  • the CD112R antigen binding protein comprises at least 2, 3, 4, 5, or 6 of the CDRs of the amino acid sequence of Table C1. CDRs of a given antibody HC or LC may be determined by any one or more methods known in the art.
  • the TIGIT antigen binding protein (e.g., an antibody or antigen binding fragment thereof) comprises a variant sequence of a HC variable region amino acid sequence or a variant sequence of a LC variable region amino acid sequence listed in Table B2 which variant sequence differs from the sequence of Table B2 by only 1 to 12 amino acids or which has at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity).
  • the TIGIT antigen binding protein comprises a variant sequence of a FL HC amino acid sequence or a variant sequence of a FL LC amino acid sequence listed in Table B2 which variant sequence differs from the sequence of Table B2 by only 1 to 46 amino acids or which has at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity).
  • the TIGIT antigen binding protein comprises a pair of HC variable region and LC variable region amino acid sequences listed in a single row of Table C2 or comprising one of the following pairs of amino acid sequences: (a) the first 100 amino acids of each of (a) SEQ ID NOs: 239 and 240; (b) the first 100 amino acids of each of SEQ ID NOs: 243 and 244; (c) the first 100 amino acids of each of SEQ ID NOs: 245 and 246; (d) the first 100 amino acids of each of SEQ ID NOs: 253 and 254; (e) the first 100 amino acids of each of SEQ ID NOs: 255 and 256; (f) the first 100 amino acids of each of SEQ ID NOs: 257 and 258, (g) the first 100 amino acids of each of SEQ ID NOs: 259 and 260, or (h) the first 100 amino acids of each of SEQ ID NOs: 261 and 262.
  • the TIGIT antigen binding protein comprises the first 105, first 106, first 107, first 108, first 109, first 110, first 111, first 112, first 113, first 114, or first 115 amino acids of SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NIO: 246, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, or SEQ ID NO: 262, and/or the first 115, first 116, first 117, first 118, first 119, first 120, first 121, first 122, first 123, first 124, first 125, first 126, or first 127 amino acids of SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NIO: 245, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, or SEQ ID NO: 261.
  • the TIGIT antigen binding protein comprises a pair of full-length (FL) HC and FL LC amino acid sequences listed in a single row of Table C2 or comprising one of the following pairs of amino acid sequences: (a) SEQ ID NOs: 239-240; (b) SEQ ID NOs: 243-244; (c) SEQ ID NOs: 245-246; (d) SEQ ID NOs: 253-254; (e) SEQ ID NOs: 255-256; (f) SEQ ID NOs: 257-258, (g) SEQ ID NOs: 259-260, or (h) SEQ ID NOs: 261-262.
  • the TIGIT antigen binding protein comprises a FL LC amino acid sequence having an odd numbered SEQ ID NO. listed in the “Engineered*” column of Table C2 and a FL HC amino acid sequence having a SEQ ID NO. one greater than the FL LC SEQ ID NO.
  • the TIGIT antigen binding protein comprises at least a portion of a sequence of any one of the SEQ ID NOs: of Table C2, wherein the portion comprises the first 100 amino acids of the amino acid sequence, the first 105 amino acids of the amino acid sequence, the first 110 amino acids of the amino acid sequence, the first 115 amino acids of the amino acid sequence, or the first 120 amino acids of the amino acid sequence.
  • the TIGIT antigen binding protein comprises at least 2, 3, 4, 5, or 6 of the CDRs of the amino acid sequence of Table C2.
  • CDRs of a given antibody HC or LC may be determined by any one or more methods known in the art.
  • TABLE C2 SEQ ID NOs: of Consensus FL HC and FL LC of TIGIT antigen binding proteins and Engineered Versions Thereof
  • the CD112R antigen binding protein e.g., an antibody or antigen binding fragment thereof
  • the CD112R antigen binding protein is bivalent comprising two antigen binding sites.
  • the TIGIT antigen binding protein comprises an antibody, antigen-binding fragment of an antibody (e.g., Fab), or an antibody protein product, e.g., an scFv.
  • the TIGIT antigen binding protein is bivalent comprising two antigen binding sites.
  • the TIGIT antigen binding protein comprises a LC variable region amino acid sequence which is highly similar to SEQ ID NO: 192 or SEQ ID NO: 222 or the first 105-115 amino acids of SEQ ID NO: 240 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity) and the LC variable region amino acid sequence comprises a glutamic acid at position 1 (Glu1), or a conservative amino acid substitution thereof, a glutamine at position 27 (Gln27), or a conservative amino acid substitution thereof, a serine at position 28 (Ser28), or a conservative amino acid substitution thereof, a serine at position 91 (Ser91), or a conservative amino acid substitution thereof, a serine at position 92 (Ser92), or a conservative amino acid substitution thereof, a serine at position 93 (Ser93),
  • Glu1 glutamic acid at position 1
  • Gln27 glutamine at position
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti- TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti- TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the amino acid residues named above are positioned about 3 angstroms to about 4 angstroms from an amino acid of TIGIT.
  • the TIGIT antigen binding protein comprises a HC variable region amino acid sequence which is highly similar to SEQ ID NO: 191 or SEQ ID NO: 221 or the first 115-127 amino acids of SEQ ID NO: 239 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)) and comprises a valine at position 32 (Val32), or a conservative amino acid substitution thereof, a tyrosine at position 33 (Tyr33), or a conservative amino acid substitution thereof, a tyrosine at position 52 (Tyr52), or a conservative amino acid substitution thereof, a tyrosine at position 54 (Tyr54), or a conservative amino acid substitution thereof, a tyrosine at position 55 (Tyr55), or a conservative amino acid substitution thereof, a serine at position 56 (S)
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • each of Tyr52, Ser56, Thr59, Phe109, Tyr55, Tyr60, or a conservative amino acid substitution thereof forms a hydrogen bond with an amino acid of TIGIT.
  • the TIGIT antigen binding protein e.g., TIGIT antibody
  • each of the amino acid residues named above are positioned about 3 angstroms to about 4 angstroms from an amino acid of TIGIT.
  • the TIGIT antigen binding protein e.g., anti- TIGIT antibody
  • Tyr52 and Thr59 form a hydrogen bond with the same amino acid of TIGIT.
  • Ser56 forms a hydrogen bond with two different amino acids of TIGIT.
  • the TIGIT antigen binding protein comprises a LC variable region amino acid sequence which is highly similar to SEQ ID NO: 122 or 212 or 2012 or the first 105-115 amino acids of SEQ ID NO: 246 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity) and comprises a glutamic acid at position 1 (Glu1), or a conservative amino acid substitution thereof, an isoleucine at position 2 (Ile2), or a conservative amino acid substitution thereof, a glutamine at position 27 (Gln27), or a conservative amino acid substitution thereof, a serine at position 28 (Ser28), or a conservative amino acid substitution thereof, a valine at position 29 (Val29), or a conservative amino acid substitution thereof, a serine at position 30 (Ser30), or a conservative amino acid substitution thereof, a serine
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the amino acid residues named above are positioned about 3 angstroms to about 4 angstroms from an amino acid of TIGIT.
  • the TIGIT antigen binding protein comprises a HC variable region amino acid sequence which is highly similar to SEQ ID NO: 121 or 211 or the first 115-127 amino acids of SEQ ID NO: 245 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity) and a glycine at position 32 (Gly32), or a conservative amino acid substitution thereof, a tyrosine at position 35 (Tyr35), or a conservative amino acid substitution thereof, a tyrosine at position 52 (Tyr52), or a conservative amino acid substitution thereof, a tyrosine at position 54 (Tyr54), or a conservative amino acid substitution thereof, a tyrosine at position 55 (Tyr55), or a conservative amino acid substitution thereof, a serine at position 56 (Ser56), or
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • each of Ser58, Asn106, Tyr107, Tyr35, Arg102, and Ser56, or a conservative amino acid substitution thereof forms a hydrogen bond with an amino acid of TIGIT.
  • each of the amino acid residues named above are positioned about 3 angstroms to about 4 angstroms from an amino acid of TIGIT.
  • the TIGIT antigen binding protein comprises a LC variable region amino acid sequence which is highly similar to SEQ ID NO: 162 or the first 105-115 amino acids of SEQ ID NO: 262 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity) and comprises an arginine at position 30 (Arg30), or a conservative amino acid substitution thereof, an arginine at position 31 (Arg31), or a conservative amino acid substitution thereof, a tyrosine at position 32 (Tyr32), or a conservative amino acid substitution thereof, a serine at position 91 (Ser91), or a conservative amino acid substitution thereof, a tyrosine at position 92 (Tyr92), or a conservative amino acid substitution thereof, a serine at position 93 (Ser93), or a conservative amino acid substitution thereof,
  • the TIGIT antigen binding protein comprises a LC CDR1 amino acid sequence comprising Arg30, or a conservative amino acid substitution thereof, Arg31, or a conservative amino acid substitution thereof, Tyr32, or a conservative amino acid substitution thereof, or any combination thereof.
  • the TIGIT antigen binding protein, e.g., anti-TIGIT antibody comprises a LC CDR3 amino acid sequence comprising Ser91, or a conservative amino acid substitution thereof, Tyr92, or a conservative amino acid substitution thereof, Ser93, or a conservative amino acid substitution thereof, Thr94, or a conservative amino acid substitution thereof, or any combination thereof.
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the amino acid residues named above are positioned about 3 angstroms to about 4 angstroms from an amino acid of TIGIT.
  • the TIGIT antigen binding protein comprises a HC variable region amino acid sequence which is highly similar to SEQ ID NO: 161 or the first 115-127 amino acids of SEQ ID NO: 261 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity) and a threonine at position 30 (Thr30), or a conservative amino acid substitution thereof, a glycine at position 31 (Gly31), or a conservative amino acid substitution thereof, a tyrosine at position 32 (Tyr32), or a conservative amino acid substitution thereof, a tyrosine at position 33 (Tyr33), or a conservative amino acid substitution thereof, a tryptophan at position 47 (Trp47), or a conservative amino acid substitution thereof, a tryptophan[ at position 50 (Trp50), or a
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • each of Leu104, Tyr33, Asn101, Tyr107, Tyr60, Ser55, Ser52, or a conservative amino acid substitution thereof forms a hydrogen bond with an amino acid of TIGIT.
  • each of the amino acid residues named above are positioned about 3 angstroms to about 4 angstroms from an amino acid of TIGIT.
  • the TIGIT antigen binding protein comprises a LC variable region amino acid sequence which is highly similar to SEQ ID NO: 132 or the first 105-115 amino acids of SEQ ID NO: 254 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity) and comprises a glutamine at position 27 (Gln27), or a conservative amino acid substitution thereof, a leucine at position 30 (Leu30), or a conservative amino acid substitution thereof, a serine at position 32 (Ser32), or a conservative amino acid substitution thereof, a serine at position 96 (Ser96), or a conservative amino acid substitution thereof, an isoleucine at position 97 (Ile97), or a conservative amino acid substitution thereof, a glutamine at position 98 (Gln98), or a conservative amino acid substitution thereof, a leu
  • the TIGIT antigen binding protein comprises a LC CDR1 amino acid sequence comprising Gln27, or a conservative amino acid substitution thereof, Leu30, or a conservative amino acid substitution thereof, Ser32, or a conservative amino acid substitution thereof, or any combination thereof.
  • the TIGIT antigen binding protein comprises a LC CDR3 amino acid sequence comprising Ser96, or a conservative amino acid substitution thereof, Ile97, or a conservative amino acid substitution thereof, Gln98, or a conservative amino acid substitution thereof, Leu99, or a conservative amino acid substitution thereof, or any combination thereof.
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • the TIGIT antigen binding protein comprises a LC variable region amino acid sequence comprising Ser32, or a conservative amino acid substitution thereof, and/or Gln98, or a conservative amino acid substitution thereof, each of which forms a hydrogen bond with an amino acid of TIGIT.
  • the amino acid residues named above are positioned about 3 angstroms to about 4 angstroms from an amino acid of TIGIT.
  • the TIGIT antigen binding protein comprises a HC variable region amino acid sequence which is highly similar to SEQ ID NO: 131 or the first 115-127 amino acids of SEQ ID NO: 253 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity) and an aspartate at position 33 (Asp33), or a conservative amino acid substitution thereof, a tyrosine at position 52 (Tyr52), or a conservative amino acid substitution thereof, a tyrosine at position 54 (Tyr54), or a conservative amino acid substitution thereof, a tyrosine at position 55 (Tyr55), or a conservative amino acid substitution thereof, a serine at position 56 (Ser56), or a conservative amino acid substitution thereof, a glycine at position 57 (Gly57), or a conservative amino acid
  • the TIGIT antigen binding protein comprises a HC CDR1 amino acid sequence comprising Asp33, or a conservative amino acid substitution thereof.
  • the TIGIT antigen binding protein comprises a HC CDR2 amino acid sequence comprising Tyr52, or a conservative amino acid substitution thereof, a Tyr54, or a conservative amino acid substitution thereof, Tyr55, or a conservative amino acid substitution thereof, Ser56, or a conservative amino acid substitution thereof, Gly57, or a conservative amino acid substitution thereof, Gly58, or a conservative amino acid substitution thereof, Thr59, or a conservative amino acid substitution thereof, Tyr60, or a conservative amino acid substitution thereof, Pro63, or a conservative amino acid substitution thereof, Lys66, or a conservative amino acid substitution thereof, or any combination thereof.
  • the TIGIT antigen binding protein e.g., anti-TIGIT antibody
  • each of Tyr52, Ser56, Thr59, Tyr60, Phe109, Tyr54, Tyr55, Lys66, or a conservative amino acid substitution thereof forms a hydrogen bond with an amino acid of TIGIT.
  • the antigen binding protein comprises a heavy chain amino acid sequence which comprises a set of charge pair mutations, as described herein.
  • the heavy chain amino acid sequence comprises charge pair mutations selected from V1, V103, and V131 charge pair mutations.
  • the antigen binding protein comprises one or more amino acid modifications, relative to the naturally-occurring counterpart, in order to improve half- life/stability or to render the antibody more suitable for expression/manufacturability.
  • the antigen binding protein is designed to prevent or reduce interaction between the Fc and Fc receptors.
  • the antigen binding protein is a Stable Effector Functionless (SEFL) antibody comprising a constant region that lacks the ability to interact with Fc ⁇ receptors. SEFL antibodies are known in the art. See, e.g., Liu et al., J Biol Chem 292: 1876-1883 (2016); and Jacobsen et al., J. Biol.
  • the SEFL antibody comprises one or more of the following mutations, numbered according to the EU system: L242C, A287C, R292C, N297G, V302C, L306C, and/or K334C.
  • the SEFL antibody comprises N297G.
  • the SEFL antibody comprises A287C, N297G, and L306C.
  • the SEFL antibody comprises R292C, N297G, and V302C (i.e., SEFL2- 2).
  • the antigen binding protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2019 optionally SEQ ID NO: 2020.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH1, VH3, or VH4 family of gene segments. In various aspects, the antigen-binding protein is an antibody comprising a HC variable region encoded by a D gene segment of the D1, D3, D5, D6 or D7 family of gene segments. In various aspects, the antigen- binding protein is an antibody comprising a HC variable region encoded by a J gene segment of the JH4 or JH6 family of gene segments.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH3 family of gene segments and a D gene segment of the D1, D3, or D6 family of gene segments, and/or a J gene segment of the JH4 or JH6 family of gene segments.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH3 family of gene segments, a D gene segment of the D1 family of gene, and a J gene segment of the JH6 family of gene segments.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH3 family of gene segments, a D gene segment of the D3 family of gene, and a J gene segment of the JH6 family of gene segments.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH3 family of gene segments, a D gene segment of the D6 family of gene, and a J gene segment of the JH6 family of gene segments.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH1 family of gene segments, a D gene segment of the D5 family of gene, and a J gene segment of the JH6 family of gene segments.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH4 family of gene segments, a D gene segment of the D7 family of gene, and a J gene segment of the JH6 family of gene segments.
  • the antigen-binding protein is an antibody comprising a HC variable region encoded by a V gene segment of the VH3 family of gene segments, a D gene segment of the D1 family of gene, and a J gene segment of the JH4 family of gene segments.
  • Polypeptides comprising, consisting essentially of, or consisting of one or more of the amino acid sequence(s) of Table A1 or Table A2 or Table B1 or Table B2 or Table C1 or Table C2.
  • the polypeptide comprises six of the CDRs of Table A1 or Table A2 with intervening amino acids.
  • the polypeptide comprises only one of the HC variable or LC variable amino acid sequences of Table B1 or Table B2. In various aspects, the polypeptide comprises both the HC variable and LC variable amino acid sequences of Table B1 or Table B2 fused as one sequence, optionally, wherein the HC variable and LC variable amino acid sequences are linked together by a linker sequence. In various instances, the polypeptide comprises two copies of the HC variable region sequence and two copies of the LC variable region sequences, optionally, linked together with linker sequences. In some aspects, the polypeptide comprises the amino acid sequence of an scFv 1 an scFv 2 or an (scFv) 2 .
  • the polypeptide comprises the amino acid sequence of a diabody, triabody, single domain antibody, single variable domain, tandem scFv, tascFvs, and the like.
  • the polypeptide comprises only one of the FL HC or FL LC amino acid sequences of Table B1 or Table B2 or Table C1 or Table C2.
  • the polypeptide comprises both the FL HC and FL LC amino acid sequences of Table B1 or Table B2 or Table C1 or Table C2 fused as one sequence, optionally, wherein the FL HC and FL LC are linked together by a linker sequence.
  • the polypeptide comprises a variant sequence of a parent sequence comprising the amino acid sequence of Table A1 or Table A2 or Table B1 or Table B2 or Table C1 or Table C2.
  • the polypeptide comprises an amino acid sequence which differs by only 1, 2, 3, 4, 5, 6, or more amino acids, relative to the parent sequence.
  • the polypeptide comprises a variant sequence which differs by only one or two amino acids, relative to the parent sequence.
  • the polypeptide comprises variant sequence having only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions, relative to the above-referenced amino acid sequence(s).
  • the polypeptide comprises an amino acid sequence which has greater than or about 30%, greater than or about 50%, or greater than or about 70% sequence identity to the parent amino acid sequence.
  • the polypeptide comprises an amino acid sequence which has at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity to the parent sequence.
  • the antigen-binding protein comprises an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity along the full- length of the parent sequence.
  • the polypeptide comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity along the full-length of the parent sequence
  • the polypeptide is lipidated (e.g., myritoylated, palmitoylated), glycosylated, amidated, carboxylated, phosphorylated, esterified, acylated, acetylated, cyclized, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated, as further described herein.
  • peptidomimetics designed to mimic a polypeptide of the present disclosure.
  • the peptidomimetics are substantially similar in structure to a polypeptide of the present disclosure but has at least one structural difference.
  • the peptidomimetic is a peptoid having one or more linkages in replacing one or more peptide linkages.
  • the peptoid comprises a side chain that is connected to the nitrogen of the peptide backbone, instead of an ⁇ -carbon as in peptides.
  • the peptoids of the present disclosure lack the amide hydrogen which is responsible for many of the secondary structure elements in peptides and proteins. See, e.g., Reyna et al., PNAS 89(20): 9367-9371 (1992).
  • the peptidomimetic is a D-peptide peptidomimetic comprising D-isomer amino acids.
  • the peptidomimetic is a peptoid in which the side chain of an amino acid is connected to the alpha nitrogen atom of the peptide backbone. Methods of making peptoids are known in the art.
  • the peptidomimetic is a ⁇ -peptide comprising ⁇ amino acids which have their amino group bonded to the ⁇ -carbon rather than the alpha carbon.
  • Methods of making ⁇ -peptides are known in the art. See, for example, Seebach et al., Helvetica Chimica Acta 79(4): 913-941 (1996).
  • Aptamers [00127] In exemplary embodiments, the antigen binding protein is an aptamer.
  • a loop structure is often involved with providing the desired binding attributes as in the case of: aptamers which often utilize hairpin loops created from short regions without complimentary base pairing, naturally derived antibodies that utilize combinatorial arrangement of looped hyper-variable regions and new phage display libraries utilizing cyclic peptides that have shown improved results when compared to linear peptide phage display results.
  • aptamers which often utilize hairpin loops created from short regions without complimentary base pairing
  • naturally derived antibodies that utilize combinatorial arrangement of looped hyper-variable regions
  • new phage display libraries utilizing cyclic peptides that have shown improved results when compared to linear peptide phage display results.
  • conjugates comprising one or more of the antigen binding proteins of the present disclosure linked to a heterologous moiety.
  • heterologous moiety is synonymous with the term “conjugate moiety” and refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) which is different from the antigen binding proteins described herein.
  • exemplary conjugate moieties that can be linked to any of the antigen binding proteins described herein include but are not limited to a heterologous peptide or polypeptide, a targeting agent, a diagnostic label such as a radioisotope, fluorophore or enzymatic label, a polymer including water soluble polymers, or other therapeutic or diagnostic agents.
  • the conjugate in some embodiments comprises one or more of the antigen binding proteins described herein and one or more of: a peptide (which is distinct from the antigen binding proteins described herein), a polypeptide, a nucleic acid molecule, another antibody or fragment thereof, a polymer, a quantum dot, a small molecule, a toxin, a diagnostic agent, a carbohydrate, an amino acid.
  • the conjugate of the present disclosure comprises an antigen binding protein as described herein and a heterologous moiety which is a polypeptide (e.g., a polypeptide distinct from any of the antigen binding proteins described herein), and the conjugate is a fusion polypeptide or fusion protein or a chimeric protein or chimeric polypeptide. Additional descriptions of such conjugates are provided herein under “Fusion proteins”.
  • the heterologous moiety is attached via non-covalent or covalent bonding to the antigen binding protein of the present disclosure.
  • the linkage between the antigen binding protein and the heterologous moiety is achieved via covalent chemical bonds, e.g., peptide bonds, disulfide bonds, and the like, or via physical forces, such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions.
  • covalent chemical bonds e.g., peptide bonds, disulfide bonds, and the like
  • physical forces such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions.
  • non- covalent coupling systems may be used, including, e.g., biotin-avidin, ligand/receptor, enzyme/substrate, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cellular adhesion molecule partners; or any binding partners or fragments thereof which have affinity for each other.
  • the antigen binding protein in exemplary embodiments is linked to a conjugate moiety via direct covalent linkage by reacting targeted amino acid residues of the antigen binding protein with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of these targeted amino acids.
  • Reactive groups on the antigen binding protein or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimido or hydrazino group.
  • Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.
  • the conjugate moieties can be linked to the antigen binding protein indirectly through intermediate carriers, such as polysaccharide or polypeptide carriers. Examples of polysaccharide carriers include aminodextran.
  • polypeptide carriers examples include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier.
  • Cysteinyl residues are most commonly reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid, chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha- bromo-E-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and
  • arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. [00137]
  • the specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O- acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • arginine and histidine free carboxyl groups
  • free sulfhydryl groups such as those of cysteine
  • free hydroxyl groups such as those of serine, threonine, or hydroxyproline
  • aromatic residues such as those of tyrosine, or tryptophan
  • the heterologous moiety is attached to the antigen binding protein of the present disclosure via a linker.
  • the linker comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long.
  • the chain atoms are all carbon atoms.
  • the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate.
  • the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell.
  • the length of the linker is long enough to reduce the potential for steric hindrance. If the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide, the entire conjugate can be a fusion protein. Such peptidyl linkers may be any length. Exemplary peptidyl linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length, and are flexible or rigid. In exemplary aspects, the linker is a peptide comprising about 2 to about 20 amino acids. In exemplary aspects, the linker is a peptide comprising about 2 to about 15 amino acid, about 2 to about 10 amino acids, or about 2 to about 5 amino acids.
  • Suitable peptide linkers are known in the art. See, e.g., Chen et al., Adv Drug Delivery Reviews 65(10): 1357-1369 (2013); Arai et al., Protein Eng Des Sel 14(8): 529-532 (2001); and Wriggers et al., Curr Trends in Peptide Science 80(6): 736-746 (2005).
  • the linker is a peptide comprising the amino acid sequence GGGGS (SEQ ID NO: 2021).
  • the antigen binding protein is linked to a polypeptide which is distinct from any of the antigen binding proteins described herein, and the conjugate is a fusion polypeptide or fusion protein or a chimeric protein or chimeric polypeptide.
  • the present disclosure provides fusion polypeptides or fusion proteins comprising an antigen binding protein of the present disclosure and a heterologous polypeptide or peptide.
  • the fusion protein of the present disclosure comprises a HC variable amino acid sequence fused to a LC variable amino acid sequence or a FL HC sequence fused to a FL LC sequence.
  • the fusion protein comprises a peptide linker between the HC variable amino acid sequence and the LC variable amino acid sequence or between the FL HC sequence and the FL LC sequence
  • Nucleic Acids [00145] The present disclosure further provides nucleic acids comprising a nucleotide sequence encoding an antigen binding protein or polypeptide or fusion protein of the present disclosure.
  • nucleic acid includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, or modified forms thereof, which can be single-stranded or double- stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered inter-nucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • the nucleic acid can comprise any nucleotide sequence which encodes any of the antigen-binding proteins or polypeptides of the present disclosure. In some embodiments, the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. In other embodiments, the nucleic acid comprises one or more insertions, deletions, inversions, and/or substitutions. [00146] In some aspects, the nucleic acids of the present disclosure are recombinant.
  • the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • the nucleic acids in some aspects are constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra; and Ausubel et al., supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5- fluorouracil, 5-bromouracil, 5-chIorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridme, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 - isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N -substituted adenine, 7-methylguanine, 5- methylammomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosyl
  • the nucleic acid comprises a nucleotide sequence encoding an antigen binding protein or polypeptide or fusion protein of the present disclosure.
  • the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence(s) having a SEQ ID NO: listed in Table A1 or Table A2 or Table B1 or Table B2 or Table C1 or Table C2.
  • the nucleotide sequence encodes an amino acid sequence comprising six of the CDRs of Table A1 or Table A2 with intervening amino acids.
  • nucleotide sequence only one of the HC variable or LC variable amino acid sequences of Table B1 or Table B2.
  • nucleotide sequence encodes both the HC variable and LC variable amino acid sequences of Table B1 or Table B2 fused as one sequence, optionally, wherein the HC variable and LC variable amino acid sequences are linked together by a linker sequence.
  • the nucleotide sequence encodes a polypeptide described herein.
  • nucleotide sequence encodes only one of the FL HC or FL LC amino acid sequences of Table B1 or Table B2 or Table C1 or Table C2.
  • nucleotide sequence encodes an amino acid sequence both the FL HC and FL LC amino acid sequences of Table B1 or Table B2 or Table C1 or Table C2 fused as one sequence, optionally, wherein the FL HC and FL LC are linked together by a linker sequence.
  • the nucleotide sequence encodes a variant sequence of a parent sequence comprising the amino acid sequence of Table A1 or Table A2 or Table B1 or Table B2 or Table C1 or Table C2.
  • the nucleotide sequence encodes an amino acid sequence which differs by only 1, 2, 3, 4, 5, 6, or more amino acids, relative to the parent sequence.
  • the nucleotide sequence encodes a variant sequence which differs by only one or two amino acids, relative to the parent sequence. In exemplary aspects, the nucleotide sequence encodes a variant sequence having only 1, 2, 3, 4, 5, 6, or more conservative amino acid substitutions, relative to the above-referenced amino acid sequence(s).
  • the nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs: 2037-2092, as shown in Table D. In various instances, the nucleotide sequence of any one of SEQ ID NOs: 2037-2092 comprises a nucleotide sequence encoding a signal sequence.
  • the nucleic acid of the present disclosure comprises a nucleotide sequence of any one of SEQ ID NOs: 2037-2092 without a nucleotide sequence encoding the signal sequence. In various aspects, the nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs: 2037-2092 without the first 60, 63, 66, or 69 nucleic acids of any one of SEQ ID NOs: 2037-2092. TABLE D [00149] Vectors [00150] The nucleic acids of the present disclosure in some aspects are incorporated into a vector. In this regard, the present disclosure provides vectors comprising any of the presently disclosed nucleic acids. In exemplary aspects, the vector is a recombinant expression vector.
  • the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors of the present disclosure are not naturally-occurring as a whole. However, parts of the vectors can be naturally- occurring.
  • the presently disclosed vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages.
  • the altered nucleotides or non-naturally occurring internucleotide linkages do not hinder the transcription or replication of the vector.
  • the vector of the present disclosure can be any suitable vector and can be used to transform or transfect any suitable host.
  • Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJoIIa, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).
  • Bacteriophage vectors such as ⁇ GTIO, ⁇ GTl 1, ⁇ ZapII (Stratagene), ⁇ EMBL4, and ⁇ NMl 149, also can be used.
  • the vector is a viral vector, e.g., a retroviral vector.
  • the vectors of the present disclosure can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
  • Replication systems can be derived, e.g., from CoIEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • the vector can include one or more marker genes, which allow for selection of transformed or transfected hosts.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the presently disclosed expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the vector can comprise a native or normative promoter operably linked to the nucleotide sequence encoding the polypeptide (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the antigen binding protein.
  • the selection of promoters e.g., strong, weak, inducible, tissue- specific and developmental- specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • the vector encodes an antibody light chain, an antibody heavy chain, or an antibody light chain and heavy chain.
  • a vector encoding an antibody light chain may be useful for producing an antibody of the present invention when expressed in a cell that further contains a vector encoding an antibody heavy chain.
  • a vector encoding an antibody heavy chain may be useful for producing an antibody of the present invention when expressed in a cell that further contains a vector encoding an antibody light chain.
  • both the light chain and the heavy chain may be encoded on a single vector, in certain embodiments the vector encodes the light chain but does not encode the heavy chain. In other embodiment, the vector encodes the heavy chain but does not encode the light chain.
  • the vector of the present comprises a nucleotide sequence encoding HC variable region or a full-length HC and a nucleotide sequence encoding LC variable region or a full- length LC.
  • the vector of the present disclosure comprises a nucleotide sequence encoding HC variable region or a full-length HC as described in Table B1, C1, B2 or C2 or a nucleotide sequence encoding LC variable region or a full-length LC as described in Table B1, C1, B2 or C2.
  • Host cells [00159] Provided herein are host cells comprising one or more nucleic acids or vectors of the present disclosure.
  • the term “host cell” refers to any type of cell that can contain the presently disclosed vector or vectors and is capable of producing an expression product encoded by the nucleic acid(s) (e.g., mRNA, protein).
  • the host cell in some aspects is an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
  • the host cell in exemplary aspects is a cultured cell or a primary cell, i.e., isolated directly from an organism.
  • the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage.
  • the cell is a eukaryotic cell, including, but not limited to, a yeast cell, filamentous fungi cell, protozoa cell, algae cell, insect cell, or mammalian cell.
  • a yeast cell including, but not limited to, a yeast cell, filamentous fungi cell, protozoa cell, algae cell, insect cell, or mammalian cell.
  • host cells are described in the art. See, e.g., Frenzel, et al., Front Immunol 4: 217 (2013).
  • the eukaryotic cells are mammalian cells.
  • the mammalian cells are non-human mammalian cells.
  • the cells are Chinese Hamster Ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3, CS9), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2/0), cells engineered to be deficient in dihydrofolatereductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293- EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinomic human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryo fibroblast cells NIH
  • the host cell is CS9 (a CHO cell line).
  • the host cell is in some aspects a prokaryotic cell, e.g., a bacterial cell.
  • a population of cells comprising at least one host cell described herein.
  • the population of cells in some aspects is a heterogeneous population comprising the host cell comprising vectors described, in addition to at least one other cell, which does not comprise any of the vectors.
  • the population of cells is a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the vector.
  • the population in some aspects is a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a vector, such that all cells of the population comprise the vector.
  • the population of cells is a clonal population comprising host cells comprising a vector as described herein.
  • the host cells comprise a first vector comprising a nucleotide sequence encoding HC variable region or a full-length HC as described in Table B1, C1, B2 or C2 and a second vector comprising a nucleotide sequence encoding LC variable region or a full-length LC as described in Table B1, C1, B2 or C2.
  • compositions comprising an antigen-binding protein (e.g., a TIGIT binding protein, a CD112R binding protein), a polypeptide, a nucleic acid, a vector, a host cell, a conjugate, a fusion protein of the present disclosure, or a combination thereof, are provided herein.
  • the compositions in some aspects comprise the antigen-binding protein, polypeptide, a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof, in isolated and/or purified form.
  • the composition comprises a single type (e.g., structure) of binding protein, polypeptide, a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure, or comprises a combination of two or more different types (e.g., different structures) of antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector or host cell of the present disclosure.
  • the composition comprises agents which enhance the chemico- physico features of the antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector, or host cell, e.g., via stabilizing, for example, at certain temperatures (e.g., room temperature), increasing shelf life, reducing degradation, e.g., oxidation protease mediated degradation, increasing half-life of the antigen binding protein, etc.
  • the composition comprises any of the agents disclosed herein as a heterologous moiety or conjugate moiety, optionally, in admixture with the antigen binding protein or polypeptide of the present disclosure.
  • the composition additionally comprises a pharmaceutically acceptable carrier, diluents, or excipient.
  • the antigen binding proteins, polypeptides, conjugates, fusion proteins, nucleic acids, vectors, or host cells as presently disclosed are formulated into a pharmaceutical composition comprising the active agent, along with a pharmaceutically acceptable carrier, diluent, or excipient.
  • the present disclosure further provides pharmaceutical compositions comprising an active agent which pharmaceutical composition is intended for administration to a subject, e.g., a mammal.
  • the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient.
  • the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%, and a pharmaceutically acceptable diluent, carrier or excipient.
  • the compositions contain an active agent at a concentration of about 0.001 to about 30.0 mg/ml.
  • the pharmaceutical compositions comprise a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
  • the pharmaceutical composition can comprise any pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents,
  • the pharmaceutical composition comprises formulation materials that are nontoxic to recipients at the dosages and concentrations employed.
  • compositions comprising an active agent and one or more pharmaceutically acceptable salts; polyols; surfactants; osmotic balancing agents; tonicity agents; anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents.
  • the pharmaceutical composition comprises one or more polyols and/or one or more surfactants, optionally, in addition to one or more excipients, including but not limited to, pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics.
  • pharmaceutically acceptable salts including but not limited to, pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • formulation materials for modifying, maintaining or preserving for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents;
  • amino acids
  • the pharmaceutical compositions can be formulated to achieve a physiologically compatible pH.
  • the pH of the pharmaceutical composition can be for example between about 4 or about 5 and about 8.0 or about 4.5 and about 7.5 or about 5.0 to about 7.5.
  • Routes of Administration [00175] With regard to the present disclosure, the active agent, or pharmaceutical composition comprising the same, can be administered to the subject via any suitable route of administration.
  • the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • parenteral means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous.
  • the active agent of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2- dimethyl- l53-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl- ⁇ -aminopropionates, and 2-alkyl -imidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • the parenteral formulations in some embodiments contain from about 0.5% to about 25% by weight of the active agent of the present disclosure in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the site of injection, such compositions can contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • HLB hydrophile-lipophile balance
  • parenteral formulations in some aspects are presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions in some aspects are prepared from sterile powders, granules, and tablets of the kind previously described.
  • injectable formulations are in accordance with the present disclosure.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B.
  • the active agents of the disclosure are believed to be useful in methods of inhibiting the biological activities that are initiated upon binding of TIGIT to CD155 or CD112R to CD112, as described herein, and are thus believed to be useful in methods of increasing an immune response, e.g., a T-cell mediated immune response and methods of treating or preventing one or more diseases, e.g., cancer.
  • the amount or dose of the active agent administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame.
  • the dose of the active agent of the present disclosure should be sufficient to treat cancer as described herein in a period of from about 1 to 4 hours, 1 to 4 days, or 1 to 4 weeks or longer, e.g., 5 to 20 or more weeks, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular active agent and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
  • an assay which comprises comparing the extent to which cancer is treated upon administration of a given dose of the active agent of the present disclosure to a mammal among a set of mammals, each set of which is given a different dose of the active agent, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which cancer is treated upon administration of a certain dose can be represented by, for example, the cytotoxicity of the active agent or the extent of tumor regression achieved with the active agent in a mouse xenograft model. Methods of measuring cytotoxicity of the antigen binding proteins and methods of assaying tumor regression are known in the art.
  • tumor xenografts may be established by subcutaneous inoculation of mice in the right flank with human tumor cells suspended in PBS pH 7.4. Tumors may be measured using a digital caliper.
  • the dose of the active agent of the present disclosure also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular active agent of the present disclosure.
  • the attending physician will decide the dosage of the active agent of the present disclosure with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, active agent of the present disclosure to be administered, route of administration, and the severity of the condition being treated.
  • the dose of the active agent of the present disclosure can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg body weight/day.
  • the active agents described herein can be modified into a depot form, such that the manner in which the active agent of the present disclosure is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No.4,450,150).
  • Depot forms of active agents of the present disclosure can be, for example, an implantable composition comprising the active agents and a porous or non-porous material, such as a polymer, wherein the active agent is encapsulated by or diffused throughout the material and/or degradation of the non-porous material.
  • the depot is then implanted into the desired location within the body of the subject and the active agent is released from the implant at a predetermined rate.
  • the pharmaceutical composition comprising the active agent in certain aspects is modified to have any type of in vivo release profile.
  • the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release, or bi- phasic release formulation.
  • Methods of formulating peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374: 46-52 (2009) and International Patent Application Publication Nos. WO 2008/130158, WO2004/033036; WO2000/032218; and WO 1999/040942.
  • compositions can further comprise, for example, micelles or liposomes, or some other encapsulated form, or can be administered in an extended release form to provide a prolonged storage and/or delivery effect.
  • the active agents described herein are administered alone, and in alternative embodiments, the active agents described herein are administered in combination with another therapeutic agent, e.g., another active agent of the present disclosure of a different type (e.g., structure). Accordingly, the present disclosure provides a combination comprising a first antigen binding protein which targets CD112R and a second antigen binding protein which targets TIGIT, each of which is an antigen binding protein according to the present disclosures.
  • the first antigen binding protein is any one of 1E1, 1E1.016, 24F1, 29E10, 24F1.001, 29E10_CONS.020, 29E10_CONS.021, 29E10_CONS.022, 29E10_CONS.025, 11E4, 31B3, 27G12, 28F9, 28H7, or 36C8 as described in Table A1 or Table B1.
  • the second antigen binding protein is any one of 55G7.041.008, 58A7.003.008.075, 4G10, 11A3, 28B8, 39D2, 43B7, 55G7, 66H9, 43B7.002.015, 58A7.003.08, 66H9.009, or 58A7 as described in Table A2 or Table B2.
  • the first antigen binding protein is 24F1, 29E10_CONS.020 or 29E10_CONS.022.
  • the second antigen binding protein is 43B7.002.015 or 66H9.009.
  • the combination comprises 24F1 and 43B7.002.015.
  • the combination comprises 24F1 and 66H9.009.
  • the combination comprises 29E10_CONS.020 and 43B7.002.015. In another aspect, the combination comprises 29E10_CONS.020 and 66H9.009. In one aspect, the combination comprises 29E10_CONS.022 and 43B7.002.015. In another aspect, the combination comprises 29E10_CONS.022 and 66H9.009. In another aspect, the combination comprises 43B7.002.015 and 1E1.016 or 24F1 or 29E10. In yet another aspect, the combination comprises 66H9.009 and 1E1.016 or 24F1 or 29E10. In exemplary aspects, the combination comprises 43B7 and 29E10 or 24F1 or 11E4.
  • the present disclosure provides the combination as a composition, e.g., pharmaceutical composition, in various instances. Accordingly, the present disclosure provides a composition, e.g., pharmaceutical composition, comprising the first antigen binding protein and the second antigen binding protein.
  • the first antigen binding protein is 24F1, 29E10_CONS.020 or 29E10_CONS.022.
  • the second antigen binding protein is 43B7.002.015 or 66H9.009.
  • the composition comprises 24F1 and 43B7.002.015.
  • the composition comprises 24F1 and 66H9.009.
  • the composition comprises 29E10_CONS.020 and 43B7.002.015.
  • the composition comprises 29E10_CONS.020 and 66H9.009. In one aspect, the composition comprises 29E10_CONS.022 and 43B7.002.015. In another aspect, the composition comprises 29E10_CONS.022 and 66H9.009. In another aspect, the composition comprises 43B7.002.015 and 1E1.016 or 24F1 or 29E10. In yet another aspect, the composition comprises 66H9.009 and 1E1.016 or 24F1 or 29E10. In exemplary aspects, the composition comprises 43B7 and 29E10 or 24F1 or 11E4. In exemplary instances, the first antigen binding protein and the second antigen binding protein are present in the composition at a ratio of about 1:1.
  • the combination or composition further comprises an additional active agent, e.g., a third antigen binding protein.
  • the third antigen binding protein binds to a negative regulator of the immune system, an immune suppressor, or an immune checkpoint protein, including but not limited to CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, or co-stimulatory receptor: ICOS, OX40, 41BB, CD27, GITR.
  • the additional active agent is a PD-1 binding protein, e.g., an anti- PD-1 antibody.
  • anti-PD-1 antibodies examples include nivolumab (BMS-936558), pembrolizumab (MK3475), BMS 936558, BMS- 936559, TSR-042 (Tesaro), Epdr001 (Novartis), and pidilizumab (CT-011).
  • the third antigen binding protein is any PD-1 antigen binding proteins described in International Patent Application No. PCT/US2019/013205, which published as WO/2019/140196 the entire contents of which is incorporated herein by reference.
  • the third antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, a HC CDR3 amino acid sequence, a LC CDR amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence of SEQ ID NOs: 352-357 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, a HC CDR3 amino acid sequence, a LC CDR amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence of SEQ ID NOs: 2027-2032, respectively.
  • the third antigen binding protein comprises a HC variable region amino acid sequence and a LC variable region amino acid sequence of SEQ ID NO: 358 and SEQ ID NO: 359 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a HC variable region amino acid sequence and a LC variable region amino acid sequence of SEQ ID NO: 2033 and SEQ ID NO: 2034, respectively.
  • the third antigen binding protein comprises a FL HC amino acid sequence and a FL LC amino acid sequence of SEQ ID NO: 360 and SEQ ID NO: 361 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a FL HC amino acid sequence and a FL LC amino acid sequence of SEQ ID NO: 2035 and SEQ ID NO: 2036, respectively.
  • the third antigen binding protein comprises a FL HC amino acid sequence and a FL LC amino acid sequence of SEQ ID NO: 2093 and SEQ ID NO: 2094, respectively.
  • the first antigen binding protein and the second antigen binding protein and the third antigen binding protein are present in the composition at a ratio of about 1:1:1.
  • the additional active agent is a PD-L1 binding protein, e.g., an anti- PD-L1 antibody.
  • the presently disclosed combination or composition comprises a CD112R antigen-binding protein, a TIGIT antigen-binding protein, and a PD-L1 antigen- binding protein.
  • the composition in exemplary aspects comprises an anti-CD112R antibody, an anti- TIGIT antibody, and an anti-PD-L1 antibody, optionally, at about a 1:1:1 ratio.
  • anti-PD- L1 antibodies useful in the methods of the present disclosure and methods of making them are described in WIPO Patent Publication Number WO 2010/077634 and US Patent No.8,217,149, both of which are incorporated herein by reference.
  • the anti-PD-L1 antibody is atezolizumab (CAS Registry Number: 1422185-06-5).
  • Atezolizumab (Genentech), also known as MPDL3280A, is an anti-PD-L1 antibody. Atezolizumab is a humanized immunoglobulin (Ig) G1 monoclonal antibody. It has been engineered to have a single amino acid substitution that leads to elimination Fc-effector function and to be a non-glycosylated antibody with minimal binding to Fc receptors.
  • Ig immunoglobulin
  • Atezolizumab comprises: (a) an HC CDR1, HC CDR2, and HC CDR3 sequence of GFTFSDSWIH (SEQ ID NO:2095), AWISPYGGSTYYADSVKG (SEQ ID NO: 2096) and RHWPGGFDY (SEQ ID NO: 2097), respectively, and (b) a LC CDR1, LC CDR2, and LC CDR3 sequence of RASQDVSTAVA (SEQ ID NO: 2098), SASFLYS (SEQ ID NO: 2099) and QQYLYHPAT (SEQ ID NO:2100), respectively.
  • Atezolizumab comprises a heavy chain and a light chain sequence, wherein (a) the heavy chain variable region sequence comprises the amino acid sequence: (SEQ ID NO: 2101), and (b) the light chain variable region sequence comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:2102).
  • Atezolizumab comprises a heavy chain and a light chain sequence, wherein (a) the heavy chain comprises the amino acid sequence:
  • the PD-L1 antigen binding protein is highly similar to or the same as atezolizumab and comprises (a) an HC CDR1, HC CDR2, and/or HC CDR3 sequence of GFTFSDSWIH (SEQ ID NO:2095), AWISPYGGSTYYADSVKG (SEQ ID NO: 2096) and RHWPGGFDY (SEQ ID NO: 2097), respectively, and (b) a LC CDR1, LC CDR2, and/or LC CDR3 sequence of RASQDVSTAVA (SEQ ID NO: 2098), SASFLYS (SEQ ID NO: 2099) and QQYLYHPAT (SEQ ID NO:2100), respectively.
  • the PD-L1 antigen binding protein comprises a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2101 and a light chain variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2102.
  • the PD-L1 antigen binding protein comprises a HC which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2103 and a LC which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2104.
  • the anti-PD-L1 antibody is avelumab (CAS Registry Number: 1537032-82-8).
  • Avelumab also known as MSB0010718C, is a human monoclonal IgGl anti-PD-L1 antibody (Merck KGaA, Pfizer).
  • Avelumab comprises a heavy chain and a light chain sequence, wherein: [00197] (a) the heavy chain comprises the amino acid sequence:
  • the light chain comprises the amino acid sequence: [00199]
  • the PD-L1 antigen binding protein is highly similar to or the same as avelumab and comprises the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of avelumab.
  • the PD-L1 antigen binding protein comprises a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the HC variable region of avelumab and/or a LC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the LC variable region of avelumab.
  • the PD-L1 antigen binding protein comprises a HC which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2105 and/or a LC which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2106.
  • the anti-PD-L1 antibody is durvalumab (CAS Registry Number: 1428935-60-7).
  • Durvalumab also known as MEDI4736, is an Fc-optimized human monoclonal IgGl kappa anti-PD-L1 antibody (Medlmmune, AstraZeneca) described in WO2011/066389 and US2013/034559 both of which are incorporated herein by reference in their entirety.
  • Durvalumab comprises a heavy chain and a light chain sequence, wherein (a) the heavy chain comprises the amino acid sequence:
  • the light chain comprises the amino acid sequence: [00202]
  • the PD-L1 antigen binding protein is highly similar to or the same as durvalumab and comprises the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of durvalumab.
  • the PD-L1 antigen binding protein comprises a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the HC variable region of durvalumab and/or a LC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the LC variable region of durvalumab.
  • the PD-L1 antigen binding protein comprises a HC which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2107 and/or a LC which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to SEQ ID NO: 2108.
  • the anti-PD-L1 antibody is MDX-1105 (Bristol Myers Squibb) or an antibody comprising a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical the HC variable region of MDX-1105 and/or a LC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the LC variable region of MDX- 1105.
  • MDX-1105 also known as BMS-936559, is an anti-PD-L1 antibody described in WIPO patent publication number WO2007/005874 which is incorporated herein by reference in its entirety.
  • the anti-PD-L1 antibody is LY3300054 (Eli Lilly), a human anti-PD-L1 antibody or an antibody comprising a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical the HC variable region of LY3300054 and/or a LC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the LC variable region of LY3300054.
  • the anti-PD-L1 antibody is STI-A1014 (Sorrento), a human anti- PD-L1 antibody or an antibody comprising a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical the HC variable region of STI-A1014 and/or a LC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the LC variable region of STI-A1014.
  • the anti-PD-L1 antigen binding protein is KN035 also known as envafolimab or ASC 22 (Suzhou Alphamab).
  • KN035 is single -domain antibody (dAB) generated from a camel phage display library.
  • the anti-PD-L1 antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by a protease in the tumor microenvironment), activates an antibody antigen binding domain to allow it to bind its antigen, e.g., by removing a non-binding steric moiety.
  • the PD-L1 antigen binding protein comprises a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical the HC variable region of KN035 and/or a LC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the LC variable region of KN035.
  • the PD-L1 antigen binding protein is CX-072 (CytomX Therapeutics), CK-301, CS-1001, SHR-1316 (also known as HTI-1088), CBT-502 (also known as TQB-2450), or BGB-A333, each of which are described in Akinleye and Rasool, J of Hematology and Oncology 12, Article No.92 (2019) (which is hereby incorporated by reference in its entirety), or a PD-L1 antigen binding protein comprising a HC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical the HC variable region of CX-072, CK-301, CS-1001, SHR-1316, CBT-502, or BGB-A333 and/or a LC variable region which is at least 85% (e.g., at least 90%, at least 95%, at least 98%) identical to the LC variable region of CX-072, CK-301,
  • the anti-PD-L1 antibody comprises the six CDR sequences (e.g., the three heavy chain CDRs and the three light chain CDRs) and/or the heavy chain variable domain and light chain variable domain from a PD-L1 antibody described in U.S. Patent Publication Number 20160108123 (Assigned to Novartis); WIPO Patent Publication Numbers WO2016/000619 (Applicant: Beigene), WO2012/145493 (Applicant: Amplimmune), WO2013/181634 (Applicant: Sorrento), and WO2016/061142 (Applicant: Novartis), and U.S.
  • the other therapeutic aims to treat or prevent cancer.
  • the other therapeutic is a chemotherapeutic agent.
  • the other therapeutic is an agent used in radiation therapy for the treatment of cancer.
  • kits comprising an antigen-binding protein, polypeptide a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof.
  • the kit in exemplary aspects comprises at least one antigen-binding protein, polypeptide a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof, in a container.
  • the at least one antigen-binding protein, polypeptide a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure is provided in the kit as a unit dose.
  • unit dose refers to a discrete amount dispersed in a suitable carrier.
  • the unit dose is the amount sufficient to provide a subject with a desired effect, e.g., treatment of cancer.
  • the kit comprises several unit doses, e.g., a week or month supply of unit doses, optionally, each of which is individually packaged or otherwise separated from other unit doses.
  • the components of the kit/unit dose are packaged with instructions for administration to a patient.
  • the kit comprises one or more devices for administration to a patient, e.g., a needle and syringe, and the like.
  • the at least one antigen-binding protein, polypeptide a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure, or a combination thereof is/are pre-packaged in a ready to use form, e.g., a syringe, an intravenous bag, etc.
  • the ready to use form is for a single use.
  • the kit comprises multiple single use, ready to use forms of the at least one antigen-binding protein, polypeptide a conjugate, fusion protein, nucleic acid, vector, or host cell of the present disclosure.
  • the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein.
  • the kit comprises more than one antigen binding protein of the present disclosure.
  • the kit comprises a first antigen binding protein which binds to CD112R and a second antigen binding protein which binds to TIGIT.
  • the first antigen binding protein is formulated with the second antigen binding protein.
  • the kit comprises a composition comprising the first antigen binding protein and the second antigen binding protein.
  • the first antigen binding protein is packaged and/or formulated separately from the second antigen binding agent.
  • the first antigen binding protein is any one of 1E1, 1E1.016, 24F1, 29E10, 24F1.001, 29E10_CONS.020, 29E10_CONS.021, 29E10_CONS.022, 29E10_CONS.025, 11E4, 31B3, 27G12, 28F9, 28H7, or 36C8 as described in Table A1 or Table B1.
  • the second antigen binding protein is any one of 55G7.041.008, 58A7.003.008.075, 4G10, 11A3, 28B8, 39D2, 43B7, 55G7, 66H9, 43B7.002.015, 58A7.003.08, 66H9.009, or 58A7 as described in Table A2 or Table B2.
  • the first antigen binding protein is 24F1, 29E10_CONS.020 or 29E10_CONS.022.
  • the second antigen binding protein is 43B7.002.015 or 66H9.009.
  • the kit comprises 24F1 and 43B7.002.015.
  • the kit comprises 24F1 and 66H9.009.
  • the kit comprises 29E10_CONS.020 and 43B7.002.015. In another aspect, the kit comprises 29E10_CONS.020 and 66H9.009. In one aspect, the kit comprises 29E10_CONS.022 and 43B7.002.015. In another aspect, the kit comprises 29E10_CONS.022 and 66H9.009. In another aspect, the kit comprises 43B7.002.015 and 1E1.016 or 24F1 or 29E10. In yet another aspect, the kit comprises 66H9.009 and 1E1.016 or 24F1 or 29E10. In exemplary aspects, the kit comprises 43B7 and 29E10 or 24F1 or 11E4.
  • the first antigen binding protein and the second antigen binding protein are present in the composition at a ratio of about 1:1.
  • the kit comprises an additional active agent, e.g., a third antigen binding protein.
  • the third antigen binding protein binds to a negative regulator of the immune system, an immune suppressor, or an immune checkpoint protein, including but not limited to CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, or co-stimulatory receptor: ICOS, OX40, 41BB, CD27, GITR.
  • the additional active agent is a PD-1 binding protein, e.g., an anti-PD-1 antibody.
  • anti-PD-1 antibodies include nivolumab (BMS-936558), pembrolizumab (MK3475), BMS 936558, BMS- 936559, TSR-042 (Tesaro), Epdr001 (Novartis), and pidilizumab (CT-011).
  • the third antigen binding protein is any PD-1 antigen binding proteins described in International Patent Application No. PCT/US2019/013205, which published as WO/2019/140196 the entire contents of which is incorporated herein by reference.
  • the third antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, a HC CDR3 amino acid sequence, a LC CDR amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence of SEQ ID NOs: 352-357 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, a HC CDR3 amino acid sequence, a LC CDR amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence of SEQ ID NOs: 2027-2032, respectively.
  • the third antigen binding protein comprises a HC variable region amino acid sequence and a LC variable region amino acid sequence of SEQ ID NO: 358 and SEQ ID NO: 359 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a HC variable region amino acid sequence and a LC variable region amino acid sequence of SEQ ID NO: 2033 and SEQ ID NO: 2034, respectively.
  • the third antigen binding protein comprises a FL HC amino acid sequence and a FL LC amino acid sequence of SEQ ID NO: 360 and SEQ ID NO: 361 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a FL HC amino acid sequence and a FL LC amino acid sequence of SEQ ID NO: 2035 and SEQ ID NO: 2036, respectively.
  • the additional active agent is a PD-L1 binding protein, e.g., an anti-PD-L1 antibody.
  • the presently disclosed kit comprises a CD112R antigen-binding protein, a TIGIT antigen-binding protein, and a PD-L1 antigen-binding protein.
  • Suitable examples of PD-L1 antibodies that may be included in the kit of the present disclosure include any of those described herein as well as an anti-PD-L1 comprising (a) an CDR-H1, CDR-H2, and CDR-H3 sequence of GFTFSDSWIH (SEQ ID NO:2095), AWISPYGGSTYYADSVKG (SEQ ID NO: 2096) and RHWPGGFDY (SEQ ID NO: 2097), respectively, and (b) an CDR-L1, CDR-L2, and CDR-L3 sequence of RASQDVSTAVA (SEQ ID NO: 2098), SASFLYS (SEQ ID NO: 2099) and QQYLYHPAT (SEQ ID NO:2100), respectively.
  • an anti-PD-L1 comprising (a) an CDR-H1, CDR-H2, and CDR-H3 sequence of GFTFSDSWIH (SEQ ID NO:2095), AWISPYGGSTYYADSVKG (SEQ ID NO: 2096) and
  • the anti-PD-L1 antigen binding protein comprises a HC variable region of SEQ ID NO: 2102 and a LC variable region of 2103.
  • each antigen binding protein is separately packaged in the kit.
  • the kit comprises a container, e.g., a vial, syringe, bag, etc. comprising at least two of the first, second, and third antigen binding proteins.
  • the kit comprises all the antigen binding proteins in the same container as an admixture.
  • the kit comprises an additional active agent, e.g., a third antigen binding protein.
  • the third antigen binding protein binds to a negative regulator of the immune system, an immune suppressor, or an immune checkpoint protein, including but not limited to CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, or co-stimulatory receptor: ICOS, OX40, 41BB, CD27, GITR.
  • the additional active agent is a PD-1 binding protein, e.g., an anti-PD-1 antibody.
  • the third antigen binding protein is any PD-1 antigen binding proteins described in International Patent Application No.
  • the third antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, a HC CDR3 amino acid sequence, a LC CDR amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence of SEQ ID NOs: 352-357 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a HC CDR1 amino acid sequence, a HC CDR2 amino acid sequence, a HC CDR3 amino acid sequence, a LC CDR amino acid sequence, a LC CDR2 amino acid sequence, and a LC CDR3 amino acid sequence of SEQ ID NOs: 2027-2032, respectively.
  • the third antigen binding protein comprises a HC variable region amino acid sequence and a LC variable region amino acid sequence of SEQ ID NO: 358 and SEQ ID NO: 359 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a HC variable region amino acid sequence and a LC variable region amino acid sequence of SEQ ID NO: 2033 and SEQ ID NO: 2034, respectively.
  • the third antigen binding protein comprises a FL HC amino acid sequence and a FL LC amino acid sequence of SEQ ID NO: 360 and SEQ ID NO: 361 of WO/2019/140196, respectively.
  • the third antigen binding protein comprises a FL HC amino acid sequence and a FL LC amino acid sequence of SEQ ID NO: 2035 and SEQ ID NO: 2036, respectively.
  • each antigen binding protein is separately packaged in the kit.
  • the kit comprises a container, e.g., a vial, syringe, bag, etc. comprising at least two of the first, second, and third antigen binding proteins.
  • the kit comprises all the antigen binding proteins in the same container as an admixture.
  • compositions of the present disclosure are useful for inhibiting TIGIT signaling and/or CD112R signaling and/or PD-1 signaling and/or PD-L1 signaling.
  • TIGIT inhibiting activity and/or CD112R inhibiting activity and/or PD-1 inhibiting activity and/or PD-L1 inhibiting activity of the compositions provided herein allow such entities to be useful in methods of enhancing T cell activity and enhancing an immune response, and, in particular, an immune response against a tumor or cancer.
  • the methods comprise administering to the subject the pharmaceutical composition of the present disclosure in an effective amount.
  • the T cell activity or immune response is directed against a cancer cell or cancer tissue or a tumor cell or tumor.
  • the immune response is a humoral immune response.
  • the immune response is an innate immune response.
  • the immune response which is enhanced is a T-cell mediated immune response.
  • compositions of the present disclosure may enhance, e.g., T cell activity or enhance an immune response, to any amount or level.
  • the enhancement provided by the methods of the present disclosure is at least or about a 10% enhancement (e.g., at least or about a 20% enhancement, at least or about a 30% enhancement, at least or about a 40% enhancement, at least or about a 50% enhancement, at least or about a 60% enhancement, at least or about a 70% enhancement, at least or about a 80% enhancement, at least or about a 90% enhancement, at least or about a 95% enhancement, at least or about a 98% enhancement).
  • a 10% enhancement e.g., at least or about a 20% enhancement, at least or about a 30% enhancement, at least or about a 40% enhancement, at least or about a 50% enhancement, at least or about a 60% enhancement, at least or about a 70% enhancement, at least or about a 80% enhancement, at least or about a 90% enhancement, at least or about a 95% enhancement, at least or about a 98% enhancement.
  • T cell activity assays are described in Bercovici et al., Clin Diagn Lab Immunol.7(6): 859–864 (2000). Methods of measuring immune responses are described in e.g., Macatangay et al., Clin Vaccine Immunol 17(9): 1452-1459 (2010), and Clay et al., Clin Cancer Res.7(5):1127-35 (2001).
  • NK natural killer
  • the methods comprise administering to the subject the pharmaceutical composition of the present disclosure in an effective amount.
  • the NK cell activity is directed against a cancer cell or cancer tissue or a tumor cell or tumor.
  • the method comprises administering to the subject the pharmaceutical composition of the present disclosure in an amount effective for treating the cancer or the solid tumor in the subject.
  • the subject has a histologically or cytologically confirmed metastatic or locally advanced solid tumor, which optionally is not amendable to curative treatment with surgery or radiation.
  • the human subject has an advanced solid tumor.
  • the solid tumor is a metastatic solid tumor and/or non-localized and/or non-resectable.
  • the solid tumor is a Stage 3 or Stage 4 tumor.
  • the subject has at least one measurable lesion as defined by modified RECIST 1.1 which has not undergone biopsy within 3 months of the screening scan.
  • the human subject has an Eastern Cooperative Oncology Group (ECOG) performance status of less than or equal to 2, optionally, 0 or 1, (Oken et al., Am J Clin Oncol 5: 649- 655 (1982).
  • ECG Eastern Cooperative Oncology Group
  • the cancer treatable by the methods disclosed herein can be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream.
  • the cancer in some aspects is one selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pan
  • the cancer is selected from the group consisting of: head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma.
  • the tumor is non-small cell lung cancer (NSCLC), head and neck cancer, renal cancer, triple negative breast cancer, and gastric cancer.
  • the subject has a tumor (e.g., a solid tumor, a hematological malignancy, or a lymphoid malignancy) and the pharmaceutical composition is administered to the subject in an amount effective to treat the tumor in the subject.
  • the tumor is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, renal cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, gastric cancer, lymphoma or leukemia, and the pharmaceutical composition is administered to the subject in an amount effective to treat the tumor in the subject.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • the pharmaceutical composition is administered to the subject in an amount effective to treat the tumor in the subject.
  • Also provided herein are methods of treating a subject comprising administering to the subject a CD112R antigen-binding protein, a TIGIT antigen-binding protein, and a PD-L1 antigen- binding protein, optionally, an anti-CD112R antibody, an anti-TIGIT antibody, and an anti-PD-L1 antibody.
  • each antigen-binding protein is separately formulated and separately administered to the subject.
  • each antigen-binding protein is administered to the subject according to a regimen specific to the antigen-binding protein and thus, the method in various aspects comprises administering one antigen-binding protein on a day or days different from the day or days on which another antigen-binding protein is administered to the subject.
  • the regimen for the CD112R antigen-binding protein is different from the regimen of at least the TIGIT antigen binding protein or the PD-L1 antigen binding protein.
  • the regimen for the CD112R antigen-binding protein is different from the regimen for the PD-L1 antigen-binding protein.
  • at least two of the antibodies are formulated together (e.g., co-formulated) and thus administered together (e.g., co-administered).
  • the anti-CD112R and anti-TIGIT antibodies are co-formulated and co-administered and the anti- PD-L1 antibody is administered to the subject separately from the co-formulation comprising the anti- CD112R and anti-TIGIT antibodies.
  • all three antibodies are formulated separately but at least two are administered together (e.g., co-administered).
  • at least the anti-CD112R and anti-TIGIT antibodies are separately formulated but are co-administered and the anti-PD-L1 antibody is administered to the subject separately from the anti-CD112R and anti-TIGIT antibodies.
  • the method comprises administering a composition comprising all three antigen-binding proteins, wherein all three are co-formulated and co-administered to the subject.
  • the CD112R antigen binding protein is any CD112R antigen-binding protein described herein including any one of 1E1, 1E1.016, 24F1, 29E10, 24F1.001, 29E10_CONS.020, 29E10_CONS.021, 29E10_CONS.022, 29E10_CONS.025, 11E4, 31B3, 27G12, 28F9, 28H7, or 36C8 as described in Table A1 or Table B1.
  • the TIGIT antigen binding protein is any TIGIT antigen-binding protein described herein including any one of 55G7.041.008, 58A7.003.008.075, 4G10, 11A3, 28B8, 39D2, 43B7, 55G7, 66H9, 43B7.002.015, 58A7.003.08, 66H9.009, or 58A7 as described in Table A2 or Table B2.
  • the CD112R antigen binding protein is 24F1, 29E10_CONS.020 or 29E10_CONS.022.
  • the TIGIT antigen binding protein is 43B7.002.015 or 66H9.009.
  • the CD112R antigen-binding protein is 24F1 and the TIGIT antigen-binding protein 43B7.002.015. In another aspect, the CD112R antigen-binding protein is 24F1 and the TIGIT antigen-binding protein is 66H9.009. In one aspect, the CD112R antigen-binding protein is 29E10_CONS.020 and the TIGIT antigen-binding protein is 43B7.002.015. In another aspect, the CD112R antigen-binding protein is 29E10_CONS.020 and the TIGIT antigen-binding protein is 66H9.009. In one aspect, the combination comprises 29E10_CONS.022 and 43B7.002.015.
  • the CD112R antigen-binding protein is 29E10_CONS.022 and the TIGIT antigen-binding protein is 66H9.009.
  • the PD-L1 antigen binding agent is any of the PD-L1 antigen binding agents described supra.
  • the PD-L1 antigen binding agent is atezolizumab.
  • the term “treat,” as well as words related thereto, do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating cancer of the present disclosure can provide any amount or any level of treatment.
  • the treatment provided by the method of the present disclosure can include treatment of one or more conditions or symptoms or signs of the cancer being treated.
  • the treatment provided by the methods of the present disclosure can encompass slowing the progression of the cancer.
  • the methods can treat cancer by virtue of enhancing the T cell activity or NK cell activity or an immune response against the cancer, reducing tumor or cancer growth, reducing metastasis of tumor cells, increasing cell death of tumor or cancer cells, and the like.
  • the methods treat by way of delaying the onset or recurrence of the cancer by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 4 months, 6 months, 1 year, 2 years, 4 years, or more.
  • the methods treat by way increasing the survival of the subject.
  • the treatment provided by the methods of the present disclosure provides a therapeutic response as per Response Evaluation Criteria in Solid Tumors (RECIST) or other like criteria.
  • RECIST is a set of criteria to evaluate the progression, stabilization or responsiveness of tumors and/or cancer cells jointly created by the National Cancer Institute of the United States, the National Cancer Institute of Canada Clinical Trials Group and the European Organisation for Research and Treatment of Cancer. According to RECIST, certain tumors are measured in the beginning of an evaluation (e.g., a clinical trial), in order to provide a baseline for comparison after treatment with a drug. The response assessment and evaluation criteria for tumors are published in Eisenhauer et.
  • the treatment provided by the methods of the present disclosure provides a therapeutic response as per a modified RECIST tumor response assessment, as follows: [00224] Accordingly, methods of slowing the progression of a cancer in a subject, enhancing the T cell activity or an immune response against a cancer in a subject, reducing tumor or cancer growth in a subject, reducing metastasis of tumor cells in a subject, increasing cell death of tumor or cancer cells in a subject, and/or delaying the onset or recurrence of a cancer in a subject are provided herein.
  • the method comprises administering to the subject a CD112R antigen-binding protein, a TIGIT antigen-binding protein, and a PD-L1 antigen-binding protein, in accordance with the present disclosures.
  • a method of treating a cancer to provide a complete response (CR), partial response (PR), or stable disease (SD), as per a modified RECIST 1.1, in a subject is provided.
  • the method comprises administering to the subject a CD112R antigen-binding protein, a TIGIT antigen-binding protein, and a PD-L1 antigen-binding protein, in accordance with the present disclosures.
  • the subject upon treatment, the subject exhibits at least SD.
  • the subject exhibits at least SD after about 2 or more cycles of treatment with the CD112R antigen-binding protein, TIGIT antigen-binding protein, and PD-L1 antigen-binding protein.
  • upon treatment the subject exhibits at least a PR.
  • the subject upon treatment, exhibits at least a PR or a CR after about 2 cycles of treatment with the CD112R antigen-binding protein, TIGIT antigen-binding protein, and PD-L1 antigen-binding protein.
  • the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal is a human.
  • Methods of Manufacture [00228] The antigen binding proteins of the present disclosure may be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides are described in, for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752. Additional exemplary methods of making the peptides of the invention are set forth herein.
  • the antigen binding proteins described herein are commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), Multiple Peptide Systems (San Diego, CA), Peptide 2.0 Inc. (Chantilly, VA), and American Peptide Co. (Sunnyvale, CA).
  • the antigen binding proteins can be synthetic, recombinant, isolated, and/or purified.
  • the antigen binding proteins are recombinantly produced using a nucleic acid encoding the amino acid sequence of the peptide using standard recombinant methods.
  • the method comprises culturing a presently disclosed host cell so as to express the antigen-binding protein and harvesting the expressed antigen-binding protein.
  • the host cell can be any of the host cells described herein.
  • the host cell is selected from the group consisting of: CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells.
  • the step of culturing a host cell comprises culturing the host cell in a growth medium to support the growth and expansion of the host cell.
  • the growth medium increases cell density, culture viability and productivity in a timely manner.
  • the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors.
  • the growth medium is a fully chemically defined media consisting of amino acids, vitamins, trace elements, inorganic salts, lipids and insulin or insulin-like growth factors.
  • the method of making an antigen binding protein of the present disclosure comprises culturing the host cell in a feed medium.
  • the method comprises culturing in a feed medium in a fed-batch mode. Methods of recombinant protein production are known in the art. See, e.g., Li et al., “Cell culture processes for monoclonal antibody production” Mabs 2(5): 466–477 (2010).
  • the method making an antigen binding protein can comprise one or more steps for purifying the protein from a cell culture or the supernatant thereof and preferably recovering the purified protein.
  • the method comprises one or more chromatography steps, e.g., affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography.
  • the method comprises purifying the protein using a Protein A affinity chromatography resin.
  • the method further comprises steps for formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds.
  • EXAMPLE 1 This example describes TIGIT family receptor and ligand expression in cancer and normal T cells.
  • Correlation analyses were performed with RNAseq data from The Cancer Genome Atlas (TCGA) on multiple tumor indications to assess co-expression of TIGIT family members with each other and with PD-1. The same analyses were performed for the ligands of the TIGIT family members and PD-1.
  • the tumor indications included Breast Invasive Carcinoma (BRCA), Kidney Renal Clear Cell Carcinoma (KIRC), -Neck Squamous Cell Carcinoma (HNSC), and Skin Cutaneous Melanoma (SKCM).
  • BRCA Breast Invasive Carcinoma
  • KIRC Kidney Renal Clear Cell Carcinoma
  • HNSC -Neck Squamous Cell Carcinoma
  • SKCM Skin Cutaneous Melanoma
  • TILs tumor infiltrating lymphocytes
  • CD112R Single cell RNAseq data from tumor infiltrating lymphocytes (TILs) from human liver carcinoma also were analyzed, and the data suggested that, while TIGIT and PD-1 expression overlap, CD112R expression was broader ( Figure 1B). Similar results were obtained from additional single cell RNA seq datasets from TILS from colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) (data not shown).
  • CD112R is the most recent addition to the TIGIT family of receptors. It was previously shown to be expressed on NK cells and activated T cells, and predominantly expressed on CD8 T cells.
  • CD112R expression was confirmed as being induced on CD8 T cell upon activation and that a significant proportion of these cells also co-expressed PD-1 and TIGIT, consistent with the pattern suggested by the scRNAseq data ( Figure 1C).
  • Figure 1C To ascertain the expression of CD112R, TIGIT, and PD-1 and the ligands CD112 and CD155 in primary human cells, the expression of these molecules on tumor-infiltrating immune cells and tumor cells from human tumor tissues was evaluated. Amongst the limited number of samples, the relative expression of the receptors detectable by FACS were highly variable (Figure 1D). Additionally, it was observed that the ligand expression on Epcam+ CD45- tumor cells versus Epcam- CD45+ immune cells was significantly different.
  • CD112 and CD155 were co-expressed at high levels in Epcam+ CD45- tumor cells but were expressed at low levels on intra- tumor immune cells, with significantly fewer cells expressing both ligands.
  • CD45+ Epcam- myeloid cells in PBMC showed very few, if any, cells co-expressing these ligands.
  • EXAMPLE 2 [00243] This example demonstrates CD112R blockade enhances T cell responses. [00244] To demonstrate the function of CD112R in T cells, an in vitro assay system using engineered CHO cells that stably express CD112 and CD3 engager was developed.
  • Purified human pan T cells were pre-activated with CD3/CD28 antibodies and then allowed to rest.
  • CD112R expressed on the surface of the T cells binds to CD112 expressed on the surface of the CHO cells, IL- 2 release is expected to be suppressed.
  • An illustration of the assay is provided in Figure 2A.
  • the T cells were confirmed as having induced CD112R expression on the cell surface ( Figure 2B).
  • the ability of tool antibodies to bind to CD112R and block IL-2 release was tested using this assay system.
  • Tool antibodies demonstrated dose-dependent binding to cells expressing huCD112R (Figure 2C) and the relative affinity/avidity of these antibodies correlated with their ability to block ligand binding ( Figure 2D).
  • Figure 2C dose-dependent binding to cells expressing huCD112R
  • Figure 2D relative affinity/avidity of these antibodies correlated with their ability to block ligand binding
  • CD112 engages CD226 to induce a costimulatory signal.
  • CD112-mediated co-stimulation of T cells in the absence of CD112R, is entirely driven by CD226 ( Figure 2F), further confirming that CD112R primarily inhibits CD226-dependent costimulatory signal by binding to the same ligand as does CD226.
  • CD112R blockade As a good strategy for enhancing the T cell response.
  • EXAMPLE 3 This example demonstrates the generation of CD112R monoclonal antibodies (mAbs).
  • mAbs monoclonal antibodies
  • Full human antibodies to human CD112R were generated as follows.
  • Fully human antibodies to human CD112R were generated by immunizing XENOMOUSE® transgenic mice (U.S. Pat.
  • HEK293 cells were mock-transfected or transiently transfected with either human or cynomolgus CD112R. Sera from immunized animals was diluted 100-fold and incubated on the transfected cells for 1 hour on ice. The cells were then washed to remove unbound antibodies and a secondary anti-human IgG Fc specific antibody labeled with Cy5 was incubated on the cells for an additional 15 minutes at 4 degrees. The cells were washed once to remove unbound secondary antibody and fluorescent signal on the cells was quantitated by FACS.
  • Hybridoma cells were removed from the flask and washed in sterile FACS buffer (2% FBS PBS).
  • Cells were then stained with the soluble human CD112R protein and incubated at 4 degrees Celsius for 1 hour. Cells were washed again in FACS buffer and stained with 1 mL of detection cocktail containing 5 ⁇ g/mL of Alexa Fluor 488 conjugated F(ab’)2 fragment goat anti-human IgG Fc (Jackson, Cat: 109-546-098) and Alexa Fluor 647 conjugated streptavidin (Jackson, Cat: 016-600- 084) then incubated at 4 degrees Celsius for 30 minutes in the dark. Cells were washed again in FACS buffer, resuspended in media and then put through a 40-micron cell strainer to remove aggregated cells.
  • Antigen specific cells were sorted using BD FACSAria 3 by gating on population exhibiting both Alexa Fluor 488 and Alexa Fluor 647 fluorescence (IgG+ and antigen binding cells). [00261] The sorted cells were cultured for a few days in hybridoma media. After confirming the successful enrichment of CD112R specific cells, the hybridomas were then single cell sorted into 384- well microtiter plates using BD FACSAria 3. After 2 weeks of culture, supernatants from the microtiter plates were collected and screened for CD112R binding.
  • FIG. 3 The order of the screening assays used to identify and select antibodies to human CD112R is shown in Figure 3.
  • Human CD112R Specificity Assay [00265] Transfected cells were used to assess an antibody's binding specificity using flow cytometry on host Human Embryonic Kidney (HEK) 293T cells as follows. Proteins were expressed on HEK 293T cells by transfection using human CD112R, murine CD112R, rat CD112R, human CD96, human CD226 or control expression vectors, GibcoTM Opti-MEM® media (Gibco, Cat. No.
  • transfected cells were resuspended in FACS buffer (PBS + 2 % Fetal Bovine Serum) and added to a 96-well plate.
  • FACS buffer PBS + 2 % Fetal Bovine Serum
  • Hybridoma supernatant samples were added such that 2.5ug/mL final, note the exception of 11E4 which was tested at 1:10 dilution final, cells were resuspended and incubated for 1 hour at 4°C. Plates were washed twice with FACS buffer, centrifuged to pellet the cells, supernatant removed and resuspended in FACS buffer to remove unbound antibody.
  • Alexa Fluor 488-goat anti-human IgG (Fc ⁇ fragment specific) secondary (Jackson ImmunoResearch, Cat. No.109-545-098) made up in FACS buffer at 5ug/mL was then added to each well, cells resuspended and incubated for 15 minutes at 4oC. Plates were washed twice with FACS buffer, centrifuged to pellet the cells, supernatant removed and resuspended in FACS buffer to remove unbound secondary antibody. Samples were then resuspended in FACS buffer and read on BD AccuriTM Flow Cytometer with an Intellicyt HyperCyt autosampler.
  • Jurkat cells stably expressing human CD112R and NFAT-luciferase reporter (generated in house using Promega’s Jurkat NFAT-luciferase cell line cat# CS176401) were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 2mM L- glutamine (Sigma), 10mM HEPES (Hyclone, GE Healthcare Life Sciences), 1X MEM NEAA (Sigma), 1X sodium pyruvate (Sigma), 500ug/mL geneticin (Invitrogen) and 0.5ug/mL puromycin (Invitrogen).
  • the Jurkat NFAT-luciferase/CD112R Clone C4 cells were stimulated by engagement of the T-cell receptor by co-culturing with Chinse Hamster Ovary (CHO)-K1 cells stably expressing human CD112 and human T-cell engager (generated in house).1 x 10 4 CHO-K1-CD112+ cells were seeded into white half area 96-well plates (Costar cat# 3688) in full growth media containing Nutrient Mixture F12 HAM (Sigma), 10 % fetal bovine serum, 10 mM HEPES, 500 ⁇ g/mL geneticin, 200 ⁇ g/mL hygromycin B (Invitrogen) and 100ug/mL zeocin (Invitrogen) overnight at 37°C/5%CO2.
  • T cells were stimulated with 5 ug/mL of anti-human CD3 clone OKT3 (eBioscience) and 1 ⁇ g/mL of anti-human CD28 (BD Pharmingen) for 72 hours at 37 °C/5% CO 2 in a plate that had been pre-coated with 5 ⁇ g/mL anti mouse IgG Fc (Pierce). After 72 hours, cells were removed, washed and suspended at a concentration of 0.5x10 6 cells/mL with 10 ng/mL of IL-2 (Pepro Tech). Cells were then incubated for another 5 days at 37 °C/5 % CO 2 .
  • cynomolgus primary cell binding assay For cynomolgus primary cell binding assay, cynomolgus PBMCs (SNBL) were thawed and suspended in a concentration between 4x10 6 and 5x10 6 cells/mL. PBMCs were stimulated with 1 ⁇ g/mL of anti-human CD3 clone SP34 (BD Pharmingen) and 1 ⁇ g/mL of anti-human CD28 (BD Pharmingen) for 72 hours at 37 °C/5 % CO 2 in a plate that had been pre-coated with 5 ⁇ g/mL anti-mouse IgG Fc (Pierce).
  • BD Pharmingen anti-human CD3 clone SP34
  • BD Pharmingen anti-human CD28
  • the panel of >1200 recombinant human binders was tested for binding to recombinant cyno CD112R transiently expressed on HEK293T cells. Two hundred seventy four antibodies of the panel were found to bind to cyno CD112R, only two (11E4 and 1E1) of which were found to bind to endogenous cyno CD112R expressed on primary cyno T-cells.
  • Second Wave and Antibody Selection A second wave of Xenomouse immunizations involving a custom-generated XMG2 CD112R knock-out (KO) mouse strain, in addition to the XMG2kl strain, was carried out. Hybridoma cells were generated as essentially described above, and the screening assays described in Figure 3 were used to identify and select antibodies to human CD112R. Representative data of Harvests 6-9 from the second wave are shown in Figure 4B and 4C. The second wave led to over 1300 human CD112R-specific antibodies, of which 350 resulted in a 3-fold or higher induction of NFAT-luciferase signal as determined by the Jurkat RGA.
  • the KinExA signal generated by antibody alone is taken as 100% Free and the % inhibited free fraction (IFF) is calculated from the signal measured in presence of antigen as follows: [00279] The antibodies that demonstrate the lowest %IFF have the highest affinity; conversely, the antibody that demonstrates highest %IFF has the lowest affinity and they can be ranked by plotting the %IFF in a graph. Monoclonal antibodies that give an IFF of 50% or less should have passed the Kd cutoff of 100pM meaning they should have a Kd of 100pM or less, respectively. [00280] The results are shown in Figure 6. Of the ten antibodies analyzed, eight exhibited a K D below 100 pM and only two exhibited a K D above 100 pM.
  • RNA total or mRNA
  • RNA total or mRNA
  • Qiagen Rneasy mini or the Invitrogen mRNA catcher plus kit Purified RNA was used to amplify the antibody heavy and light chain variable region (V) genes using cDNA synthesis via reverse transcription, followed by a polymerase chain reaction (RT-PCR).
  • RT-PCR polymerase chain reaction
  • the fully human antibody gamma heavy chain was obtained using the Qiagen One Step Reverse Transcriptase PCR kit (Qiagen).
  • This method was used to generate the first strand cDNA from the RNA template and then to amplify the variable region of the gamma heavy chain using multiplex PCR.
  • the 5' gamma chain-specific primer annealed to the signal sequence of the antibody heavy chain, while the 3' primer annealed to a region of the gamma constant domain.
  • the fully human kappa light chain was obtained using the Qiagen One Step Reverse Transcriptase PCR kit (Qiagen). This method was used to generate the first strand cDNA from the RNA template and then to amplify the variable region of the kappa light chain using multiplex PCR.
  • the 5’ kappa light chain-specific primer annealed to the signal sequence of the antibody light chain while the 3’ primer annealed to a region of the kappa constant domain.
  • the fully human lambda light chain was obtained using the Qiagen One Step Reverse Transcriptase PCR kit (Qiagen). This method was used to generate the first strand cDNA from the RNA template and then to amplify the variable region of the lambda light chain using multiplex PCR.
  • the 5’ lambda light chain-specific primer annealed to the signal sequence of light chain while the 3’ primer annealed to a region of the lambda constant domain.
  • the amplified cDNA was purified enzymatically using exonuclease I and alkaline phosphatase and the purified PCR product was sequenced directly. Amino acid sequences were bioinformatically deduced from the corresponding nucleic acid sequences. Two additional, independent RT-PCR amplification and sequencing cycles were completed for each hybridoma sample to confirm that any mutations observed were not a consequence of the PCR. The derived amino acid sequences were then analyzed to determine the germline sequence origin of the antibodies and to identify deviations from the germline sequence. The amino acid sequences corresponding to complementary determining regions (CDRs) of the sequenced antibodies were aligned and these alignments were used to group the clones by similarity.
  • CDRs complementary determining regions
  • the sequences were also analyzed for “hotspots” (residues that were computationally predicted or empirically determined to negatively impact the molecule's expression, purification, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and/or immunogenicity).
  • the results of the sequence analysis are shown in Figure 7A.
  • Figure 7B lists the antibodies analyzed for sequence diversity and indicates the VH germline and HC CDR3 residues.
  • the IC50 (nM) as determined by the Jurkat RGA for each antibody is also listed.
  • the inhibitory activity of the six antibodies were comparable to those of two reference anti-CD112R antibodies, PL-52575 and PL- 52577, having IC50s as 0.12 nM and 0.10 nM, respectively.
  • Competition-based binning for the lead panel of CD112R antibodies [00288] Select human CD112R-binding hybridoma supernatants were tested for competition-based binning by utilizing the Octet HTX platform.
  • Antibodies were loaded on Anti-HuFc (kinetic) biosensors ForteBio cat 18-5064 at 2ug/mL for two minutes in an assay buffer comprising 10mM Tris, 0.1%Triton, 150mM NaCl, 1mM CaCl2, 0.1mg/mL BSA, Ph7.4.
  • the biosensors were subsequently blocked with 50 ⁇ g/mL irrelevant HuIgG2 in assay buffer for five minutes.
  • CD112R at 1 ⁇ g/mL was bound for two minutes in assay buffer, then the biosensors were dipped in assay buffer for one minute to establish a baseline signal.
  • Binding confirmation to Primary Human and Cyno T-cells [00292] Primary human and cyno T-cells were prepared and analyzed as essentially described above. Exhausted hybridoma culture supernatants containing the antibodies 1E1, 11E4, 27G12, 29E10, 31B3 and 24F1 were quantitated, then titrated for binding on the surface of primary T-cells. Curve fitting analysis using Prism allowed determination of an EC50 value for binding to the primary cells. [00293] For all antibodies 1E1, 11E4, 27G12, 29E10, 31B3 and 24F1, the EC50 values for binding to human T-cells was within 10-fold for binding to the cyno PBMCs.
  • 11B1 which is a structurally close to 11E4 was used in the assays as there was not enough 11E4 ESN.
  • FCS Express was used to obtain Geo Means and Screener was used to determine fold over Isotype control, titration curves and EC50 values.
  • Figures 10A and 10B The results of the assay using cyno PBMCs and human T-cells are shown in Figures 10A and 10B, respectively, and Table 1.
  • the graphs of Figures 10A and 10B plot the fold over isotype control signal plotted as a function of the log concentration of the indicated antibody. Table 1 provides the EC50 (ng / ⁇ l) for each antibody.
  • 1E1 exhibited the highest binding affinity for CD112R expressed by primary human and cyno cells.
  • the EC50s of 29E10 and 24F1 were very similar between the two species and to each other.
  • 11B1 had a high EC50 of 0.108 ng/ ⁇ L for Human but a low EC50 of 0.0281 ng/ ⁇ L for Cyno.
  • FIG. 11A provides a graph of the NFAT luciferase induction plotted as a function of log concentration of the indicated antibody. As shown in Figure 11, all antibodies tested demonstrate potency as CD112R antagonists.
  • the anti- CD112R antibodies were additionally engineered to remove “hotspots,” or residues that were computationally predicted or empirically determined to negatively impact the molecule's expression, purification, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and/or immunogenicity.
  • hotspots or residues that were computationally predicted or empirically determined to negatively impact the molecule's expression, purification, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and/or immunogenicity.
  • a variety of amino acid mutations at these hotspots were designed based on conservation, co-variation, chemical similarity, predictions from structural modeling, and prior knowledge from other antibody engineering campaigns.
  • Engineered antibodies were designed that included both single mutations and combinations of mutations.
  • Engineered variants were cloned by ordering synthetic DNA fragments comprising the designed variable domains and inserting these using Golden Gate cloning methods into a stable mammalian expression vector containing the constant HC domains under puromycin selection or the constant LC domains under hygromycin selection.
  • Antibodies were expressed by co-transfecting HCs and LCs in CHO-K1 cells and selecting for stable expression using puromycin and hygromycin.
  • Antibodies were purified by Protein A affinity chromatography using AmMag TM Protein A Magnetic Beads (GenScript). The identity of each molecule was confirmed by intact mass spectrometry.
  • the expression titer in conditioned medium was measured by ForteBio Octet (Pall Life Sciences) using Protein A sensors.
  • the percent of high molecular weight (% HMW) material present after Protein A affinity chromatography was measured by analytical size exclusion chromatography, and the purity was measured by % main peak in non-reduced microcapillary electrophoresis using a LabChip GXII (Perkin Elmer).
  • the Tm of the first melting transition (Tm1) and the onset temperature of aggregation (Tagg) were measured by DSF using a Prometheus (Nanotemper).
  • Antibody activity was measured by the CD112R Jurkat reporter gene assay as described above, averaging two independent measurements.
  • Anti-CD112R antibody 11E4 (comprising HC variable region sequence and LC variable region sequence of SEQ ID NOs 101 and 102, respectively) was engineered for improved manufacturability by grafting the CDRs of each antibody into selected alternate human frameworks with a preference for well-behaved VH1, VH3, VH5, VK1, and VK3 germlines. The alternate frameworks were selected by considering sequence similarity.
  • Framework engineered variants were cloned by ordering synthetic DNA fragments comprising the designed variable domains and inserting these using Golden Gate cloning methods into a stable mammalian expression vector containing the constant HC or LC domains and purified by Protein A affinity chromatography using AmMag ⁇ Protein A Magnetic Beads (GenScript). The identity of each variant was confirmed by intact mass spectrometry. For each variant, the expression titer in conditioned medium was measured by ForteBio Octet (Pall Life Sciences) using Protein A sensors.
  • % HMW percent of high molecular weight
  • % HMW percent of high molecular weight
  • the Tm of the first melting transition was measured by DSF using a Prometheus (Nanotemper).
  • Purified samples were incubated at 40°C for 2 weeks and the change in % main peak was determined by analytical size exclusion chromatography.
  • Antibody activity was measured by the CD112R Jurkat reporter gene assay as described above, averaging two independent measurements. The results of the analyses for engineered variants are shown in Table 3 of Figure 18.
  • Anti-CD112R antibodies 31B3, 11E4, 29E10, 1E1.016, and 24F1 were engineered for improved manufacturability and for increased binding to CD112R through yeast display.
  • Library designs for 11E4 and 1E1.016 used a CDR walk in triplicate across the CDRs to cover all CDR amino acids including the chemical hotspot liabilities.
  • the other 4 antibodies had libraries designed specifically around their chemical hotspot liabilities and residues predicted to contribute to poor surface properties, determined through homology modeling and patch analysis by the BioLuminate computational modeling software package (Schrödinger, LLC, New York, NY, 2020).
  • EXAMPLE 4 This example demonstrates preliminary biochemical and functional experiments conducted to characterize tool TIGIT antibodies.
  • Competitive receptor-ligand (R-L) binding assays were performed on TIGIT antibodies 10A7 (Genentech TIGIT antibody) and MBSA43 (eBioscienceTM cat#16-9500-85) using CHO-S cells transiently expressing human TIGIT. Briefly, CHO-S cells expressing human TIGIT were mixed with an antibody sample and incubated for 1 hour at 4°C in FACS Buffer (1X PBS pH7.4 + 2% Fetal Bovine Serum).
  • Figure 12A is a graph of the % inhibition of binding of TIGIT to CD155-Fc plotted as a function of tool TIGIT antibody concentration.
  • Figure 12B is a graph of the % inhibition of binding to CD112-Fc as a function of antibody concentration.
  • the results shown in Figures 12A and 12B demonstrate that 10A7 and MBSA43 tool antibodies inhibit both CD155 and CD112 interactions with TIGIT. [00309] Additional binding assays were performed to determine whether tool TIGIT antibodies could inhibit the binding interactions between CD226 and CD155 endogenously expressed by 293T cells. 293T cells endogenously express high levels of CD155.
  • the 293T cells were transiently transfected with human TIGIT (TIGIT-293 cells) or were mock transfected (Mock-293 or 293T Mock).
  • TIGIT-293 cells human TIGIT
  • Mock-293 or 293T Mock mock transfected
  • CD226 is blocked from binding to endogenous CD155 by TIGIT expressed by TIGIT-293 cells but not blocked in the context of Mock-293 cells (which do not transiently express TIGIT).
  • TIGIT expressed by TIGIT-293 cells is bound by tool antibodies and is blocked from binding to CD155, thereby allowing CD226 to interact with CD155.
  • Figure 12C provides a schematic of the assay.
  • TIGIT-293 cells or Mock-293 cells were mixed with the antibody sample with and without human CD226-Fc at 10ug/mL (R&D, 666-DN) and incubated for 1 hour at 4°C in FACS Buffer (1X PBS Ph7.4 + 2% Fetal Bovine Serum). Cells were washed 2X with FACS Buffer and then incubated with Gt anti Hu IgG-Fc Alexa (Jackson, 109-605-098) and 7-AAD (Sigma, 9400-5MG) for 15 minutes at 4°C. Cells were washed 1X with FACS Buffer and resuspended in FACS buffer.
  • FIGS 12D – 12E The results are shown in Figures 12D – 12E.
  • the data in Figure 12D demonstrates that huCD226 can bind 293T Mock cells (open circles) endogenously expressing huCD155.
  • huTIGIT closed squares
  • CD226 binding is blocked from cis-interaction with huCD155.
  • Figure 12E shows that treatment with TIGIT tool antibodies 10A7 (open circles) or MBSA43 (closed squares) restore the ability of CD226 to bind to CD155, as the tool antibodies block TIGIT-CD155 cis-interactions.
  • IFN release assays were carried out to determine the functional effects of the tool TIGIT antibodies. In the IFN release assays, IFN ⁇ released by primary human CD8+ memory T-cells stimulated with CHO-K1-huCD155 activator cells was measured. The results, shown in Figure 12F, demonstrate that only the MBSA43 tool antibody was able to induce IFN ⁇ release.
  • the 10A7 antibody was unable activate IFN ⁇ release by stimulated T cells.
  • Luciferase activity assays under the control of the IL-2 promoter were also performed to determine the functional effects of the tool TIGIT antibodies.
  • Jurkat T cells were stimulated for IL-2 promoter – driven expression of the luciferase reporter gene in the presence of tool antibodies or an isotype matched antibody control by CHO cells expressing human CD155 and a CD3-activator.
  • Tool TIGIT antibodies were tested for their ability to bind cyno TIGIT using primary cyno monkey T-cells and cyno TIGIT transiently expressed on CHO and 293T cells.
  • 293T cells transiently expressing cyno TIGIT or vector alone were mixed with a sample comprising tool antibody MBSA43, 10A7 or 1F4 (Genentech TIGIT antibody) and incubated for 1 hour at 4°C in FACS Buffer.
  • Figure 12H shows that each of 1F4 (closed circles), 10A7 (closed squares) and MBSA43 (closed triangles) binds cyno TIGIT transiently expressed in 293T cells.
  • An increase in GeoMean fold difference from the vector alone control is observed with increasing antibody concentration while no binding is seen with the IgG isotype control (open diamonds).
  • MBSA43 and 1F4 demonstrated binding to primary activated cyno T-cells (Figure 12I).
  • the FACS plot for 10A7 overlapped with the FACS plot for an isotype control antibody suggesting that 10A7 does not bind to primary activated cyno T-cells ( Figure 12I).
  • EXAMPLE 5 This example demonstrates the generation of TIGIT monoclonal antibodies (mAbs).
  • mAbs monoclonal antibodies
  • Fully human antibodies to human TIGIT were generated by immunizing XENOMOUSE® transgenic as essentially described above. Animals from the XMG4 and XMG2 XENOMOUSE® strains were used for these immunizations. Multiple immunogens and routes of immunization were used to generate anti-human TIGIT immune responses.
  • TIGIT-specific serum titers were monitored by live-cell FACS analysis on an Accuri flow cytometer (BD Biosciences) using transiently transfected suspension CHO cells. Animals with the highest antigen-specific serum titers against human TIGIT were sacrificed and used for hybridoma generation (Kohler and Milstein, 1975). Hybridomas were generated as essentially described in Example 3. [00319] Selection of TIGIT Specific Binding Antibodies [00320] Several screening assays were employed to identify and select anti-human TIGIT antibodies ( Figure 13). [00321] TIGIT-specific serum titers were monitored by live-cell FACS analysis.
  • Exhausted hybridoma supernatants were tested for binding to human or cyno TIGIT transiently expressed on CHO-S samples and analyzed using a BD AccuriTM Flow Cytometer and an Intellicyt HyperCyt autosampler or by Cell Insight.
  • CHO-S cells were transiently transfected with a mammalian expression construct encoding either human or cynomolgus TIGIT using PEI MAX. 3 hours post transfection, 5mM sodium butyrate was added and incubated for 24 hours. The following day, 15 ⁇ L of exhausted hybridoma media was added to each well of a 384 well FMAT plate.
  • Transfected and mock transfected CHO-S cells were mixed with the exhausted hybridoma sample and incubated for 1 hour at 4°C in FACS Buffer. After 1 hour, cells were washed 2X with FACS Buffer (1X PBS Ph7.4 + 2% FBS) and then incubated with Gt anti Hu IgG-Fc Alexa-647 (Jackson, 109-605-098) or Gt anti Mu IgG-Fc Alexa-647 (Jackson 115-605-071) and 7-AAD (Sigma, 9400-5MG) for 15 minutes at 4°C. Cells were washed 1X with FACS Buffer and resuspended in FACS buffer.
  • CHO-S or HEK293T cells were transiently transfected with a mammalian expression construct encoding TIGIT using PEI MAX or 293Fectin respectively. The following day, 15 ⁇ L of exhausted hybridoma media was added to each well of a 384 well FMAT plate.
  • the transfected cells (0.27 million/mL), the nuclear stain Hoechst 33342 (7.5 ⁇ g/mL) and a secondary detection antibody (0.75 ⁇ g/mL – Goat anti Human IgG (H+L) Alexa 488 (Jackson ImmunoResearch)) were mixed and 30 ⁇ L of this mixture was added to each well of a 384 well FMAT plate. After ⁇ 3 hours, the supernatant was aspirated using an AquaMax plate reader and 30 ⁇ L of FACS buffer was added to each well using a multidrop instrument. The plates were placed on a Big Bear Plate shaker to evenly distribute the cells in the well and then read on the Cell Insight platform using the Cell Health Bio-App.
  • the RGA was very similar to that described in Example 2, except that Jurkat cells stably expressing human TIGIT (not CD112R) and IL-2-luciferse reporter (Promega cat# CS191103A) were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 2mM L-glutamine (Sigma), 10mM HEPES (Hyclone, GE Healthcare Life Sciences), 1X MEM NEAA (Sigma), 1X sodium pyruvate (Sigma), 500ug/mL geneticin (Invitrogen) and 200ug/mL hygromycin B (Invitrogen).
  • the GloResponseTM IL-2-luc2P/TIGIT/Jurkat reporter cell line (Promega cat# CS191103A) was stimulated by engagement of T-cell receptor by co-culturing with Chinse Hamster Ovary (CHO)-K1 cells stably expressing human CD155 and human T-cell engager (Promega cat# CS191104A).1 x 10E4 CHO-K1-CD155+ cells were seeded into white half area 96-well plates (Costar cat# 3688) in full growth media containing Nutrient Mixture F12 HAM (Sigma), 10 % fetal bovine serum, 10 mM HEPES, 500 ⁇ g/mL geneticin and 200 ⁇ g/mL hygromycin B overnight at 37°C/5%CO2.
  • Figure 14A shows a schematic of the RGA. Following overnight incubation, growth media was replaced by 5 x 10 4 Jurkat IL-2Luc/TIGIT cells in the presence of hybridoma supernatants or antibodies, with respective controls, in Assay Media (RPMI 1640 medium supplemented with 1% fetal bovine serum, 2mM L-glutamine, 10mM HEPES) and incubated at 37°C/5%CO 2 for 18hrs. Reporter signal in each well was determined using Bio-GloTM Luciferase Assay System (Promega cat# G7940) according to the manufacturer’s recommendation. Luminescence was detected using EnVision Plate Reader (Perkin Elmer). [00325] A selection of active antibodies is shown in Figure 14B.
  • TIGIT antibodies 43B7, 11A3, 39D2, 66H9, 28B8, 55G7, 4G10, and 48B7 performed better than tool antibody MBSA43. These antibodies were run with titrations to determine the potency of anti-TIGIT antibodies. The activity of these anti-TIGIT antibodies in the Jurkat human TIGIT/IL-2-luciferase RGA is shown in Table 7. TABLE 7 [00326] TIGIT functional blocking assay (Primary T-cell Assay) [00327] To screen purified anti-TIGIT antibodies capable of enhancing T-cell activity by blocking TIGIT-CD155 interaction, IFN- ⁇ release from primary human CD8 memory T-cells was employed.
  • CD8 memory T-cells were isolated using EasySepTM HumanCD8+ve MEMORY T-cell Enrichment Kit (StemCell cat# 19159) from purified primary human T-cells (Biological Specialty Corp. cat# 215- 01-10) and pre-stimulated with 1 ⁇ g/mL immobilized anti CD3 (eBioscience cat# 16-0037) + 1 ⁇ g/ml anti CD28 (BD PharMingen cat# 555725) + 10ng/mL rhIL-2 (R & D Systems cat# 202-IL-050/CF) for 7 days at 37°C/5%CO2.
  • TIGIT Primary Cell Binding Assays for TIGIT
  • T cells were stimulated with 5 ⁇ g/mL of anti-human CD3 clone OKT3 (eBioscience) and 1 ⁇ g/mL of anti-human CD28 (BD Pharmingen) for 72 hours at 37 °C/5% CO2 in a plate that had been pre-coated with 5 ⁇ g/mL anti mouse IgG Fc (Pierce). After 72 hours, cells were removed, washed and suspended at a concentration of 0.5x10 6 cells/mL with 10 ng/mL of IL-2 (Pepro Tech). Cells were then incubated for another 48 to 72 hours at 37 °C/5 % CO 2 .
  • cynomolgus primary cell binding assay For cynomolgus primary cell binding assay, cynomolgus PBMCs (SNBL) were thawed and suspended in a concentration between 4x10 6 and 5x10 6 cells/mL. PBMCs were stimulated with 1 ⁇ g/mL of anti- human CD3 clone SP34 (BD Pharmingen) and 1 ⁇ g/mL of anti-human CD28 (BD Pharmingen) for 72 hours at 37 °C/5 % CO 2 in a plate that had been pre-coated with 5 ⁇ g/mL anti-mouse IgG Fc (Pierce).
  • BD Pharmingen anti-human CD3 clone SP34
  • BD Pharmingen anti-human CD28
  • cells were removed, washed and suspended at a concentration of 0.5x10E6 cells/mL with 20 ng/mL of IL-2 (Pepro Tech). Cells were then incubated for another 48 to 72 hours at 37 °C/5 % CO 2 . After the final incubation, cells were prepared for flow cytometry by incubation with normalized hybridoma supernatants, positive control antibodies and isotype control antibodies at 1 ⁇ g/mL final concentration.
  • TIGIT-binding hybridoma supernatants were then tested for their ability to block TIGIT from binding ligand.
  • Competitive binding assays were performed on the antigen-specific hybridoma supernatant samples using FACS on CHO-s cells transiently expressing human TIGIT as follows.
  • CHO-S cells expressing human TIGIT were mixed with the antibody sample (hybridoma supernatants specific for TIGIT) and incubated for 1 hour at 4°C, and then washed twice. Cells with bound sample were then incubated with huCD155-Fc-Alexa 647 (generated & labelled in house) or CD112-Fc- Alexa 647 (Sino Biological 10005-H02H; labelled in house) for 45 minutes at 4°C. The 7-AAD cell viability stain was then added and the cells incubated for a further 15 minutes at 4°C, washed twice and resuspended in FACS buffer.
  • TIGIT affinity determination [00335] Affinity determination of anti-TIGIT mAbs was performed using an OCTET biosensor. Human TIGIT mAbs were captured on anti-huIgG FC Capture (AHC) tips for kinetics (Cat#18-5060) at 2 ⁇ g/mL.
  • Mouse anti-TIGIT control mAb MBSA43 (Cat#16-9500-85) was captured on anti-mouse IgG Fc Capture tips at 2 ⁇ g/ml. Binding of both the huTIGIT (PL44403) and cynoTIGIT (PL40461) was assessed by titrating the proteins 2-fold from either 200 nM or 100 nM starting concentration down to 3 nM. Because the huTIGIT contains a mono-Fc tag, a blocking step containing 50 ⁇ g/ml irrelevant IgG2 was utilized to block the anti-Fc capture reagent on the Octet tips.
  • association was monitored for 5 min with dissociation being monitored for 20 min.
  • association was monitored for 5 min and dissociation for 5 min.
  • the assay buffer used in these experiments was 10 mM Tris, 150 mM NaCL, 1 mM CaCl 2 , 0.1 mg/ml Bovine Serum Albumin (BSA), 0.13 % Triton X-100, pH 7.6.
  • BSA Bovine Serum Albumin
  • the data in Table 10 represents the relative affinities of TIGIT mAbs to human or cynomolgus TIGIT. Due to very poor fits, no values were provided on cynoTIGIT for 2 mAbs.
  • TIGIT antibodies were among those that performed better than tool TIGIT antibodies MBSA43 and 10A7.
  • a comparison of the selected TIGIT antibodies and tool antibodies is provided in Table 11. As shown in this table and as supported by Figures 14A and 14B, the selected TIGIT antibodies exhibited better activity compared to tool antibodies.
  • Hotspot Engineering of TIGIT Antibodies [00342] Select anti-TIGIT antibodies from the XenoMouse campaign were engineered to remove “hotspots,” or residues that were computationally predicted or empirically determined to negatively impact the molecule's expression, purification, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and/or immunogenicity, as essentially described in Example 3.
  • Hotspot engineered variants were cloned, expressed transiently by co-transfecting HCs and LCs in ExpiCHO cells (Life Technologies) and were purified by Protein A affinity chromatography. Antibody activity was measured by the TIGIT Jurkat reporter gene assay as described above.
  • Anti-TIGIT antibodies 55G7.041 (comprising LC variable region sequence and HC variable region sequence of SEQ ID NOs 1923 and 1924, respectively), 66H9.009 (comprising HC variable region sequence and LC variable region sequence of SEQ ID NOs 221 and 222, respectively), 43B7.002.015 (comprising HC variable region sequence and LC variable region sequence of SEQ ID NOs 201 and 202, respectively) and 58A7.003.008 (comprising HC variable region sequence and LC variable region sequence of SEQ ID NOs 211 and 212, respectively) were engineered for improved manufacturability by grafting the CDRs of each antibody into selected alternate human frameworks with a preference for well-behaved VH1, VH3, VH5, VK1, VK3, and VL2 germlines.
  • Framework engineered variants were cloned by ordering synthetic DNA fragments comprising the designed variable domains and inserting these using Golden Gate cloning methods into a stable mammalian expression vector containing the constant HC or LC domains. Antibodies were expressed by co-transfecting HCs and LCs in CHO-K1 cells and purified by Protein A affinity chromatography using MabSelect SuRe resin (GE Healthcare Life Sciences).
  • each variant was confirmed by intact mass spectrometry.
  • the expression titer in conditioned medium was measured by ForteBio Octet (Pall Life Sciences) using Protein A sensors.
  • the percent of high molecular weight (% HMW) material present after Protein A affinity chromatography was measured by analytical size exclusion chromatography, and the purity was measured by % main peak in non-reduced microcapillary electrophoresis using a LabChip GXII (Perkin Elmer).
  • the Tm of the first melting transition was measured by DSF using a Prometheus (Nanotemper). Purified samples were incubated at 40°C for 2 weeks and the change in % main peak was determined by analytical size exclusion chromatography.
  • Antibody activity was measured by the TIGIT Jurkat reporter gene assay as described above, averaging two independent measurements. The results of the analyses for engineered variants are shown below in Table 13. [00347] Yeast Display Optimization of TIGIT Antibodies [00348] Anti TIGIT antibodies 43B7.002.015 (comprising HC variable region sequence and LC variable region sequence of SEQ ID NOs 201 and 202, respectively) and 58A7.003.008 (comprising HC variable region sequence and LC variable region sequence of SEQ ID NOs 211 and 212, respectively) were engineered for improved manufacturability and for increased binding to TIGIT through yeast display.
  • libraries were generated in which every possible adjacent pair of residues in the all six CDRs were simultaneously mutated to all possible amino acids through use of degenerate NNK codons.
  • the libraries were displayed on the surface of yeast derivative of BJ5464, wherein the Fd domain was fused to the N-terminus of alpha-agglutin and the LC was not fused to the yeast surface. Efficiency of display was measured by binding of Alexafluor 647 conjugated anti-Fab antibody.
  • Libraries were sorted using fluorescence activated cell sorting (FACS) for high binding to biotin conjugated recombinant TIGIT extracellular domain (ECD) using streptavidin PE as fluorescence secondary.
  • FACS fluorescence activated cell sorting
  • variable domains present in the sorted binding/display double positive pools and display positive pools were amplified with primers specific to the FW1 and FW4 domains of the HC and LC and submitted to NGS analysis on an Illumina MiSeq for a 2x 300bp run. Mutations were selected after processing the data through a common frequency analysis where the ratio of positive binding amino acid frequencies are divided by positive display amino acid frequencies which is then normalized to the parental sequence ratio. The sequences where the enrichment values were greater than or equal to the parental sequence were considered beneficial or tolerated diversity and were used for additional rational antibody engineering post affinity maturation.
  • Affinity maturation yeast display libraries for each HC and LC were designed using a combination of structure guided (58A7.003.008), model guided proximity pairs (43B7.002.015), and CDR NNK scanning libraries (43B7.002.015 and 58A7.003.008). After these HC or LC libraries were sorted for TIGIT binding equal to or greater than parental, the 4 libraries were combined in a chain shuffle library. Here all library designs were mixed together to be agnostic between design approaches. The libraries were displayed on the surface of yeast as described above and sorting for high binding to biotin-labeled recombinant TIGIT ECD with increased stringency in each consecutive round.
  • Clones with high binding were selected and screened as individual clones and the clones with the highest ratios of binding/display were selected for sequencing. Variable domains were amplified using vector and constant domain primers and the PCR products were submitted for Sanger Sequencing. Sequences which introduced chemical liabilities were dropped from the panel design. Chemical liabilities and charge surface patches identified by the BioLuminate modeling suite (Schrödinger, LLC ) were mutated using information from the NGS enrichment sort data described above. The final engineered sequences were selected for further characterization as recombinant expressed monoclonal antibodies.
  • Top display engineered variants were cloned by ordering synthetic DNA fragments comprising the designed variable domains and inserting these using Golden Gate cloning methods into a stable mammalian expression vector containing the constant HC domains under puromycin selection or the constant LC domains under hygromycin selection.
  • Antibodies were expressed by co- transfecting HCs and LCs in CHO-K1 cells and selecting for stable expression using puromycin and hygromycin.
  • Antibodies were purified by Protein A affinity chromatography using AmMag ⁇ Protein A Magnetic Beads (GenScript). The identity of each molecule was confirmed by intact mass spectrometry.
  • % HMW percent of high molecular weight
  • KO cells were used in a Jurkat RGA and compared to CD226-expressing Jurkat cells expressing TIGIT and an IL-2-Luciferase construct.
  • Jurkat cells were co-cultured with CHO-K1-CD155+ cells as described herein, and luciferase activity was measured.
  • the antibodies used in this experiment included tool antibody MBSA43 and engineered TIGIT antibodies (as described above) AB1 (43B7.002.015) and AB2 (66H9.010).
  • AB1 43B7.002.0105
  • AB2 66H9.010
  • TIGIT antibodies block TIGIT-CD226 cis interactions to prevent TIGIT-CD226-mediated signal suppression.
  • CD226 involvement contributes to a stronger TCR response.
  • Anti TIGIT mAbs AB1 and AB2 (closed circles and closes squares, respectively) block TIGIT-CD226 interactions better than MBSA43 (closed diamonds) as max activity is not completely reached.
  • MBSA43 closed diamonds
  • Binding of anti-TIGIT antibodies to human TIGIT SEQ ID NO: 1 and cynomolgus TIGIT SEQ ID NO: 2024, and binding of anti-CD112R antibodies to human CD112R SEQ ID NO: 3 and CD112R (N81D) SEQ ID NO: 2026 were characterized on Biacore T200 using Surface Plasmon Resonance (SPR) technology.
  • SPR Surface Plasmon Resonance
  • an anti-human Fab antibody from Fab capture kit (GE Healthcare Life Sciences) was immobilized on all four flow cells of a CM5 chip to approximately 6000 – 9000 RU using standard amine coupling reagents (GE Healthcare Life Sciences). PBS plus 0.005% P20 was used as instrument running buffer throughout the assays.
  • the first flow cell with immobilized anti-human Fab antibody only was used as background control.
  • Anti-TIGIT or anti- CD112R antibodies were captured on the second, third and fourth flow cells to approximately 100 – 190 RU.
  • Binding data of anti-TIGIT antibodies to human and cynomolgus TIGIT, anti-CD112R antibodies to human CD112R and CD112R (N81D) were analyzed using Biacore T200 Evaluation Software 3.0 (GE Healthcare Life Sciences). All the data were double referenced by subtracting the blank control surface and the blank cycle injecting sample buffer only.
  • on rate (k a ), off rate (k d ), equilibrium dissociation constant (K D ) and maximum binding response (R max ) were calculated from global fitting using the 1:1 kinetic binding model.
  • FIG. 16A A schematic of the cell assay system is shown in Figure 16A.
  • Three different TIGIT antibodies 43B7.002.015, 66H9.009 and 58A7.002.008 were tested and demonstrated a mild effect as single agents, but when used in combination with a CD112R blocking antibody, T cell activity was strongly enhanced in this assay (Figure 16B).
  • CD112R and TIGIT blockade by 1:1 antibody mixture was compared to blockade by a bispecific antibody (bsAb) where both antibodies were present in an IgG-scFv format in this assay.
  • bsAb bispecific antibody
  • Table 20 The effects on T cell activity of the monoclonal antibody mixtures are shown in Table 20. These effects on T cell activity were comparable to those achieved with the bsAb, suggesting that these receptors independently contribute to T cell activation (data for bsAb not shown).
  • the pp65 peptide-specific CTL response to peptide-pulsed tumor cells for dual blockade of CD112R and TIGIT, triple blockade (3x), single blockade of TIGIT, and single blockade of CD112R is shown in Figure 17B.
  • the pp65 peptide-specific CTL response to peptide-pulsed tumor cells for dual blockade of PD-1 and CD112R, triple blockade (3x), single blockade of CD112R, and single blockade of PD-1 is shown in Figure 17C. Expected and observed outcomes are shown in Figure 17D.
  • the pp65 peptide-specific CTL response to peptide-pulsed tumor cells was slightly enhanced with single blockade of TIGIT, CD112R, or PD-1 (relative to isotype control), and each case of dual blockade (TIGIT+CD112R, TIGIT+PD-1, CD112R+PD-1) further enhanced the pp65 peptide-specific CTL response.
  • triple blockade demonstrated the most significant increase in T cell activity against the tumor cells. Whether the responses of the different combinations represented strictly additive or synergistic effects were evaluated by comparing the calculated expected vs.
  • composition performed as well as the two monoclonal antibodies formatted into a bispecific molecule. Both modalities significantly enhanced primary human T cell activity in in vitro assays and that the overall activities were comparable.
  • triple blockade using the monoclonal CD112R and TIGIT antibodies in combination with PD-1 blocking antibody further enhanced T cell activity, better than any single or double combinations from the three.
  • CD112R and TIGIT co-blockade presents a promising approach for a combination treatment with PD-1/PD-L1, since triple blockade can engage two non-redundant costimulatory pathways to enhance T/NK cell activity.
  • EXAMPLE 10 [00369] This example describes the materials and methods used in the experiments of Examples 1- 2 and 7-8. [00370] TCGA correlation plot generation in Array Studio [00371] The log (FPKM) values representing transcript levels of genes of interest were extracted from the TCGA dataset and plotted in scatter plots to show pairwise correlation. The analysis was performed for TIGIT family receptors and PD-1 and their ligands for multiple cancer indications.
  • CD112R binding assay and FACS staining [00375] To evaluate binding of anti-CD112R tool antibodies (PL-52576, PL-52575, PL-52577) to human CD112R, CHO K1 cells transfected to express CD112R were incubated with anti-CD112R antibody, CD112 ligand Fc (Sino biological), or isotype control for 1 hour at 4C, and the bound antibody was detected with APC conjugated anti-human IgG secondary antibody (R&D systems) or PE conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch).
  • APC conjugated anti-human IgG secondary antibody R&D systems
  • PE conjugated anti-mouse IgG secondary antibody Jackson Immunoresearch
  • Pan T cells and memory CD8 T cells were purified from previously frozen PBMCs (Cepheus Bioscience) using magnetic bead based human Pan T cell (miltenyi Biotec) and memory CD8 T cell isolation kits (STEMCELL technologies) using manufacturers recommended protocol.
  • Pan T cells were cultured with human anti-CD3/anti-CD28 dynabeads (Life technologies) at 1:1 ratio in the presence of 50IU/ml recombinant human IL2 (R&D systems) for 3 days and then, after removal activation beads, were rested for 3 days without supplemental IL2.
  • Memory CD8 T cells were cultured with human anti-CD3/anti-CD28 dynabeads (Life technologies) at 1:1 ratio in presence of 50IU/ml recombinant human IL2 (R&D systems) for 7 days, IL2 was replenished every 2-3 days, and then, after removal of activation beads, were rested for 2 days in presence of 50IU/ml recombinant IL2.
  • CMVpp65 (495-503) reactive CD8+ T cells For expanding CMVpp65 (495-503) reactive CD8+ T cells, PBMC from HLA-A2+, CMVpp65 (495-503) reactive CD8+ donors were cultured with CMVpp65 (495-503) (Anaspec) peptide-pulsed monocyte derived dendritic cells, generated using monocyte derived dendritic cell differentiation kit (R&D sytems), in the presence of 50IU/ml recombinant IL2 (R&D Systems), 10ng/ml recombinant IL7 (R&D System), 1000IU/ml recombinant IL4 (R&D systems) for 14 days in a Grex-10 flask (Wilson wolf manufacturing).
  • R&D sytems monocyte derived dendritic cell differentiation kit
  • RPMI complete media RPMI 1640 supplemented with Glutamax (GIBCO) + 10% FBS (GIBCO)+ Pen/strep + NEAA + Sodium Pyruvate + ⁇ -ME + HEPES (GIBCO)).
  • Glutamax Glutamax
  • FBS Glutamax
  • Pen/strep NEAA + Sodium Pyruvate + ⁇ -ME + HEPES (GIBCO)
  • CHO K1 cell line expressing anti CD3 scfv was (obtained from internal Amgen sources) used for transfection to induce CD112 and CD155 expression.
  • CHO cell line expressing ‘low’ anti- CD3 scfv was transfected with pcDNA3.1_human CD112 var delta_zeo vector (obtained from internal Amgen source) using Lipofectamine 2000 transfection kit (Invitrogen) using manufacturer’s recommended protocol. After transfection, CHO cells expressing CD112 were sorted as single cell clones and allowed to outgrow in selection media (Ham’s F12 media containing (GIBCO) 1X glutamax (GIBCO), 10% heat inactivated FBS (GIBCO), 200ug/ml hygromycin, and 100ug/ml zeocin and (Thermo Fischer)).
  • selection media Ham’s F12 media containing (GIBCO) 1X glutamax (GIBCO), 10% heat inactivated FBS (GIBCO), 200ug/ml hygromycin, and 100ug/ml zeocin and (Thermo Fischer)).
  • CHO cells expressing CD112, anti-CD3scfv, and CD155 were sorted into single cell clones and allowed to expand in selection media (Ham’s F12 media containing (GIBCO) 1X glutamax (GIBCO), 10% heat inactivated FBS (GIBCO), 200ug/ml hygromycin, 15ug/ml puromycin, and 100ug/ml zeocin and (Thermo Fischer)).
  • selection media Ham’s F12 media containing (GIBCO) 1X glutamax (GIBCO), 10% heat inactivated FBS (GIBCO), 200ug/ml hygromycin, 15ug/ml puromycin, and 100ug/ml zeocin and (Thermo Fischer)).
  • CHO K1 cells were transfected with vector Pmscv_FLAG_human CD112R (N81D)(41-326)_IRES_EGFP, using Lipofectamine 2000 kit (Invitrogen) using manufacturer recommended protocol.
  • CHO cells either transfected to express CD112 or with an empty vector along with aCD3scfv, were co-cultured with previously expanded Pan T cells and soluble anti-CD28 (Biolegend) in the presence of anti-CD112R antibody or isotype control, with or without anti-CD226 (Abcam).
  • Cell culture supernatant was harvested after 24 hours and evaluated for IL2 using standard ELISA kit (R&D Systems) as a readout for T cell function.
  • CHO cells expressing CD112 and CD155 were co-cultured with previously expanded memory CD8 T cells and soluble anti-CD28 (Biolegend) in the presence of the blocking antibodies and supernatant was harvested after 24 hours and analyzed using MSD human IFN ⁇ V-plex detection kit.
  • SKMEL30 cells were pretreated with 100ng/ml recombinant IFN ⁇ for 24 hours, pulsed with 1ug/ml CMVpp65 (495-503) peptide and co-cultured with previously expanded CMVpp65 (495-503) reactive CD8+ T cells for 72 hours in the presence of single, double, or triple combinations of anti-TIGIT, anti-CD112R, and anti-PD-1 antibodies in a 1:1:1 ratio in RPMI complete media (RPMI 1640 supplemented with Glutamax (GIBCO), 10% FBS (GIBCO), Pen/strep, NEAA, Sodium Pyruvate, ⁇ -ME and HEPES (GIBCO)).
  • RPMI complete media RPMI 1640 supplemented with Glutamax (GIBCO), 10% FBS (GIBCO), Pen/strep, NEAA, Sodium Pyruvate, ⁇ -ME and HEPES (GIBCO)
  • the tissues were processed by first cutting into small pieces, followed by enzymatic digestion and mechanical dissociation with GentleMacs in digestion buffer (DMEM/F12 supplemented with 10% FBS, Penn/strep, L-Glutamine, HEPES, 1.5mg/ml collagenase type II, 10ug/ml hyaluronidase type iv, 10uM Y-27632 and Dnase I) at 37C. After digestion, cell suspension was filtered through 70um mesh filter, treated with RBC lysis buffer and resuspended in culture media.
  • digestion buffer DMEM/F12 supplemented with 10% FBS, Penn/strep, L-Glutamine, HEPES, 1.5mg/ml collagenase type II, 10ug/ml hyaluronidase type iv, 10uM Y-27632 and Dnase I
  • TIL stimulation assay was performed by culturing 250,000 dissociated tumor cells per well in a 96-well plate, in the presence of combinations of anti-CD112R, TIGIT, PD-1, and/or isotype control antibodies, each at 10ug/ml for 30ug/ml total antibodies per sample in RPMI complete media at 37°C. Supernatant was harvested on day 3 and day 6 and analyzed for IFN ⁇ by MSD.
  • EXAMPLE 11 [00387] This example demonstrates formulations comprising CD112R mAbs and TIGIT mAbs.
  • Formulations comprising anti-TIGIT mAbs, anti-CD112R mAbs, or both (140 mg/mL total antibody concentration) were prepared and characterized for viscosity and stability.
  • the anti- TIGIT mAbs used in these studies included 43B7 and 66H9, and the anti-CD112R mAbs used were 1E1, 29E10 and 24F1. The results are shown in Table 21.
  • TABLE 21 p p g [00389] As shown in Table 21, formulations comprising both anti-TIGIT mAbs, anti-CD112R mAbs demonstrated acceptable viscosities of less than 15 cP.
  • the anti-PD-1 mAb concentration was fixed at 0.5 nM (for the formulations comprising 1.5 nM total mAb concentration) and 10 nM (for the formulations comprising 30 nM total mAb concentration).
  • Table 23 summarizes the amounts of each antibody in the formulations. TABLE 23 [00395] As shown in Figure 19A, all formulations comprising TIGIT mAb and CD112R mAb at the total Ab amount of 1.5 nM (Bars 1-6) performed better than those formulations lacking both TIGIT mAb and CD112R mAb (Bars 7-12).
  • peripheral CD8+ T and NK cells expressed more comparable levels of CD112R and TIGIT compared to TILs (Figure 20B).
  • Tumor cells expressed varying levels of the ligands CD155 and CD112 ( Figure 20C).
  • At least some of the CD112R and TIGIT mAbs of the present disclosure exhibit similar range of affinities to their respective targets and nearly identical pharmacokinetic profiles. Because CD112R and TIGIT expression is variable from donor to donor in tumor versus blood, as is that of the ligands, target saturation via dosing with mAb combinations at 1:1 ratio is reasonable and an elegant approach to cover targets that ultimately converge on the same downstream pathway.
  • EXAMPLE 14 This example demonstrates the in vivo pharmacokinetics (PK) of CD112R mAb and TIGIT mAb in non-human primates (NHP).
  • PK pharmacokinetics
  • An exploratory toxicology study was performed to evaluate the toxicity and PK characteristics of a formulation comprising anti-CD112R mAb (24F1), anti-TIGIT mAb (43B7.002.015), or both antibodies.
  • IV intravenous
  • groups of male cynomolgus monkeys (3 animals per group) were dosed as follows: Group 1 received sequential administration of 0.5 mg/kg anti-CD112R mAb and 0.5 mg/kg anti-TIGIT mAb.
  • Group 2 received 5 mg/kg anti-TIGIT mAb, while Group 3 received 5 mg/kg anti-CD112R mAb.
  • Mean concentration- time profiles are provided in Figure 21. As shown in this figure, after a single IV administration, exposure increased approximately dose proportionally between 0.5 and 5 mg/kg for TIGIT mAb and slightly higher than dose proportionally for CD112R mAb at the same dose levels. Mean peak concentrations and areas under the concentration-time curve were similar CD112R mAb and TIGIT mAb at both dose levels, suggesting PK profiles are suitable for coformulation at a 1:1 ratio.
  • EXAMPLE 15 [00400] This example demonstrates the CD112R mAb + TIGIT mAb activity in primary human Natural Killer (NK) cells.
  • NK Natural Killer
  • CD112R and TIGIT also regulate NK cell activity (Li et al, 2020; and Stanietsky et al, 2013)
  • the activities of anti-CD112R mAb (24F1) and anti-TIGIT mAb (43B7.002.015), and co- formulations comprising a 1:1 ratio of the anti-CD112R mAb (24F1) and anti-TIGIT mAb (43B7.002.015) were evaluated in an NK cell activation assay.
  • primary NK cells were purified from blood from healthy donors, and then co-cultured with tumor cells. NK cell response as represented by IFN ⁇ production and tumor cell killing was measured.
  • the purified NK cells expressed high levels of CD226, TIGIT, and CD112R but very low levels of PD-1 (Figure 22A).
  • the target tumor cells endogenously expressed the ligands CD112, CD155, and PD-L1 ( Figure 22B).
  • Figures 22C – 22D each of the CD112R mAbs and TIGIT mAbs individually induced NK cell activation.
  • the combination of the two Abs provided a further increase in tumor cell killing (Figure 22C) and IFN production ( Figure 22D).
  • a single-cell suspension from dissociated tumor tissue was cultured in the presence of anti-TIGIT mAb, anti- CD112R mAb, anti-PD-1 mAb, or combinations thereof without any exogenous antigens.
  • the idea behind the assay was that the endogenous tumor cells supply tumor-derived antigens to T and/or NK cells.
  • T and/or NK cells Out of 10 different samples representing 4 different solid tumor indications (pancreatic (PANC), colorectal (CRC), triple negative breast cancer (TNBC), and gastrointestinal (GIST) cancer), a response in 6 samples, as measured by IFN ⁇ on day 3, was detected.
  • PANC pancreatic
  • CRC colorectal
  • TNBC triple negative breast cancer
  • GIST gastrointestinal
  • mice Each weighed approximately 17-24 g at first measurement.
  • the strains of mice used in the study included: Balb/c TIGITxCD112R double KO, Balb/c CD112R-GFP KI, Balb/c TIGIT KO, Balb/c TIGITxCD112R WT/WT; C57BL/6 TIGITxCD112R double KO, C57BL/6 CD112R-GFP KI, C57BL/6 TIGIT KO, C57BL/6 TIGITxCD112R WT/WT; NOD SCID IL2rg (NSG).
  • Balb/c and C57BL/6 were sourced from Charles River Labs and the NSG mice were obtained from Jackson Labs.
  • mice were 4 to 12 weeks old.
  • Balb/c mice were CT26 (colon carcinoma) tumor- bearing, C57BL/6 were B16F10 (melanoma) tumor-bearing, and NSG mice were CMV-SKMEL30 (melanoma) tumor-bearing.
  • Test articles included a co-formulation comprising a 1:1 ratio of anti-TIGIT mAb (43B7.002.015) and anti-CD112R mAb (24F1), anti-muPD-1 Clone 29F.1A12, anti-human PD-1 and control articles included vehicle control (100% DPBS), mIgG1 N297G Isotype Control, hIgG1 Isotype Control.
  • CT26, B16F10 and CMV-SKMEL30 cells were obtained from the Cancer Pharmacology Cell Bank (Amgen, Thousand Oaks). CT26 cells were maintained at 37qC in RPMI-1640 supplemented with 10 ⁇ FBS, 1% NaPyr, 1% HEPES and 1% NEAA. B16F10 cells were maintained at 37qC in DMEM supplemented with 10 ⁇ FBS. CMV-SKMEL30 cells were maintained at 37qC in RPMI-1640 supplemented with 10 ⁇ FBS, 1% NaPyr, 1% NEAA, 1% GlutaMAX, 5 ug/mL Blasticidin and 1 ug/mL puromycin.
  • mice were determined to be free of contamination with mycoplasma as well as a panel of murine vial pathogens in addition to being authenticated.
  • T225 tissue culture flasks of cells were harvested and viable cells were quantified by trypan blue exclusion on the Vi-Cell XR (Beckman Coulter, Brea, CA).
  • Female mice were injected with 3 ⁇ 10 5 CT26 or B16F10 cells in 0.1 mL serum-free media, or 1 ⁇ 10 6 CMV-SKMEL30 cells in 0.1 mL 1:1 RPMI:Matrigel (BD Biosciences, Bedford, MA) subcutaneously in the right flank.
  • mice were also injected with 2.5 ⁇ 10 6 CMV-expanded CTLs intravenously. Animals were randomized into groups such that every group had similar average tumor volumes of approximately 100 mm 3 . [00412] In CT26 and B16F10 syngeneic models, animals were dosed with isotype control or anti- murine PD-1 antibody at 100 ⁇ g/mouse.
  • mice were dosed with isotype control antibodies, the anti-TIGIT-mAb/anti-CD112R mAb co-formulation (at 200 ⁇ g / mouse) and/or anti-PD-1 antibodies (at 300 ⁇ g / mouse) intraperitoneally (IP) twice weekly (2QW).
  • Animals were weighed, and tumor volumes were measured twice per week. Tumor measurements were calculated from the length, width and height of tumors measured with a PRO-MAX electronic digital caliper (Japan Micrometer Mfg. Co. LTD). The tumor volume was calculated as [L ⁇ W ⁇ H] and expressed in mm 3 .
  • mice In a first model, knockout mouse strains lacking CD112R or TIGIT, or both, and were generated, and the effects of target deficiency together with or without PD-1 blocking mAb in syngeneic tumor models were evaluated.
  • a xenograft model where immunodeficient NSG mice were implanted with human melanoma tumor cell line (SKMEL30) that were engineered to express CMV antigen, along with CMV Ag-specific CD8 T cells was generated. After tumors were established, mice were dosed with human PD-1 mAb, CD112R and TIGIT mAb combination, or PD-1+TIGIT+CD112R mAb triple combination.
  • Tumor growth was measured over the course of study, and the percent tumor growth inhibition (TGI) at study termination was calculated to compare the activity of PD-1 single blockade, TIGIT+CD112R combination (double knockout or combination mAb blockade), or TIGIT+CD112R+PD-1 triple combination (TIGIT+CD112R double KO or mAb combination, plus PD-1 mAb) against isotype control-treated group.
  • TGI percent tumor growth inhibition
  • TGI tumor growth inhibition
  • triple blockade with anti-TIGIT mAb/antiCD112R mAb combination formulation plus anti-PD-1 mAb led to the largest decrease in tumor volume relative to tumor volume of isotype control treated mice (black).
  • CD112R+TIGIT+PD-1 triple blockade inhibits tumor growth better than double combination or single blockade.
  • the data of Figures 24A-24C support that triple blockade led to the most robust inhibition of tumor growth compared to groups treated with PD- 1 mAb alone, or the TIGIT+CD112R combination. These results support that TIGIT+CD112R+PD-1 triple blockade is efficacious in inhibiting tumor growth in vivo.
  • each formulation was 140 mg/mL.
  • Formulations comprising both anti- CD112R mAb and an anti-TIGIT mAb comprised ⁇ 70 mg/mL each at a 1:1 ratio.
  • the formulations were then stored at -30° C, 4° C, 25° C, or 40° C for up to 4 weeks. Samples of each formulation were then analyzed by size exclusion-ultra high performance liquid chromatography (SE-UHPLC) and reduced capillary electrophoresis-sodium dodecyl sulfate (Rce-SDS) to evaluate the stability of each formulation. Stability was determined by calculating the percentage of each separated component as compared to the total integrated area.
  • SE-UHPLC size exclusion-ultra high performance liquid chromatography
  • Rce-SDS reduced capillary electrophoresis-sodium dodecyl sulfate
  • Proteins are detected by a photodiode array (PDA) detector as they pass through a UV detection window. Stability is evaluated by determining the percent corrected peak area of reach component.
  • the Rce-SDS method separates the heavy chain (HC), light chain (LC), non-glycosylated HC (NGHC), and other minor peak species and groups under reducing conditions. As shown in Table 24, all formulations were relatively stable, forming about 4% LMW+HMW peaks after storage -30° C, 4° C, 25° C, or 40° C for up to 4 weeks.
  • the formulations comprising TIGIT-12 mAb however, associated with higher %ages. Without being bound to theory, the higher % is caused by increased clipping of this antibody.
  • the activity of dual blockade of two of TIGIT, CD112R, and PD-L1 compared to triple blockade is evaluated in an antigen-specific CTL assay using peptide-pulsed SK-MEL tumor cells expressing PD-L1 as the antigen presenting cells (APCs).
  • the antigen-specific CTL assay is similar to the antigen-specific CTL assay of Example 7 (for which the methodology is described in Example 10), with a few exceptions, including the use of anti-PD-L1 antibodies in place of anti-PD-1 antibodies and the use of SKMEL30 cells engineered to express human PD-L1 as the APCs.
  • SKMEL30 cells expressing human PD-L1 are pulsed with 1 ⁇ g/ml CMVpp65 (495- 503) peptide and co-cultured with previously-expanded CMVpp65 (495-503) reactive CD8+ T cells for 72 hours in the presence of dual blockade antibody solution, triple blockade antibody solution, single blockade antibody solution or a control antibody solution, wherein each antibody solution comprises the concentration of antibodies as noted in Table 30 in RPMI complete media (RPMI 1640 supplemented with Glutamax (GIBCO), 10% FBS (GIBCO), Pen/strep, NEAA, Sodium Pyruvate, ⁇ - ME and HEPES (GIBCO)).
  • RPMI complete media RPMI 1640 supplemented with Glutamax (GIBCO), 10% FBS (GIBCO), Pen/strep, NEAA, Sodium Pyruvate, ⁇ - ME and HEPES (GIBCO)

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Abstract

L'invention concerne des compositions comprenant une protéine de liaison à l'antigène CD112R, une protéine de liaison à l'antigène TIGIT et une protéine de liaison à l'antigène PD-L1. La composition dans des aspects donnés à titre d'exemple comprend un anticorps anti-CD112R, un anticorps anti-TIGIT et un anticorps anti-PD-L1, éventuellement, à environ un rapport 1:1:1. L'invention concerne également des méthodes de traitement d'un patient comprenant l'administration au sujet d'une protéine de liaison à l'antigène CD112R, d'une protéine de liaison à l'antigène TIGIT et d'une protéine de liaison à l'antigène PD-L1, éventuellement, d'un anticorps anti-CD112R, d'un anticorps anti-TIGIT et d'un anticorps anti-PD-L1. Dans des cas donnés à titre d'exemple, chaque protéine de liaison à l'antigène est formulée séparément et administrée au sujet. Dans d'autres aspects, au moins deux des anticorps sont formulés ensemble et ainsi administrés ensemble. Facultativement, au moins les anticorps anti-CD112R et anti-TIGIT sont co-formulés et co-administrés et l'anticorps anti-PD-L1 est administré séparément. Dans des cas donnés à titre d'exemple, la méthode comprend l'administration d'une composition comprenant toutes les trois protéines de liaison à l'antigène.
PCT/US2023/010775 2022-01-14 2023-01-13 Triple blocage de tigit, cd112r et pd-l1 Ceased WO2023137161A1 (fr)

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