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WO2024263692A1 - α-BUTYROPHILIN2A1 ANTIBODIES - Google Patents

α-BUTYROPHILIN2A1 ANTIBODIES Download PDF

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Publication number
WO2024263692A1
WO2024263692A1 PCT/US2024/034693 US2024034693W WO2024263692A1 WO 2024263692 A1 WO2024263692 A1 WO 2024263692A1 US 2024034693 W US2024034693 W US 2024034693W WO 2024263692 A1 WO2024263692 A1 WO 2024263692A1
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Prior art keywords
seq
amino acid
acid sequence
cdr
sequence identity
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French (fr)
Inventor
Erin J. ADAMS
Anthony A. KOSSIAKOFF
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University of Chicago
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University of Chicago
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2827—Immunoglobulins [IGs], 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 B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/35—Valency
    • 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
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/75—Agonist effect on 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the antibody comprises an antigen binding fragment (Fab).
  • kits comprising an antibody or antibody fragment described herein.
  • a BTN2A1 -related disorder comprises an autoimmune disorder, an inflammatory disorder, or transplant rejection.
  • FIGS. 1A-1D show engineering of 20.1 mAb reveals lack of 3A1 clustering in Vy9V62 activation.
  • FIG. 1A Schematic representing the putative effects of increasing 20.1 inter-Fab distance on BTN3A1 membrane organization.
  • FIG. IB Schematic representing key features of modified mAb constructs with hinge-regions lengthened using Glycine-Serine linkers.
  • FIG. 1C Activation of G115-TCR expressing Jurkat Jrt-3.3 cells after co-incubation with Daudi AAAVS1 cells pre-incubated with 20.1 hinge variants at increasing concentrations or control (Buffer).
  • FIGS. 2A-2D show engineering of 103.2 mAb reveals lack of 3A1 conformational constraint in Vy9V52 antagonism.
  • FIG. 2A Structural alignment of BTN3A1 in complex with the 103.2 single-chain-variable-fragment (scFv) (PDB ID: 4F9P) with 2 mouse IgGl Fragment antibodies (Fab) (PDB ID: 1BAF) and ColabFold models of the murine IgGl Hinge regionREF.
  • Distance between heavy-chain cysteine residues forming each terminal Fab disulfide bond (yellow) is denoted by *.
  • FIGS. 4A-4G show structure of 2A1.9 Fab in complex with BTN2A1 ectodomain provides insight into TCR-competition mechanism of V , y9V62 Antagonism.
  • FIG. 4A Structure and surface representation of BTN2A1 ectodomain complexed with antagonistic 2A1.9 Fab as determined by X-ray Crystallography (PDB ID: 8VC7).
  • FIG. 4B CFG Face of BTN2A1 ectodomain showing key residues implicated in Vy9V82 activation (Karunakaran, M.M., et al., Immunity, 2020) or TCR binding (Fulford, T.S., et al.,.
  • FIG. 4C Surface representation of BTN2A1 ectodomain with residues contacting 2A1.9 Fab (orange), Vy9V52 TCR (PDB ID: 8DFW, green) or both (olive) highlighted.
  • FIG. 4D Surface representation of BTN2A1 ectodomain with residues contacting 2A1.9 Fab (orange), for Vy9V82 activation as assessed by mutagenesis (red) or both (pink) highlighted.
  • FIGS. 5A-5D show docking orientations of 2A1.4 and 2A1.11 Q-BTN2A1 Fabs on BTN2A1 are similar to 2A1.9.
  • FIG. 5A Models of Fabs 2A1.9 (left), 2A1.4 (middle) and 2A1.11 (right) in complex with the BTN2A1 ectodomain dimer fitted into volume maps of 7.4, 7.5, and 6.5A resolution, respectively.
  • Models were generated by fitting the BTN2A1 dimer (PDB ID: 8DFW; Chain A,B) and a muIgGl Fab complexed with an a-Fab elbow nanobody (PDB ID: 7PIJ) into volume maps acquired by single-particle cryo-electron microscopy.
  • FIG. 5B Alignment of 2A1.9 and 2A1.4 with 2A1.11 Fab Complex volume maps.
  • FIG. 5C Docking angles of models of the 2A1.9, 2A1.4 and 2A1.11 Fabs on the BTN2A1 ectodomain aligned by the BTN2A1 IgV domain with nanobodies and one Fab removed for clarity. Docking angles were calculated using the CA of BTN2A1 Cys97, GlnlOO and Fab heavy -chain Cys22.
  • FIG. 5D Alignment of a-BTN2Al mAb CDRH3 loop sequences.
  • FIG. 9 shows inhibition of activation of Vg9Vd2 T-cells expanded from Peripheral Blood Mononuclear Cells (PBMCs) with Daudi target-cells.
  • Target cells were incubated with mAbs +/- phosphoantigen (pAg) and then coincubated with T-cells.
  • CD107a expression on primary Vg9Vd2 cells was assayed and activation was compared to + (103.2 antagonist mAb) and - (MOPC isotype, Buffer) controls.
  • the term “and/or” includes any and all combinations of listed items, including any of the listed items individually.
  • “A, B, and/or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and/or C.”
  • compositions, system, or method that do not materially affect the basic nature of the composition, system, or method.
  • Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
  • BTN2A1 has its general meaning in the art and refers to human BTN2A1 polypeptide, such as:
  • the term “subject” broadly refers to any animal, including human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.).
  • the term “patient” typically refers to a subject that is being treated for a disease or condition.
  • Random mutagenesis of CDR and/or framework residues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91: 3809- 3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994- 2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319 (1995); and Hawkins et al, J. Mol. Biol., 226: 889-896 (1992).
  • Selective mutation at selective mutagenesis positions and at contact or hypermutation positions with an activity-enhancing amino acid residue is described in U.S. Patent No. 6,914,128 Bl.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site.
  • Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.
  • Antibody fragment refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (z.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody.
  • antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodics, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.
  • Bispecific antibody is used herein to refer to a full-length antibody that is generated by quadroma technology (see Milstein et al., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628- 631 (1985)), or by knob-into-hole or similar approaches, which introduce mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14): 6444-6448 (1993)), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody.
  • a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC).
  • a bispecific antibody has two distinct antigen-binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds to.
  • CDR is used herein to refer to the “complementarity determining region” within about an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted “CDR1", “CDR2", and “CDR3”, for each of the variable regions.
  • CDR set refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region.
  • a polypeptide comprising a single CDR may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen-antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues a e directly and most substantially involved in influencing antigen binding.
  • Epitope refers to a site(s) on any molecule that is recognized and can bind to a complementary site(s) on its specific binding partner.
  • the molecule and specific binding partner are part of a specific binding pair.
  • an epitope can be on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, glycolipids, glycoproteins or lipopolysaccharides), or a polysaccharide.
  • Its specific binding partner can be, for example, an antibody.
  • Label and “detectable label” as used herein refer to a moiety attached to an antibody or an analyte to render the reaction between the antibody and the analyte detectable, and the antibody or analyte so labeled is referred to as “detectably labeled.”
  • a label can produce a signal that is detectable by visual or instrumental means.
  • Various labels include signal-producing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like.
  • Representative examples of labels include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein.
  • the moiety itself may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.
  • the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, U lin, 1251, 1311, 177Lu, I66H0, and 153Sm), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6- carboxyfluorescein, 3 ’6-carboxy fluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tet
  • a radioactive label such as 3H, 14C, 32P
  • An acridinium compound can be used as a detectable label in a homogeneous chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Let. 14: 3917-3921 (2004); and Adamczyk et al., Org. Let. 5: 3779-3782 (2003)).
  • BTN3A1 Butyrophilin
  • the antibodies comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61, and a heavy chain comprising at least 80% sequence identity (e
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 37 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 38.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 41 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 42.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 43 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 44.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 48.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 49 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 50.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 51 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 52.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 53 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 54.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 56.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • SEQ ID NO: 34 arc antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 58.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 59 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 60.
  • antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 61 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 62.
  • antibodies that bind to BTN2A1 that comprise a light chain variable region and a heavy chain variable region.
  • the antibodies comprise a heavy chain variable region comprising one or more CDRs set forth in Table 1.
  • the antibodies further comprise a light chain variable region comprising a CDR set forth in Table 1.
  • antibodies that bind to BTN2A1 that comprise a CDR- Hl, CDR-H2, and CDR-H3 set forth in Table 1 and a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g.
  • SEQ ID NO: 33 SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
  • SEQ ID NO: 33 SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61.
  • BTN2A1 that comprise a CDR-L3, CDR-H1, CDR-H2, and CDR
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.1 See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.1 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.2 See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.2 (See Table 1). In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.3 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.3 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.4 (See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.4 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.5 (See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.5 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.6 (See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.6 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.7 (Sec Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.7 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.8 See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.8 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.9 See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.9 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.10 See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.10 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.11 See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.11 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.12 See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.12 (See Table 1). In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.13 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.13 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.14 See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.14 (See Table 1).
  • antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.15 See Table 1).
  • the antibody comprises the CDR-L3 of Fab 2A1.15 (See Table 1).
  • the antibody further comprises a detectable label.
  • the detectable label may be any suitable detectable label.
  • Antibodies may be prepared by any of a variety of techniques.
  • antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies via conventional techniques, or via transfection of antibody genes, heavy chains, and/or light chains into suitable bacterial or mammalian cell hosts, in order to allow for the production of antibodies.
  • Antibodies can be expressed in either prokaryotic or eukaryotic host cells. However, expression of antibodies in eukaryotic cells is preferable, and most preferable in mammalian host cells.
  • Exemplary mammalian host cells for expressing the recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.
  • DHFR selectable marker e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NSO myeloma cells, COS cells, and SP2 cells.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in a process to purify the antibodies.
  • the proteolytic enzyme papain may be used to preferentially cleave IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)i fragment, which comprises both antigen-binding sites.
  • Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure may be performed. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies. In addition, bifunctional antibodies may be produced.
  • Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g., as available from various commercial vendors such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martin sreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos.
  • SAM selected lymphocyte antibody method
  • An affinity matured antibody may be produced by any one of a number of procedures that are known in the art. For example, see Marks et al., BioTechnology, 10: 779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas et al., Proc. Nat. Acad. Set. USA, 91: 3809- 3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994- 2004 (1995); Jackson et al., J.
  • kits comprising at least one antibody or a composition comprising an antibody as described herein.
  • the kit comprises an antibody described herein (e.g. an antibody that binds BTN2A1), along with instructions for use of the kit. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they arc not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure.
  • kits comprise additional reagents for performing an assay to detect one or BTN2A1, including containers (e.g. tubes, microtiter plates, strips, etc.), buffers, stabilizers, preservatives, controls, and the like.
  • containers e.g. tubes, microtiter plates, strips, etc.
  • buffers e.g. buffers, stabilizers, preservatives, controls, and the like.
  • the antibodies, compositions, and kits comprising the same find use in methods for detecting BTN2A1 in a sample.
  • a method for detecting BTN2A1 in a sample comprises contacting the sample with an antibody described herein.
  • the antibody is a Fab.
  • the antibody is detectably labeled.
  • the method may comprise contacting the sample with an antibody described herein, and detecting a signal from the detectable label. The signal from the detectable label may be indicative of the presence and/or amount of BTN2A1 in the sample.
  • the method detects BTN2A1 in the sample, but does not detect a BTN3 polypeptide or BTN3A1. In some embodiments, the method detects BTN2A1 in the sample, but does not detect a BTN2A2. Accordingly, in some embodiments the methods may be used to differentiate between BTN2A1 and other related polypeptides in a sample.
  • the antibodies described herein additionally find use in various diagnostic and therapeutic methods.
  • the antibodies herein have in vitro and in vivo diagnostic and therapeutic utilities.
  • these antibodies can be administered to cells (e.g., in culture (in vitro or in vivo) or in a subject (e.g., in vivo)) to treat, prevent or diagnose a variety of disorders.
  • the methods are particularly suitable for treating, preventing or diagnosing BTN2A1 -related disorders and/or autoimmune, inflammatory disorders, and transplant rejection.
  • a “BTN2A1 -related disorder” includes conditions associated with or characterized by aberrant BTN2A1 levels and/or diseases or conditions that can be treated by modulating BTN2A1 induced signaling activity in a subject (e.g., in human blood cells), for example, by inhibiting the production of IFNy or TNFa of activated of Vy9V52 T cells and/or the cytolytic function of activated Vy9V52 T cells. These include inflammatory conditions, autoimmune diseases and organ or tissue transplant rejection.
  • autoimmune diseases which may be treated include but are not limited to rheumatoid arthritis (RA), insulin dependent diabes mellitus (Type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, pemphigus vulgaris, pemphigoid, addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, coeliac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic leucopenia, idiopathic thrombocytopenic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, phacogenic uveitis, primary biliary cirrhosis, primary
  • An object of the present invention relates to a method of inhibiting an immune response in a subject, in particular inhibiting the cytolytic property of Vy9V82 T cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody described herein.
  • the disclosure also pertains to the methods of manufacturing a medicament for use in the treatment of BTN2A1 -related disorders (e.g., inflammatory conditions, autoimmune diseases and organ or tissue transplant rejection), said medicament comprising an anti-BTN2Al antibody of the present disclosure as described herein.
  • BTN2A1 -related disorders e.g., inflammatory conditions, autoimmune diseases and organ or tissue transplant rejection
  • said medicament comprising an anti-BTN2Al antibody of the present disclosure as described herein.
  • a method of diagnosing a BTN2A1 -related disorder e.g., inflammatory conditions, autoimmune diseases and organ or tissue transplant rejection
  • the method comprises contacting a sample obtained from the subject with an antibody described herein.
  • the method may comprise contacting the sample with an antibody that binds to BTN2A1 described herein.
  • the antibodies may be conjugated to a therapeutic agent for use in treating one or more conditions in a subject (e.g., a BTN2A1 -related disorder).
  • a therapeutic agent for use in treating one or more conditions in a subject (e.g., a BTN2A1 -related disorder).
  • an antibody-drug conjugate comprising an antibody described herein conjugated to an immunosuppressive or immunomodulating agents or other anti-inflammatory agents be useful for treating a BTN2A1 -related disorder in a subject.
  • the antibodies or antibody-drug conjugates described herein may be administered to the subject by any suitable route.
  • the antibody or antibody-drug conjugate may be formulated into a suitable composition for administration to the subject.
  • the formulation of the composition e.g. liquid or solid
  • the composition may comprise additional reagents including pharmaceutically acceptable excipients such as buffers, stabilizers, preservatives, and the like.
  • the antibody, antibody-drug conjugate, or the composition comprising the same may be provided (e.g. administered) to the subject by any suitable method.
  • Suitable routes of administrating the composition described herein include, for example, topical, parenteral (e.g. by injection), and oral forms of administration.
  • the composition is administered parenterally, such as by subcutaneous, intramuscular, intraarterial, or intravenous injection.
  • the antibodies or antigen-binding domains thereof described herein may be used in cellbased therapies.
  • the antibodies or antigen-binding domains thereof may be used in methods of adoptive cell therapy, including tumor infiltrating lymphocyte (TIL), T-cell receptor (TCR), and chimeric antigen receptor (CAR)-based cell therapies.
  • TIL tumor infiltrating lymphocyte
  • TCR T-cell receptor
  • CAR chimeric antigen receptor
  • the antibodies described herein may be used as engagers for T and NK-cell based therapies.
  • the term “engager” refers to an antibody or antibody fragment that enhances interactions between a T-cell or an NK cell and tumor cells. Accordingly, an “engager” may enhance anticancer activity of a T-cell or an NK cell.
  • an antibody fragment set forth in Table 1 may be used as an engager for NK cell or a T-cell based immunotherapy.
  • the antigen binding domains of the antibodies described herein may be incorporated into a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the immune cells are a T cell (including CD4 + T cell, CD8 + T cells) a B cell, a natural killer (NK) cell, a natural killer T cell (NKT) cell, a monocyte cell or a dendritic cell.
  • a chimeric antigen receptor typically comprises an extracellular domain able to bind an antigen (i.c. an antigen binding domain), a transmembrane domain, optionally a hinge domain, and at least one intracellular domain.
  • the antigen binding domain comprises an oligopeptide or polypeptide that binds to a target antigen.
  • the antibodies or fragments thereof i.e. antigen binding portions thereof may be used as the antigen binding domain in a CAR, including for use in a CAR-T cell or a CAR-NK cell.
  • a chimeric antigen receptor comprising a Fab fragment set forth in Table 1.
  • transmembrane domain means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signaling domains. This may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Typically, the transmembrane domain denotes a single transmembrane alpha helix of a transmembrane protein, also known as an integral protein.
  • a chimeric antigen receptor may optionally comprise a “hinge domain” which serves as a linker between the extracellular and transmembrane domains.
  • the terms “hinge”, “spacer”, or “linker” refers to an amino acid sequence of variable length typically encoded between two or more domains of a polypeptide construct to confer for example flexibility, improved spatial organization and/or proximity.
  • the terms “intracellular domain”, “internal domain”, “cytoplasmic domain” and “intracellular signaling domain” are used interchangeably herein and mean any oligopeptide or polypeptide known to function as a domain that transmits a signal that causes activation or inhibition of a biological process in a cell.
  • an immune cell comprising a chimeric antigen receptor described herein may be particularly useful for immunotherapy, such as including for treating a BTN2A1 -related disorder.
  • VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT SEQ ID NO: 44
  • Target cells trigger Vy9V52 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vy9V52 T cell receptor (TCR).
  • BTN phospho-antigen metabolites
  • TCR Vy9V52 T cell receptor
  • mAb a-BTN3Al and a-BTN2Al antibody
  • Modified a-BTN3Al mAbs with increased inter-Fab distances establish that tight clustering of BTN3A1 is not necessary to stimulate Vy9V82 T cell activation, and that antagonism may occur through occlusion of a binding interaction between BTN3A1 and a yet unknown co-receptor.
  • a panel of additional a- BTN2A1 antagonists utilize different biophysical mechanisms to compete with Vy9V62 TCRs for BTN2A1 binding.
  • the complex structures of BTN2A1 ectodomain and Fabs from three antagonist antibodies provide molecular insights into BTN2A1 epitopes involved in pAg- signaling.
  • U-BTN2A1 Fabs with a human-IgGl Herceptin scaffold were cloned into the RH2.2 vector.
  • Fab Heavy and Light chains were on the same plasmid.
  • Fab overexpression was induced in Escherichia coli BL21 Gold (DE3) cells in 2xYT media using ImM isopropyl p-d-1- thiogalactopyranoside (IPTG) for 4 hr at 37C.
  • IPTG ImM isopropyl p-d-1- thiogalactopyranoside
  • Bacterial cells were centrifuged, resuspended and lysed by homogenization and sonication. Bacterial lysate was spun at 20,000 x g for 45’.
  • Fabs were purified from the periplasmic fraction with Protein Gal or G-F resin (Kossiakoff Lab, U.Chicago), eluted with 0.1 M Glycine pH 2.3 and neutralized with a 1:5 volume ratio of 1 M Tris Buffer pH 8.0. Fabs were then dialyzed into Phosphate Buffered Saline (PBS).
  • PBS Phosphate Buffered Saline
  • 0.-BTN3A1 Fab 20.1 and 103.2 Heavy and Light chains with a hybrid murine-IgGl Fab IgV and human-IgGl Herceptin Fab IgC scaffold were cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site, acid- and basic-zipper, respectively, and hexa-histidine tag.
  • Baculovirus was generated by transfection of plasmid and linearized baculovirus DNA into Sf9 insect cells using Cellfectin transfection reagent. Baculovirus was then added to High-Five insect cells and proteins were expressed for 60-68 hrs at 27°C.
  • U-BTN3A1 mAbs with a murine-IgGl scaffold were cloned into the AbVec vector.
  • MAbs were expressed in Expi293 cells at 37C using the ExpiFectamineTM transfection system with 0.5 pg each of Heavy and Light chain plasmids per 1 mL of culture. 7 days after transfection, supernatant was isolated by centrifugation at 3000 x g for 10’.
  • mAbs were then purified using Protein G or Protein Gal resin, eluted with 0.1 M Glycine pH 2.3 and neutralized with a 1:5 volume ratio of 1 M Tris Buffer pH 8.0. MAbs were then dialyzed into PBS.
  • BTN2A1 ectodomain (residues 1-219) and BTN2Al ectodomain C219S (residues 1 -219) were cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site and hexa-histidine tag. Proteins were expressed in High-Five insect cells with the baculovirus expression system. Proteins were purified using Ni-NTA resin and eluted with Hanks Buffered Saline (HBS) + 200 mM and 500 mM imidazole.
  • HBS Hanks Buffered Saline
  • Proteins were de-glycosylated for 2 hrs at 37C using Endo-F3 in HBS + 15mM imidazole at a concentration of ⁇ 1 mg/mL. Proteins were repurified using Ni-NTA resin and HBS + 200 mM imidazole elution to remove Endo-F3 and incubated with 3C protease overnight in HBS + 75 mM imidazole for the removal of the hexa-histidine tag. Proteins were purified further using anion-exchange chromatography over the MonoQ column in 20 mM Tris pH 8.0 with a 20 mL gradient of 30-700 mM NaCl. Proteins were subsequently utilized for structural determination.
  • BTN2A1 ectodomain (residues 1-217) and BTN3Al ectodomain (residues 1-217) were cloned into the pAcGP67a Vector containing a C-terminal BirA biotinylation sequence, human rhinovirus 3C protease cleavage site and hexa-histidine tag. Proteins were expressed in High- Five insect cells with the baculovirus expression system. Proteins were purified using Ni-NTA resin. Proteins were then incubated with 3C protease overnight in HBS + 75mM Imidazole for the removal of the hexa-histidine tag.
  • Proteins were buffer-exchanged to HBS + 15 mM Imidazole with 0.05 M bicine buffer pH 8.3, 10 mM ATP, 10 mM MgOAc and 50 pM d-biotin and biotinylated overnight using the BirA protein. Proteins were then purified using sizeexclusion chromatography (SEC) over the S200 column in HBS and verified for biotinylation by size-shift on an SDS-PAGE gel following coincubation with Traptavidin.
  • SEC sizeexclusion chromatography
  • the y chain of the G115 or DP-alpha constant-region TCR was cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site, acidic- zipper and hexa-histidine tag.
  • the 8 chain of the G115 or DP-beta constant-region TCR was cloned into the pAcGP67a Vector containing a C-terminal BirA biotinylation sequence, human rhinovirus 3C protease cleavage site, basic-zipper and hexa-histidine tag.
  • TCRs were expressed in High-Five insect cells with the baculovirus expression system.
  • TCRs were purified using Ni- NTA resin and eluted. TCRs were then incubated with 3C protease overnight in HBS and 75 mM Imidazole for the removal of the hexa-histidine tag.
  • TCR was buffer exchanged to HBS + 15 mM Imidazole HBS + 15 mM Imidazole with 0.05 M bicine buffer pH 8.3, 10 mM ATP, 10 mM MgOAc and 50 pM d-biotin and biotinylated overnight using the BirA protein.
  • Unbiotinylated and biotinylated TCR were then purified using SEC over the S200 column in HBS and verified for biotinylation by size-shift on an SDS-PAGE gel following coincubation with Traptavidin, if applicable.
  • Biotinylated TCR was tetramerized by co-incubation with Streptavidin-PE at a ratio of 1 : 1.1 streptavidin monomer to biotinylated TCR monomer.
  • the cells were harvested by sonication in 25 mM TRIS, pH 8.0, 300 mM NaCl and 10% glycerol. After centrifugation, the supernatant was passed over a lOmL HisTrap HP column and eluted with 25 mM TRIS, pH 8.0, 300 mM NaCl, 150 mM imidazole and 10% glycerol.
  • the protein was further purified by SEC in 20 mM HEPES, pH 7.4, and 200 mM NaCl.
  • Biotinylated BTN2A1 was immobilized onto streptavidin-coated paramagnetic beads for five rounds of phage selection.
  • 1 pM of BTN2A1 was immobilized on 200 pl SA magnetic beads and was incubated with 1 mL phage library (10 10 CFU) for 1 hour at room temperature with gentle shaking. The beads were washed three times to remove nonspecific phage, added to log phase E. coli XL-1 blue cells and incubated for 20 minutes at room temperature.
  • media containing 100 pg/mL ampicillin and 10 9 p.f.u./mL of M13K07 helper phage was added for overnight phage amplification at 37°C.
  • the amplified phage was precipitated in 20% PEG/2.5 M NaCl for 20 minutes on ice for subsequent rounds.
  • the phage pool was negatively selected against empty paramagnetic beads for 30‘ with shaking to eliminate nonspecific binders.
  • the final antigen concentration was dropped systematically from 1 pM to 10 nM from the first to the fifth round (2nd round: 200 nM, third round: 50 nM, fourth round 20 nM, and fifth-round 10 nM).
  • the beads were subjected to five washing rounds with 0.5% BSA/PBST.
  • the bound phages were eluted using 0.1 M Glycine, pH 2.6, and neutralized with TRIS-HC1, pH 8. Then, the phage eluate was used for E. coli infection and phage amplification, as described above. Additional selection pressure using 1 pM of not biotinylated BTN3A1 in all washes was applied to ensure specificity to BTN2A1.
  • the infected cells were plated on ampicillin agar and 192 colonies were picked to produce phage clones for single-point phage ELISA assay. The promising clones demonstrating high specificity were sequenced and reformatted into a RH2.2 expressing vector.
  • Daudi cells were resuspended in PBS + 2% FBS and incubated with increasing concentrations of U-BTN2A1 mAb.
  • ct-BTN2Al mAbs were stained with 1:200 fluorescently-tagged secondary mAb and staining was assessed by flow-cytometry.
  • Single-point phage ELISA was used to test specificity of O-BTN2A1 Fabs for BTN2A1 binding.
  • 50 nM of BTN2A1 or BTN3A1 protein was directly immobilized on high-binding experimental wells for 30', followed by extensive blocking with 2% BSA for 1 hour. After 15' of incubation with phage, the wells were extensively washed three times with 0.5% BSA/PBST and incubated with Protein L-HRP (1:5000 dilution in HBST) for 20’. The plates were again washed and developed with TMB substrate and quenched with 10% H3PO4, followed by the absorbance at A450 determination.
  • biotinylated BTN3A1 ectodomain in HBS was immobilized on Streptavidin biosensor tips until Binding reached between 3 and 4 nm. Tips were blocked with 1 pM biotin for 30”. After baseline was established in PBS for 30”, the biosensor tip was exposed to 1.3 pM mAb for 90”. Dissociation in PBS was then assessed for 120”.
  • biotinylated BTN2A1 ectodomain in HBS was immobilized for 150” on Streptavidin biosensor tips. Tips were blocked with 1 pM biotin in HBS for 30s followed by 2 mg/mL BSA for 180”. The biosensor tip was exposed to 66 pM a-BTN2Al Fab concentration for 120”. Tips were immediately placed into 66 pM U-BTN2A I Fab + 80 pM Vy9V62 TCR for 120”, in HBS. Dissociation in HBS was then assessed for 120”.
  • the y and 8 chain of the G115 Vy9V82 TCR clone were cloned into the pMSC-V vector with puromycin and zeocin selection genes, respectively.
  • Jurkat JRT3.3 cells were transfected with these plasmids and selected first for y chain expression for 7-14 days using 1 mg/mL puromycin. Cells were then selected for ⁇ 4 weeks with 200 mg/mL zeocin. Cells were stained with a-y9 and a-82 antibodies and sorted on TCR expression at both 1 and 4 weeks.
  • Daudi-Cas9 AAAVS1, ABTN2A1, ABTN3A1 cell lines were generated as described (Mamedov, M.R., et al., Nature, 2023.
  • Daudi-Cas9 AAAVS1 were generated with a CRISPR guide RNA targeting AAVS1, a safe harbor site that acted as a CRISPR cutting control.
  • JRT3.3 Jurkat cells expressing the G115 TCR, Daudi AAAVS1 , Daudi A2A1 , and Daudi A3A1 were cultured in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 10 U/mL Penicillin/Streptomycin (R10). Cells were split to 0.3E6 cells/mL daily and split 24 hours prior to the start of an activation assay.
  • FBS Fetal Bovine Serum
  • R10 Penicillin/Streptomycin
  • Peripheral blood was isolated from healthy donors through the IRB 13-0666 protocol and subjected to density gradient centrifugation using Ficoll. Lymphocytes were isolated and brought to 1E6 cells/mL in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 0.1% P-me, 0.5X non-essential amino acids (Coming), 1 mM sodium pyruvate and 10 U/mL Penicillin/Streptomycin (R10+). Cells were pulsed with 5 pM Zoledronate and 100 U/mL IL-2 and incubated at 37°C.
  • IL-2 100 U/mL IL-2 was supplemented into the media every 2-3 days and the culture volume was doubled on day 6 with fresh R10+ media supplemented with 100 U/mL IL-2.
  • 100 U/mL IL-2 was pulsed into the media.
  • Vy9V82 T cells were utilized on Day 9-10 for activation assays and assayed via flow-cytometry for expansion at such time.
  • 0.05E6 Daudi target cells were incubated with PBS, mAb, or Fab +/- HMBPP, Pamidronate or MOPC isotype mAb in RIO for 2 hours in 100 pL.
  • Target cells were washed with 200 pL warm PBS and centrifuged at 300 x g for 5' 3 X and co-incubated with 0.1E6 Jurkat or Primary Vy9V52 T cells per condition overnight in 200 pL R10 or R10+, respectively.
  • 1.6 pg/mL PHA was added to wells with T cells alone prior to overnight incubation as a positive control for activation. T Cell activation was assessed via flow-cytometry.
  • BTN2A1 Full-length BTN2A1 was cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site and hexa-histidine tag.
  • BTN2A1 was expressed in High-Five insect cells with the baculovirus expression system.
  • BTN2A1 -expressing Insect cells were seeded at 1.5E5 cells/well in 96-well round-bottom plates with edge- wells containing 200 pL PBS and incubated with 150nM a-BTN2Al mAb for 30’. Insect cells were washed with 200 pL PBS and centrifuged at 300 x g for 5’. Insect cells were then sequentially stained with DAPI followed by additional washes and stained with 120 nM G115 tetramer labeled with the PE- fluorophore. MAb:tetramer competition was assessed via flow-cytometry.
  • T cell activation assays target and T cells from each condition were resuspended in PBS + 2% FBS. Cells were stained with Live-Dead fluorescent dye, ct-CD19 to gate target-cells, ct-Vy9 to gate T cells and a-CD69 fluorophore-conjugated mAbs as a marker for T cell activation.
  • target and T cells from each condition were stained with 1:1500 dilution Live/dead Fixable Near-IR Dead Cell Stain, 1:50 dilution of a- Vy9, a-V52 to gate T cells, and 1:50 dilution of a-CD25, a-CD107a and a-CD69 fluorophore- conjugated mAbs as markers for activation.
  • mAb and TCR-tetramer competition cells were resuspended in PBS + 2% FBS and stained with DAPI and fluorescently-labeled G115 TCR tetramers. Data were collected on either the Aurora or AttuneNxt.
  • X-ray datasets were collected at the Stanford SSRL beamline 14-1 on a Dectris Pilatus 6M at 100K. A complete dataset was collected at a wavelength of 1.1950E-10 m.
  • XDS, truncate, pointless, freeR, and aimless were used for data reduction and scaling.
  • An initial molecular replacement solution was obtained using PHASER through Phenix (Liebschner, D., et al., Section D, Structural biology, 2019. 75: p. 861-877) with PDB ID code 8DFW (Fulford, T.S., et al., bioRxiv, 2023: p.
  • Chain B and 4RRP (Kuzin, A., et al., Northeast Structural Genomics Consortium (NESG) Target PdR16. 2014): Chains D,J with CDR loop atoms deleted (D:25-33, 49-61, 90-96. J:28-34, 50-58, 95-100).
  • the initial model was improved using iterative rounds of manual building with Coot followed by refinement with Phenix.
  • a second round of phasing was performed using Chains C,D and J from the refined BTN2A1-Fab complex structure and followed by subsequent rounds of refinement.
  • CCP4-Contact was used to determine van der Waals interactions with a distance cut-off of 4.001.
  • PDB-ePISA was used to find salt-bridge and hydrogen-bond interactions between Fab and BTN2Al.
  • Rational engineering ofa-BTN3A mAb 20.1 provides insights into the role ofBTN3A clustering in pAg-signaling
  • the wildtype (WT) 20.1 mAb is a murine IgGl antibody in which 1 glycine separates the heavychain cysteine residues forming the final disulfide bond with the Fab light chain and the first of 3 inter-heavy-chain disulfide bonds.
  • Glycine-serine (GS) linkers ranging from 4 to 29 amino acids were added to this glycine in the 20.1 heavy-chain hinge region to generate 20.1 mAb hinge variants (20.1 5 , 20.1 10 , 20.1 15 , 20.1 30 ) (Fig. IB).
  • Each additional hinge-linker amino acid added -3.5A to the radius of diffusion between two 20.1 Fab:BTN3A complexes. This incrementally increased the radius of diffusion in multimerized BTN3A, from -155 A with the WT 20.1 mAb to -360A with the 20.1 30 mAb hinge variant, by a maximum factor of -2.3X (Fig. IB).
  • GS linkers in these engineered variants did not affect protein stability nor affinity for biotinylated BTN3A1 ectodomain as assessed by SDS-PAGE gel electrophoresis and Bio-Layer Interferometry (BLI), respectively.
  • Increasing concentrations of the WT 20.1 mAb, the 20.1 mAh hinge variants or the 20.1 Fab were added to Daudi-Cas9- AAVS1 (Daudi AAAVS1 ), a B-cell lymphoma target-cell line that readily activates Vy9V82 T cells, or Daudi-Cas9-ABTN3A1 (Daudi A3A1 ) .
  • the 20.1 Fab has an infinite radius of diffusion and retains the ability to activate Vy9V82 T cells with reduced potency (Fig. 1C-D).
  • the WT 20.1 mAb, 20.1 mAb hinge variants and the 20.1 Fab were mildly agonistic when incubated with Daudi A3A1 target cells likely due to interactions with BTN3A2 and BTN3A3 expression of which was retained in this cell line.
  • BTN3A1 residues bound by both the 20.1 agonist mAb and 0.-BTN3A agonist mAb CTX2026 are directly adjacent to a recently reported pAg-signaling hotspot.
  • a-BTN3A mAb 103.2 inhibits Vy9V32 activation through bi-valent engagement ofBTN3A ectodomains
  • the engineering of the 20.1 mAb provided insights into the role of cellular reorganization of BTN3A during pAg signaling.
  • the role of BTN3A conformational change in pAg signaling was investigated by similarly engineering the antagonist 103.2 mAb.
  • Original analysis of the complex structure between the 103.2 scFv and BTN3A (PDB ID: 4F9P) used a model derived from IgGs that had long, flexible linkers between the Fab and Fc domains (Palakodeti, A., et al., Journal of Biological Chemistry, 2012. 287(39): p. 32780-32790).
  • the distance between the heavy-chain cysteines that form the final inter-chain disulfide bond of the Fab are between 79-113A apart (Fig. 2A), a distance that the hinge region must bridge in a bi- valent mAb. Therefore, there is either significant flexibility in the BTN3A monomers between their IgV and IgC domains, or the 103.2 mAb engages the BTN3A dimer with a 1:2 stoichiometry (one 103.2 mAb to 2 BTN3A dimers).
  • the IgV domains of BTN3A have negligible structural flexibility when dimerized
  • the IgV domains can rotate up to ⁇ 20 A at the point of the linker between the IgV and IgC domains (Fig. 2A).
  • this model positions the 103.2 mAb just within the calculated threshold of monovalent binding to BTN3A with a limited overall flexibility of -8.5A. If monovalent (1:1) binding between 103.2 and BTN3A occurs in physiological contexts, substantial torsional force on the BTN3A IgV domains would be required, potentially locking them in an inactive conformation.
  • bivalent 103.2 binding to BTN3A may also influence the conformational flexibility of BTN3A dimers, preventing the propagation of intracellular pAg- binding information through JMs to BTN3A extracellular domains, ultimately influencing BTN3A interactions between heterodimers or with other proteins such as BTN2A1.
  • 103.2 variants with longer hinge regions (103.2 5 , 1O3.2 10 , 103.2 15 , 1O3.2 30 ) were generated to reduce conformational torsion on BTN3A extracellular domains (Fig. 2B), potentially altering the potency of 103.2 antagonism of Vy9V82 T cell activation.
  • GS linkers in the 103.2 mAb hinge valiants did not affect protein stability as assessed by SDS-PAGE gel electrophoresis although a minor increase in their relative affinity for biotinylated BTN3A1 ectodomain was noted as assessed by BLI.
  • the 20.1 and 103.2 mAbs provide valuable insight into the cellular and molecular changes in BTN3A during pAg- signaling.
  • a panel of 15 O-BTN2A1 Fabs were generated using phage display selection with the BTN2A1 ectodomain as the target antigen. Negative selection was carried out using the BTN3A1 ectodomain as the target antigen and Fab selectivity was verified using phage-enzyme linked immunosorbent assay (ELISA) (FIG. 6).
  • U-BTN2A1 Fabs bound the biotinylated, monomeric BTN2A1 ectodomain with varying biophysical affinities in the pico- nanomolar range as assessed by Surface Plasmon Resonance (SPR) (FIG. 7).
  • Full-length mAb constructs bound endogenous BTN2A1 on Daudi AAAVS1 with a range of cellular affinities measured by ECso of binding and had negligible background staining on Daudi-Cas9 cells where BTN2A1 expression had been knocked out (Daudi A2A1 ) (FIG. 8).
  • O.-BTN2A1 mAbs were incubated with Daudi AAAVS1 or Daudi A2A1 at a concentration of 6.8nM (Ipg/mL) in the presence and absence of 5pM HMBPP.
  • G115 Jurkats were then co-incubated with these mAb- pulsed target-cells overnight and assessed for CD69 expression via flow-cytometry. CD69 expression was assessed relative to a no mAb control group.
  • Both in the presence and absence of exogenous pAg, O.-BTN2A 1 mAbs antagonized Vy9V62 activation with varying strength (Fig.
  • the potency of antagonism for selected mAbs was tested using primary Vy9V82 T cells expanded from Peripheral Blood Mononuclear Cells (PBMCs) derived from healthy donors, (Fig. 3B) in activation assays (FIG. 9).
  • 2A1.9 was the strongest antagonist of primary Vy9V82 T cell activation, exceeding even 103.2, reducing CD107a, CD25 and CD69 expression below baseline expression levels (Fig. 3B).
  • 2A1.11 and 2A1.12 had no effect on primary Vy9V82 T cell activation (Fig. 3B).
  • 2A1.12 also had greater variation in antagonism potency than other tested mAbs, suggestive of biochemical differences in its molecular interactions with BTN2A1.
  • a-BTN2Al mAb antagonists block Vy9Vd2 TCR engagement with BTN2A1 ectodomain
  • BTN2A1 is a binding partner of the Vy9V82 T cell Receptor (TCR) and binding of its CFG-IgV face to the HV4 germline-encoded region of the Vy9V82 TCR y-chain is essential but not sufficient for pAg- signaling.
  • TCR Vy9V82 T cell Receptor
  • mAb-TCR competition was assessed by using High-Five insect cells expressing full-length BTN2A1 on their cell surface (FIG. 10). These cells were incubated with 150nM saturating concentrations of O-BTN2A 1 mAbs and were then stained with 120nM fluorescently tagged tetramers of the G115 Vy9V82 TCR clone. TCR tetramer staining intensity was assessed relative to a no mAb, full TCR staining control group.
  • G115 tetramer staining of BTN2A1 was significantly reduced to varying degrees in the presence of every O-BTN2A1 mAb (Fig. 3C).
  • Addition of certain mAbs (2A1.9, 2A1.14, 2A1.8 and 2A1.7) had a greater effect on TCR tetramer staining of BTN2A1 (Fig. 3C), potentially indicative of a larger overlapping mAb:TCR epitope on BTN2A1.
  • the G115 tetramer had no background staining on High-five insect cells expressing the control transmembrane protein ADLRG3 (Fig. 10).
  • mAbs based on their potency of Vy9V82 antagonism and TCR competition (Fig. 3D).
  • the third class of mAbs weakly blocked TCR binding to BTN2A1 but had neutral effects on Vy9V82 activation (Fig. 3D) represented by the 2A1.11 mAb.
  • the mAb:TCR competition for BTN2A1 binding was validated for representative Fabs using BLI (Fig. 3E, Fig. 10).
  • the BTN2A1 ectodomain was immobilized and exposed first (1) to 0.-BTN2A1 Fab at 66pM for 150 seconds (s) followed by (2) 66pM Fab mixed with 80pM monomeric G115 Vy9V82 TCR clone for 150s.
  • Fabs were included in the TCR association step (2) to minimize Fab dissociation.
  • the 20.1 Fab, as control, was used to determine maximum TCR binding (FIG. 10).
  • the total Buried Surface Area (BSA) on the BTN2A1 ectodomain was 964.6 A, with O 0 the Fab heavy and light chains contributing 658.6 A and 306.0 A, respectively.
  • the 2A1.9 Fab formed 15 Hydrogen-bonds (HB) and salt-bridges (SB) with BTN2A1 ectodomain, 7 of which involved residues in the CDRH3 loop.
  • HB Hydrogen-bonds
  • SB salt-bridges
  • vdW van der Waals
  • the 2A1.9 binding footprint also included critical residues for pAg-signaling (Fig. 4C) identified through rational mutagenesis based on structural models of the BTN2A1:TCR complex.
  • the binding footprints of the 2A1.9 Fab and the Vy9V62 TCR (PDB ID: 8DFW) were then mapped on the BTN2A1 ectodomain (Fig. 4D).
  • the epitopes of the 2A1.9 Fab and the Vy9V52 TCR overlapped substantially, with the 2A1.9 Fab sequestering all but 3 residues in the interface between the Vy9V82 TCR and the BTN2A1 ectodomain (Fig. 4D).
  • BTN2A1 IgV domain in the complex structure was aligned with that in the structure of the BTN2A1 ectodomain in complex with the V 9V32 TCR (PDB: 8DFW).
  • the angle between the BTN2A1 ectodomain IgV and IgC domains was altered 7.3° when bound to the 2A1.9 Fab versus the Vy9V82 TCR (Fig. 4D).
  • Critical BTN2A1 residue side chain orientations and molecular contacts with the 2A1.9 Fab were then assessed.
  • the 2A1.9 Fab residue R103 forms a network of HB and vdW interactions with BTN2A1 residues R96 and E107 and alters the position of E107 (Fig. 4E), likely contributing to the high affinity of the 2A1.9 Fab for BTN2A1 as well as its antagonism potency.
  • the 2A1.9 Fab contacts BTN2A1 Y98, Q100, and Y105 residues through G104 and Y105 in its CDRH3 loop via main and sidechain HB and vdW interactions (Fig. 4F).
  • the 2A1.9 Fab uses side- and main-chain atoms in CDRH3 and CDRH1 residues to sequester BTN2A1 residues F43 and S44 through extensive vdW interactions (Fig. 4G). Altogether these data establish that the mechanism by which 2A1.9 antagonizes Vy9V52 activation is by direct competition with the Vy9V82 TCR for BTN2A1 binding.
  • BTN3A1 Upon binding to a pAg metabolite, BTN3A1 is responsible for initiating a series of cellular and molecular events on the target cell membrane involving BTN2A1 to signal distress to Vy9V62 T cells.
  • BTN3A1 antibodies against these key players in pAg signaling were developed and rationally modified to resolve long-standing questions in the understanding of the Vy9V82 activation mechanism.
  • the 20.1 mAb or pAg binding to BTN3A1 causes BTN3A immobilization, the formation of BTN3A puncta on the cell membrane and BTN3A localization to the target cell - T cell interface, potentially creating BTN3A clusters that seed early immunological synapses.
  • an antigenic hotspot on BTN3A1 involved in pAg signaling to the MOP clone of the Vy9V82 TCR lies directly adjacent to the 20.1 epitope on BTN3A1. Binding of the 20.1 agonist to BTN3A1 may alter or occlude this antigenic hotspot enabling the recruitment of a Vy9V82 TCR ligand or binding to the Vy9V82 TCR itself.
  • the 103.2 mAb loses antagonistic function when modified into a Fab or scFv, both of which do not inhibit Vy9V82 activation.
  • the bivalent 103.2 mAb may conformationally constraint BTN3A cctodomain dimers to inhibit Vy9V82 activation. It was predicted that mAb hinge lengths greater than 10 amino acids would alleviate torsional force of monovalent 103.2 mAb binding to BTN3A dimers, potentially rescuing Vy9V82 activation. The data presented herein shows that the 103.2 mAb likely does not antagonize Vy9V82 activation by locking BTN3A ectodomains in an inactive conformation.
  • the 103.2 mAb may instead sterically occlude access to epitopes on the rest of the BTN3A ectodomain as it binds to the top of the IgV domain of BTN3A dimers. While the bulkiness of the Fc domain in the 103.2 mAb may be necessary for steric hindrance, valency may be key to 103.2 antagonism due to its overall low binding affinity for BTN2A1 of 15 nM.
  • BTN2A1 antibodies either antagonized or had no effect on Vy9V82 antagonism. This suggests that the primary role BTN2A1 serves is to coordinate the formation of the pAg signaling complex as a central protein covered in critical epitopes for pAg signaling, one of which binds the Vy9V82 TCR (Fig. 4).
  • Vy9V82 T cells have been implicated in autoimmune disorders including psoriasis, inflammatory bowel disease, celiac disease, and multiple sclerosis. Inhibiting the activity of Vy9V82 T cells in such contexts may alleviate symptoms through the management of excessive inflammation.
  • BTN3A1 In the context of cancer, data surrounding the association between BTN expression in tumors and clinical outcomes are complicated and tumor dependent. High expression of BTN3A1 in ovarian and pancreatic cancers and BTN2A1 in metastatic renal cell carcinoma has been associated with reduced patient survival. Indeed, BTN3A1 has been shown to suppress c/.p T cell TCR signaling in tumors by preventing the segregation of CD45 from the immune synapse. Thus, the role of tumor-infiltrating Vy9V82 T cells, as well as how BTN3A and BTN2A1 operate outside of pAg signaling, is poorly understood. In contexts where Vy9V32 T cells or BTNs play immunosuppressive roles, targeting BTN2A1 with antagonistic antibodies may sensitize tumors to immunotherapy.

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Abstract

Provided herein are antibodies that bind α-Butyrophilin2A1 (BTN2A1), and diagnostic, therapeutic and research methods of use thereof. In particular, BTN2A1 antibodies are provided with substantially no cross-reactivity with α-Butyrophilin3A1 (BTN3A1), and in certain embodiments exhibiting BTN2A1 antagonism and/or inhibition of Vγ9Vδ2 T-cell activation.

Description

a-BUTYROPHILIN2Al ANTIBODIES
PRIORITY STATEMENT
This application claims priority to U.S. Provisional Application No. 63/509,152, filed June 20, 2023, the entire contents of which are incorporated herein by reference for all purposes.
SEQUENCE LISTING
The text of the computer readable sequence listing filed herewith, titled “UCHI_42158_601_SQL”, created June 20, 2023, having a file size of 62,311 bytes, is hereby incorporated by reference in its entirety.
FIELD
Provided herein are antibodies that bind a-Butyrophilin2A 1 (BTN2A 1 ), and diagnostic, therapeutic and research methods of use thereof. In particular, BTN2A1 antibodies are provided with substantially no cross -reactivity with a-Butyrophilin3Al (BTN3A1), and in certain embodiments exhibiting BTN2A1 antagonism and/or inhibition of Vy9V82 T-cell activation.
SEQUENCE LISTING
The text of the computer readable sequence listing filed herewith, titled “UCHI- 42158_101_SEQUENCE_LISTING”, created June 20, 2023, having a file size of 62,311 bytes, is hereby incorporated by reference in its entirety.
BACKGROUND
Vy9/V62 T cells are important effectors of the immune defense. Vy9/V52 T cells directly lyse pathogen infected or abnormal cells. In addition, Vy9/V82 T cells regulate immune responses by inducing dendritic cell (DC) maturation as well as the isotypic switching and immunoglobulin production. This important cell platform of the immune system is strictly regulated by surface receptors, chemokines and cytokines. Vy9/V82 T cells are activated by nonpeptidic phosphorylated isoprenoid pathway metabolites, referred to as phosphoagonists (PAg). Vy9V82 T cells have also been reported to be dysregulated is several autoimmune diseases. For example, Vy9V82s were first discovered to respond to tuberculosis infection and proliferate polyclonally in cases of bacterial, parasitic, and viral infection including malaria, listeria, leishmaniosis, and tularemia. Vy9V82s are also capable of recognizing cells undergoing malignant transformation and arc the subject of early-stage clinical trials aimed at treating a range of solid and liquid tumors including acute myeloid leukemia, renal cell carcinoma, breast, lung, and liver cancers. Vy9V82 T cells (Vy9V82s) are a unique, innate-like subset of the adaptive immune system with the potential to be developed into an off-the-shelf immunotherapy. However, incomplete understanding of the molecular underpinnings of Vy9V82 activation precludes realization of their immunotherapeutic potential. As such, a deeper understanding of the molecular mechanism of Vy9V82 activation, and the development of moieties that modify Vy9V82 T-cell signaling/activity, are needed.
SUMMARY
Provided herein are antibodies (including antibody fragments) that bind a- Butyrophilin2Al (BTN2A1), and diagnostic, therapeutic and research methods of use thereof. In particular, BTN2A1 antibodies are provided with substantially no cross-reactivity with a- Butyrophilin3Al (BTN3A1), and in certain embodiments exhibiting BTN2A1 antagonism and/or inhibition of Vy9V82 T-cell activation. Antibodies that bind to BTN2A1 with no crossreactivity with BTN3A1 are referred to herein as antibodies that “bind specifically” to BTN2A1.
In some embodiments, provided herein are antibodies that bind specifically BTN2A1 comprising a light chain comprising a light chain variable region and a heavy chain comprising a heavy chain variable region. In some embodiments, the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 set forth in Table 1; and the light chain variable region comprises a CDR-L3 set forth in Table 1.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 5 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 9 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 18, and SEQ ID NO: 19, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 20, SEQ ID NO: 7, and SEQ ID NO: 21, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 2, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 24, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 25, respectively. In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 3, and SEQ ID NO: 26, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 27 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 28, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 29 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 6, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.
In some embodiments, the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 32, respectively.
In some embodiments, provided herein are antibodies or antibody fragments that binds specifically to a-Butyrophilin2Al (BTN2A1), the antibody comprising a light chain and a heavy chain, wherein: the light chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61; and the heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
In some embodiments, an antibody comprises: (a) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 33 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 34; (b) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36; (c) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 37 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 38; (d) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 39 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 40; (e) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 41 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4420; (f) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 43 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 44; (g) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 46; (h) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 48; (i) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 49 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 50; (j) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 51 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52; (k) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 53 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 54; (1) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 56; (m) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 58; (n) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 59 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 60; or (o) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 61 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62. In some embodiments, an antibody binds to BTN2A1. In some embodiments, the antibody does not bind (e.g., significantly, above background, etc.) to BTN3A1.
In some embodiments, the antibody comprises an antigen binding fragment (Fab).
In some embodiments, the antibody comprising a detectable label.
In some embodiments, provided herein is a composition (e.g., pharmaceutical composition) comprising an antibody or antibody fragment described herein.
In some embodiments, provided herein are kits comprising an antibody or antibody fragment described herein.
In some embodiments, provided herein are methods comprising contacting a sample with an antibody or antibody fragment herein and detecting a signal from a detectable label, wherein detection of a signal is indicative of the presence and/or amount of BTN2Alin the sample.
In some embodiments, provided herein are methods of inhibiting/antagonizing Vy9V82 T-cell activation in a subject, comprising providing to the subject an antibody or antibody fragment herein. In some embodiments, the subject has or is at risk of having a BTN2A1 -related disorder. Inhibition of Vy9V62 T-cell activation may be verified by a suitable method, including measuring levels of a marker of Vy9V82 T-cell activation such as CD69, CD25, CD107a, and the like. A decreased level of a marker of Vy9V82 T-cell activation (e.g. decreased percentage of CD69 positive cells) is indicative of effective inhibition of Vy9V82 T-cell activation in the subject.
In some embodiments, provided herein are methods of diagnosing a BTN2 A 1 -related disorder in a subject, the method comprising contacting a sample obtained from the subject with the antibody or antibody fragment herein.
In some embodiments, provided herein are methods of treating a BTN2A1 -related disorder in a subject, the method comprising providing to the subject the antibody or antibody fragment herein or an antibody-drug conjugate thereof.
In some embodiments, a BTN2A1 -related disorder comprises an autoimmune disorder, an inflammatory disorder, or transplant rejection.
BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1D show engineering of 20.1 mAb reveals lack of 3A1 clustering in Vy9V62 activation. (FIG. 1A) Schematic representing the putative effects of increasing 20.1 inter-Fab distance on BTN3A1 membrane organization. (FIG. IB) Schematic representing key features of modified mAb constructs with hinge-regions lengthened using Glycine-Serine linkers. (FIG. 1C) Activation of G115-TCR expressing Jurkat Jrt-3.3 cells after co-incubation with DaudiAAAVS1 cells pre-incubated with 20.1 hinge variants at increasing concentrations or control (Buffer). CD69 expression as a marker of T cell activation was assessed by flow-cytometry (gated on isotype antibody). Means + SD and non-linear regression with variable slope - four parameters (n=3). (FIG. ID) EC50 of agonism and 95% confidence intervals for 20.1 hinge variants was calculated by non-linear regression with variable slope - four parameters (R2= 0.9706, 0.9920, 0.9744, 0.8868, 0.9843, 0.9857, respectively).
FIGS. 2A-2D show engineering of 103.2 mAb reveals lack of 3A1 conformational constraint in Vy9V52 antagonism. (FIG. 2A) Structural alignment of BTN3A1 in complex with the 103.2 single-chain-variable-fragment (scFv) (PDB ID: 4F9P) with 2 mouse IgGl Fragment antibodies (Fab) (PDB ID: 1BAF) and ColabFold models of the murine IgGl Hinge regionREF. Distance between heavy-chain cysteine residues forming each terminal Fab disulfide bond (yellow) is denoted by *. First hinge-region inter heavy-chain cysteine residues shown in blue. Range of orientations of BTN3A1 IgV in relation to BTN3A1 IgC and Fab IgV in relation to Fab IgC determined by structural alignment are indicated. (FIG. 2B) Schematic representing key features of modified mAb constructs with hinge-regions between Fab and hinge disulfide bonds lengthened using Glycine-Serine linkers. (FIG. 2C) Inhibition of activation of G115-TCR expressing Jurkat Jrt-3.3 cells after co-incubation with Daud1AAAVS1 cells pre-incubated with 5 pM HMBPP and 103.2 hinge variants at increasing concentrations or controls (Buffer + HMBPP). CD69 expression as a marker of T cell activation was assessed by flow-cytometry (gated on isotype antibody). Means + SD and non-linear regression with variable slope - four parameters (n=3). (FIG. 2D) IC50 of inhibition and 95% confidence intervals for 103.2 hinge variants was calculated by non-linear regression with variable slope - four parameters (R2= 0.9501, 0.9508, 0.9273, 0.9396, 0.8086, 0.3131 respectively).
FIGS. 3A-3E show U-BTN2A1 mAbs antagonize Vy9V82 activation through different mechanisms, including blocking BTN2A1-TCR binding. (FIG. 3A) Inhibition of activation of G115-TCR expressing Jurkat Jit-3.3 cells after co-incubation with DaudiAAAVS1 cells preincubated with 5 pM HMBPP and 6.8nM (1 pg/mL) U-BTN2A1 mAb or controls (MOPC isotype, 20.1 mAb, 103.2 mAb or Buffer + 5 pM HMBPP). CD69 expression as a marker of T cell activation was assessed by flow-cytometry (gated on isotype antibody) and normalized to Buffer + 5 pM HMBPP condition (Value-Baseline/Baseline). Mean + SD (n=6, 2 independent experiments). Dunnett test pairwise comparison to - mAb control: ****p<0.0001, ***p<0.0002. (FIG. 3B) Inhibition of activation of Vy9V52 T cells expanded from primary PBMCs after coincubation with DaudiAAAVS1 cells pre-incubated with 5 pM HMBPP and increasing concentrations of U-BTN2A1 mAb or controls (MOPC isotype, 103.2 mAb or Buffer + HMBPP). CD25 (top), CD107a (middle) and CD69 (bottom) expression as markers of T cell activation were assessed by flow-cytometry (gated on isotype antibody). Mean + SD (n=3).
(FIG. 3C) Competition between Q-BTN2A I mAbs and the Vy9V52 TCR for binding to BTN2A1 ectodomain assessed in High-Five insect cells expressing full-length BTN2A1 stained with 150nM a-BTN2Al mAbs or controls (20.1, 103.2, MOPC isotypc or Buffer) followed by 120nM fluorescently-tagged Vy9V62-TCR tetramers (G115 clone). TCR tetramer staining was assessed by flow-cytometry (gated on no TCR) and normalized to TCR tetramer, - mAb condition (Value-Baseline/Baseline). Mean + SD (n=6, 2 independent experiments). Dunnett test pairwise comparison to Buffer control: ****p<0.0001. (FIG. 3D) Correlation between 0.-BTN2A1 mAb Vy9V52 antagonism (Baseline-corrected CD69 expression) and Vy9V82 TCR-competition (Baseline-corrected TCR tetramer staining) as calculated by simple linear regression (R2 = 0.136, p=0.1944). Strong and weak TCR blocking designated as lower and upper half of the range of TCR tetramer staining, respectively. TCR inhibition classified based on p values described in (FIG. 3A). (FIG. 3E) Vy9V52 TCR competition with 0.-BTN2A1 Fab or control (20.1 Fab) for binding to BTN2A1 ectodomain assessed by BLI, normalized. Immobilized biotinylated BTN2A1 ectodomain was exposed to 1. 66pM Fab followed by 2. 66pM Fab + 80pM TCR (G115 clone). Step 2 Binding (nm) of TCR to biotinylated BTN2A1 ectodomain in the presence of Fab was normalized to Fab alone binding (nm) at the end of Step 1.
FIGS. 4A-4G show structure of 2A1.9 Fab in complex with BTN2A1 ectodomain provides insight into TCR-competition mechanism of V,y9V62 Antagonism. (FIG. 4A) Structure and surface representation of BTN2A1 ectodomain complexed with antagonistic 2A1.9 Fab as determined by X-ray Crystallography (PDB ID: 8VC7). (FIG. 4B) CFG Face of BTN2A1 ectodomain showing key residues implicated in Vy9V82 activation (Karunakaran, M.M., et al., Immunity, 2020) or TCR binding (Fulford, T.S., et al.,. bioRxiv, 2023: p. 2023.08.30.555639), and docking orientation of 2A1.9 Fab CDR loops. (FIG. 4C) Surface representation of BTN2A1 ectodomain with residues contacting 2A1.9 Fab (orange), Vy9V52 TCR (PDB ID: 8DFW, green) or both (olive) highlighted. (FIG. 4D) Surface representation of BTN2A1 ectodomain with residues contacting 2A1.9 Fab (orange), for Vy9V82 activation as assessed by mutagenesis (red) or both (pink) highlighted. (FIG. 4E- FIG. 4G) Molecular contacts of 2A1.9 Fab with BTN2A1 residues implicated in Vy9V62 activation22 or TCR binding. Highlighted residues of BTN2A1 IgV domain bound to 2A1.9 Fab (light grey) or Vy9V82 TCR (dark grey) are shown. Orientation and molecular interactions of selected BTN2A1 residues (FIG. 4E) Glul07 and Arg96 (FIG. 4F) Tyr98, GlnlOO and Tyrl05 (FIG. 4G) Phe43 and Ser44. HB (distance <3.5 A) and SB (distance <4.5A) depicted in black, vdW (distance <4A) depicted in yellow. If residue contact involves both HB/SB and vdW only HB/SB is shown.
FIGS. 5A-5D show docking orientations of 2A1.4 and 2A1.11 Q-BTN2A1 Fabs on BTN2A1 are similar to 2A1.9. (FIG. 5A) Models of Fabs 2A1.9 (left), 2A1.4 (middle) and 2A1.11 (right) in complex with the BTN2A1 ectodomain dimer fitted into volume maps of 7.4, 7.5, and 6.5A resolution, respectively. Models were generated by fitting the BTN2A1 dimer (PDB ID: 8DFW; Chain A,B) and a muIgGl Fab complexed with an a-Fab elbow nanobody (PDB ID: 7PIJ) into volume maps acquired by single-particle cryo-electron microscopy. (FIG. 5B) Alignment of 2A1.9 and 2A1.4 with 2A1.11 Fab Complex volume maps. (FIG. 5C) Docking angles of models of the 2A1.9, 2A1.4 and 2A1.11 Fabs on the BTN2A1 ectodomain aligned by the BTN2A1 IgV domain with nanobodies and one Fab removed for clarity. Docking angles were calculated using the CA of BTN2A1 Cys97, GlnlOO and Fab heavy -chain Cys22. (FIG. 5D) Alignment of a-BTN2Al mAb CDRH3 loop sequences.
FIG. 6 shows a panel of O-BTN2A1 Fabs generated using phage display selection with the BTN2A1 (2A1 ) ectodomain as the target antigen. Negative selection was carried out using the BTN3A1 (3A1) ectodomain as the target antigen and Fab selectivity was verified using phage-enzyme linked immunosorbent assay (ELISA). FIG. 7 shows affinity of Fabs for BTN2A1 ectodomain as determined by Surface Plasmon Resonance (SPR) with immobilized BTN2A1 ectodomain. Fabs have a range of affinities in the nM-pM range with diverse on and off rates.
FIG. 8 shows fabs were modified into full-length hlgGl-mlgGl chimera antibodies and cellular staining affinity (EC50) was determined on Daudi cells (WT and BTN2A1 KO).
FIG. 9 shows inhibition of activation of Vg9Vd2 T-cells expanded from Peripheral Blood Mononuclear Cells (PBMCs) with Daudi target-cells. Target cells were incubated with mAbs +/- phosphoantigen (pAg) and then coincubated with T-cells. CD107a expression on primary Vg9Vd2 cells was assayed and activation was compared to + (103.2 antagonist mAb) and - (MOPC isotype, Buffer) controls.
FIG. 10 shows a-BTN2Al mAb competition for Vg9Vd2 TCR epitope of BTN2A1 ectodomain. High-Five insect cells expressing full-length BTN2A1 were incubated with saturating concentrations of unlabeled a-BTN2Al mAbs. Cells were washed and stained with the fluorcsccntly-taggcd, tctramcrizcd Vg9Vd2 TCR (G115 clone). TCR staining was assayed by flow cytometry. Experiments were repeated in BTN2A1- cells and no TCR binding was seen.
DEFINITIONS
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the embodiments described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply. As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide” is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “and/or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and/or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and/or C.”
As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
As used herein the term “BTN2A1” has its general meaning in the art and refers to human BTN2A1 polypeptide, such as:
BTN2A isoform 1 precursor (Homo sapiens): ESAAALHFSRPASLLLLLLSLCALVSAQFIVVGPTDPILATVGENTTLRCHLSPEKNAEDE VRFRSQFSPAVFVYKGGRERTEEQMEEYRGRTTFVSKDISRGSVALVIHNITAQENGTYR CYFQEGRSYDEAILHLVVAGLGSKPLISMRGHEDGGIRLECISRGWYPKPLTVWRDPYG GVAPALKEVSMPDADGLFMVTTAVIIRDKSVRNMSCSINNTLLGQKKESVIFIPESFMPS VSPCAVALPIIVVILMIPIAVCIYWINKLQKEKKILSGEKEFERETREIALKELEKERVQKE EELQVKEKLQEELRWRRTFLHAVDVVLDPDTAHPDLFLSEDRRSVRRCPFRHLGESVPD NPERFDSQPCVLGRESFASGKHYWEVEVENVIEWTVGVCRDSVERKGEVLLIPQNGFW TLEMHKGQYRAVSSPDRILPLKESLCRVGVFLDYEAGDVSFYNMRDRSHIYTCPRSAFS VPVRPFFRLGCEDSPIFICPALTGANGVTVPEEGLTLHRVGTHQSL (SEQ ID NO: 63)
As used herein, the term “subject” broadly refers to any animal, including human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.
“Affinity matured antibody” is used herein to refer to an antibody with one or more alterations in one or more CDRs, which result in an improvement in the affinity (i.e., KD, kd or ka) of the antibody for a target antigen compared to a parent antibody, which does not possess the alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. A variety of procedures for producing affinity matured antibodies is known in the art, including the screening of a combinatory antibody library that has been prepared using bio-display. For example, Marks et al., BioTechnology, 10: 779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91: 3809- 3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994- 2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319 (1995); and Hawkins et al, J. Mol. Biol., 226: 889-896 (1992). Selective mutation at selective mutagenesis positions and at contact or hypermutation positions with an activity-enhancing amino acid residue is described in U.S. Patent No. 6,914,128 Bl.
As used herein, the term “antibody” and “antibodies” are used in the broadest sense and refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, antibody fragments, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dualdomain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001/058956, the contents of each of which are herein incorporated by reference), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.
“Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (z.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodics, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.
“Bispecific antibody” is used herein to refer to a full-length antibody that is generated by quadroma technology (see Milstein et al., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628- 631 (1985)), or by knob-into-hole or similar approaches, which introduce mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14): 6444-6448 (1993)), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC). By this definition, a bispecific antibody has two distinct antigen-binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds to.
“CDR” is used herein to refer to the “complementarity determining region” within about an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted "CDR1", "CDR2", and "CDR3", for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen-antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues a e directly and most substantially involved in influencing antigen binding.
The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as "Kabat CDRs". Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as "LI", "L2", and "L3", or "Hl", "H2", and "H3", where the "L" and the "H" designate the light chain and the heavy chain regions, respectively. These regions may be referred to as "Chothia CDRs", which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995), and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat- or Chothia-defined CDRs.
“Epitope,” or “epitopes,” or “epitopes of interest” refer to a site(s) on any molecule that is recognized and can bind to a complementary site(s) on its specific binding partner. The molecule and specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, glycolipids, glycoproteins or lipopolysaccharides), or a polysaccharide. Its specific binding partner can be, for example, an antibody.
The terms “fragment antigen-binding fragment” or “antigen binding fragment” or “Fab” or “Fab fragment” are used interchangeably herein to refer to a fragment of an antibody that binds to antigens and that contains one antigen-binding site, one complete light chain, and part of one heavy chain. Fab is a monovalent fragment consisting of the VL, VH, CL and CHI domains. Fab is composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain contains the paratope (the antigen-binding site), comprising a set of complementarity determining regions, at the amino terminal end of the monomer. Each arm of the Y thus binds an epitope on the antigen. Fab fragments can be generated such as has been described in the art, e.g., using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant means.
“F(ab')2 fragment” as used herein refers to antibodies generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa. Divalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules and enable a better penetration into tissue thus facilitating better antigen recognition in immunohistochemistry. The use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A/G. F(ab')2 fragments can both bind and precipitate antigens.
“Framework” (FR) or “Framework sequence” as used herein may mean the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems (for example, see above), the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3, or FR4, a framework region, as referred by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
Human heavy chain and light chain FR sequences are known in the art that can be used as heavy chain and light chain "acceptor" framework sequences (or simply, "acceptor" sequences) to humanize a non-human antibody using techniques known in the art. In one embodiment, human heavy chain and light chain acceptor sequences are selected from the framework sequences listed in publicly available databases such as V-base (hypertext transfer protocol://vbase.mrc-cpe. cam.ac.uk/) or in the international ImMunoGeneTics® (IMGT®) information system (hypertext transfer protocol://imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/).
“Humanized antibody” is used herein to describe an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like,” i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or a variant, derivative, analog, or fragment thereof, which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In an embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.
A humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including without limitation IgGl, IgG2, IgG3, and IgG4. A humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the ail.
The framework regions and CDRs of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion, and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In a preferred embodiment, such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). A "consensus immunoglobulin sequence" may thus comprise a "consensus framework region(s)" and/or a "consensus CDR(s)". In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.
“Identical” or “identity,” as used herein in the context of two or more polypeptide or polynucleotide sequences, can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.
“Label” and “detectable label” as used herein refer to a moiety attached to an antibody or an analyte to render the reaction between the antibody and the analyte detectable, and the antibody or analyte so labeled is referred to as “detectably labeled.” A label can produce a signal that is detectable by visual or instrumental means. Various labels include signal-producing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. In some embodiments, the moiety itself may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.
Any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, U lin, 1251, 1311, 177Lu, I66H0, and 153Sm), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6- carboxyfluorescein, 3 ’6-carboxy fluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immuno-polymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. A fluorescent label can be used in FPIA (see, e.g., U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entireties). An acridinium compound can be used as a detectable label in a homogeneous chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Let. 14: 3917-3921 (2004); and Adamczyk et al., Org. Let. 5: 3779-3782 (2003)).
As used herein, the term “sample” is used in the broadest sense and is inclusive of many sample types that may be obtained from a subject. Samples may be obtained from animals (including humans) and encompass fluids (e.g. urine, blood, blood products such as plasma and serum, sputum, saliva, etc.), cells, solids, tissues, and gases.
DETAILED DESCRIPTION
Butyrophilins constitute a family of transmembrane proteins comprising butyrophilin (BTN), BTN-like (BTNL), and selection and upkeep of intraepithelial T cell (SKINT) proteins. Their extracellular moieties contain IgV and IgC2 domains exhibiting homology to the corresponding domains of B7 co- stimulatory molecules, and butyrophilins are thus considered to be members of the extended B7 or 1g superfamily.
The anti-tumor response of Vy9V82 T cells involves the sensing of accumulated phosphoantigens (pAgs). Vy9V62s are triggered by intracellular increases in the concentration of phosphoantigen metabolite (pAg) byproducts of isoprenoid biosynthesis in target cells. Vy9V82s do not employ canonical T cell antigen-recognition as target cells do not directly present pAgs to Vy9V32s via the Major Histocompatibility Complex (MHC). Rather, pAg- accumulation is indirectly signaled to Vy9V82s through heterodimers of Butyrophilin (BTN)3A1 and its isoforms BTN3A2 and BTN3A3 by a mechanism that is not fully defined. Indeed, the cellular and molecular dynamics of how intracellular pAg- BTN3A1 binding is translated through the juxtamembrane regions of BTN3A2 or BTN3A3 and coupled to extracellular BTN2A1-TCR binding to stimulate Vy9V82 activation remains to be determined. BTN3A has been explored as an immunotherapy adjuvant but a lack of understanding about the mechanism by which it mimics pAg-induced Vy9V82 activation hinders its development. For BTN2A1, the available antibody reagents are limited and their access challenging, restricting the ability to fully probe the role of BTN2A1 in Vy9V82 biology. To address these challenges, O-BTN3A antibodies and a-BTN2Al antibodies were engineered to modulate and gain deeper insight into the cellular organization and molecular architecture of the pAg-signaling complex. Provided herein are antibodies that bind a-Butyrophilin2Al (BTN2A1), and diagnostic, therapeutic and research methods of use thereof. In particular, BTN2A1 antibodies are provided with substantially no cross -reactivity with a-Butyrophilin3Al (BTN3A1) (e.g. antibodies that bind specifically to BTN2A1), and in certain embodiments exhibiting BTN2A1 antagonism and/or inhibition of Vy9V82 T-cell activation.
BTN2A1 plays a role in the cytotoxic response of primary V 9V82 T cells against cancer cells. BTN2A1 gene polymorphisms have been associated with multiple human diseases, including myocardial infarction, metabolic syndrome, hypertension, dyslipidemia, and hepatitis C. Experimental evidence indicated the involvement of V 9V82 T cells in infectious diseases and cancer. Vy9V62 T cells have been reported to be dysregulated is several autoimmune and inflammatory diseases. As such, the antibodies and antibody fragments herein may find use in the diagnosis, treatment, and prevention of any of the aforementioned conditions and/or other BTN2A1 -related disorders. Additionally, the BTN2A1 antibodies and antibody fragments herein may find use as a research or drug development tool, for example, to identify anti-cancer therapies, drugs useful for the treatment of BTN2A1 -related disorders, and/or to study BTN2A1 signaling or BTN2A1 -related disorders.
In some embodiments, the BTN2A1 antibodies and antibody fragments herein are capable of inhibiting production of IFN-y and TNF-a by activated Vy9/V82 T cells. In some embodiments, the BTN2A1 antibodies and antibody fragments herein are capable of inhibiting cytolytic function of activated Vy9/V82 T cells, and/or the proliferation of activated Vy9/V62 T cells.
In some embodiments, the BTN2A1 antibodies and antibody fragments herein bind to BTN2A1 without cross-reactivity with BTN3A1.
In some embodiments, the BTN2A1 antibodies and antibody fragments herein exhibit antagonistic properties relative to the activity of BTN2A1.
In some aspects, provided herein are antibodies. In some embodiments, provided herein are antibodies that bind to BTN2A1. In some embodiments, provided herein are antibodies that differentiate between BTN2A1 and other butyrophilins (e.g., BTN3A1) . Such antibodies are referred to herein are antibodies that “bind specifically” to peptide- BTN2A1.
In some embodiments, the antibodies comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61, and a heavy chain comprising at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
In some embodiments, provided herein arc antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 33 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 34
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 36.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 37 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 38.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 39 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 40. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 41 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 42.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 43 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 44.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 46.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 48.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 49 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 50.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 51 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 52.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 53 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 54.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 56.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein arc antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 58.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 59 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 60.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 61 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 34. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain set forth in SEQ ID NO: 33 and a heavy chain set forth in SEQ ID NO: 62.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a light chain variable region and a heavy chain variable region. In some embodiments, the antibodies comprise a heavy chain variable region comprising one or more CDRs set forth in Table 1. In some embodiments, the antibodies further comprise a light chain variable region comprising a CDR set forth in Table 1. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a CDR-H1, CDR-H2, and CDR-H3 set forth in Table 1. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a CDR- Hl, CDR-H2, and CDR-H3 set forth in Table 1 and a light chain comprising an amino acid sequence having at least 80% sequence identity (e.g. at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at last 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) with SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61. In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise a CDR-L3, CDR-H1, CDR-H2, and CDR-H3 set forth in Table 1.
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.1 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.1 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.2 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.2 (See Table 1). In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.3 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.3 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.4 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.4 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.5 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.5 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.6 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.6 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.7 (Sec Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.7 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.8 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.8 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.9 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.9 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.10 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.10 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.11 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.11 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.12 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.12 (See Table 1). In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.13 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.13 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.14 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.14 (See Table 1).
In some embodiments, provided herein are antibodies that bind to BTN2A1 that comprise the CDR-H1, CDR-H2, and CDR-H3 of Fab 2A1.15 (See Table 1). In some embodiments, the antibody comprises the CDR-L3 of Fab 2A1.15 (See Table 1).
In some embodiments, the antibody further comprises a detectable label. The detectable label may be any suitable detectable label.
Antibodies may be prepared by any of a variety of techniques. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies via conventional techniques, or via transfection of antibody genes, heavy chains, and/or light chains into suitable bacterial or mammalian cell hosts, in order to allow for the production of antibodies. Antibodies can be expressed in either prokaryotic or eukaryotic host cells. However, expression of antibodies in eukaryotic cells is preferable, and most preferable in mammalian host cells. Exemplary mammalian host cells for expressing the recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77 : 4216-4220 (1980)), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NSO myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard protein purification methods. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in a process to purify the antibodies. The proteolytic enzyme papain may be used to preferentially cleave IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)i fragment, which comprises both antigen-binding sites.
Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure may be performed. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies. In addition, bifunctional antibodies may be produced.
Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g., as available from various commercial vendors such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martin sreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternative methods rely upon immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161) that are capable of producing a repertoire of human antibodies, as known in the art and/or as described herein. Such techniques, include, but are not limited to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody producing technologies (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052, Wen et al. (1987) J. Immunol. 17:887- 892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol. 13:787-790); B- cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).
An affinity matured antibody may be produced by any one of a number of procedures that are known in the art. For example, see Marks et al., BioTechnology, 10: 779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas et al., Proc. Nat. Acad. Set. USA, 91: 3809- 3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155: 1994- 2004 (1995); Jackson et al., J. Immunol., 154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896 (1992). Selective mutation at selective mutagenesis positions and at contact or hypermutation positions with an activity enhancing amino acid residue is described in U.S. Patent No. 6,914,128 Bl.
In some aspects, provided herein are compositions comprising an antibody described herein. In some aspects, provided herein are kits. In some embodiments, the kit comprises at least one antibody or a composition comprising an antibody as described herein. In some embodiments, the kit comprises an antibody described herein (e.g. an antibody that binds BTN2A1), along with instructions for use of the kit. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they arc not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions. In some embodiments, the kit comprises additional reagents for performing an assay to detect one or BTN2A1, including containers (e.g. tubes, microtiter plates, strips, etc.), buffers, stabilizers, preservatives, controls, and the like.
In some embodiments, the antibodies, compositions, and kits comprising the same find use in methods for detecting BTN2A1 in a sample. In some embodiments, provided herein is a method for detecting BTN2A1 in a sample. The method comprises contacting the sample with an antibody described herein. In some embodiments, the antibody is a Fab. In some embodiments, the antibody is detectably labeled. The method may comprise contacting the sample with an antibody described herein, and detecting a signal from the detectable label. The signal from the detectable label may be indicative of the presence and/or amount of BTN2A1 in the sample.
In some embodiments, the method detects BTN2A1 in the sample, but does not detect a BTN3 polypeptide or BTN3A1. In some embodiments, the method detects BTN2A1 in the sample, but does not detect a BTN2A2. Accordingly, in some embodiments the methods may be used to differentiate between BTN2A1 and other related polypeptides in a sample.
The antibodies described herein additionally find use in various diagnostic and therapeutic methods. The antibodies herein have in vitro and in vivo diagnostic and therapeutic utilities. For example, these antibodies can be administered to cells (e.g., in culture (in vitro or in vivo) or in a subject (e.g., in vivo)) to treat, prevent or diagnose a variety of disorders. The methods are particularly suitable for treating, preventing or diagnosing BTN2A1 -related disorders and/or autoimmune, inflammatory disorders, and transplant rejection.
As used herein, a “BTN2A1 -related disorder” includes conditions associated with or characterized by aberrant BTN2A1 levels and/or diseases or conditions that can be treated by modulating BTN2A1 induced signaling activity in a subject (e.g., in human blood cells), for example, by inhibiting the production of IFNy or TNFa of activated of Vy9V52 T cells and/or the cytolytic function of activated Vy9V52 T cells. These include inflammatory conditions, autoimmune diseases and organ or tissue transplant rejection.
Examples of autoimmune diseases which may be treated include but are not limited to rheumatoid arthritis (RA), insulin dependent diabes mellitus (Type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, pemphigus vulgaris, pemphigoid, addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, coeliac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic leucopenia, idiopathic thrombocytopenic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, phacogenic uveitis, primary biliary cirrhosis, primary myxoedema, Reiter's syndrome, stiff man syndrome, thyrotoxicosis, ulcertitive colitis, and Wegener's granulomatosis.
The antibodies of the invention may be administered as the sole active ingredient or in conjunction with, e.g. as an adjuvant to or in combination to, other drugs e.g. immunosuppressive or immunomodulating agents or other anti-inflammatory agents, e.g. for the treatment or prevention of diseases mentioned above.
An object of the present invention relates to a method of inhibiting an immune response in a subject, in particular inhibiting the cytolytic property of Vy9V82 T cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody described herein.
The disclosure also pertains to the methods of manufacturing a medicament for use in the treatment of BTN2A1 -related disorders (e.g., inflammatory conditions, autoimmune diseases and organ or tissue transplant rejection), said medicament comprising an anti-BTN2Al antibody of the present disclosure as described herein.
In some embodiments, provided herein is a method of diagnosing a BTN2A1 -related disorder (e.g., inflammatory conditions, autoimmune diseases and organ or tissue transplant rejection) in a subject. The method comprises contacting a sample obtained from the subject with an antibody described herein. For example, the method may comprise contacting the sample with an antibody that binds to BTN2A1 described herein.
In some embodiments, the antibodies may be conjugated to a therapeutic agent for use in treating one or more conditions in a subject (e.g., a BTN2A1 -related disorder). For example, an antibody-drug conjugate comprising an antibody described herein conjugated to an immunosuppressive or immunomodulating agents or other anti-inflammatory agents be useful for treating a BTN2A1 -related disorder in a subject.
The antibodies or antibody-drug conjugates described herein may be administered to the subject by any suitable route. The antibody or antibody-drug conjugate may be formulated into a suitable composition for administration to the subject. The formulation of the composition (e.g. liquid or solid) may depend on the intended route of administration. The composition may comprise additional reagents including pharmaceutically acceptable excipients such as buffers, stabilizers, preservatives, and the like.
The antibody, antibody-drug conjugate, or the composition comprising the same may be provided (e.g. administered) to the subject by any suitable method. Suitable routes of administrating the composition described herein include, for example, topical, parenteral (e.g. by injection), and oral forms of administration. In some embodiments, the composition is administered parenterally, such as by subcutaneous, intramuscular, intraarterial, or intravenous injection.
The antibodies or antigen-binding domains thereof described herein may be used in cellbased therapies. For example, the antibodies or antigen-binding domains thereof may be used in methods of adoptive cell therapy, including tumor infiltrating lymphocyte (TIL), T-cell receptor (TCR), and chimeric antigen receptor (CAR)-based cell therapies. In some embodiments, the antibodies described herein may be used as engagers for T and NK-cell based therapies. As used herein, the term “engager” refers to an antibody or antibody fragment that enhances interactions between a T-cell or an NK cell and tumor cells. Accordingly, an “engager” may enhance anticancer activity of a T-cell or an NK cell. For example, an antibody fragment set forth in Table 1 may be used as an engager for NK cell or a T-cell based immunotherapy.
For example, the antigen binding domains of the antibodies described herein may be incorporated into a chimeric antigen receptor (CAR). The term “CAR”, or “chimeric antigen receptor” used interchangeably herein to refer to engineered receptors which may be grafted onto immune cells, including T- cells (CAR-T cells) or natural killer cells (CAR-NK cells). In some embodiments, the immune cells are a T cell (including CD4+T cell, CD8+T cells) a B cell, a natural killer (NK) cell, a natural killer T cell (NKT) cell, a monocyte cell or a dendritic cell.
A chimeric antigen receptor typically comprises an extracellular domain able to bind an antigen (i.c. an antigen binding domain), a transmembrane domain, optionally a hinge domain, and at least one intracellular domain. In some embodiments, the antigen binding domain comprises an oligopeptide or polypeptide that binds to a target antigen. The antibodies or fragments thereof (i.e. antigen binding portions thereof) may be used as the antigen binding domain in a CAR, including for use in a CAR-T cell or a CAR-NK cell. For example, in some embodiments provided herein is a chimeric antigen receptor comprising a Fab fragment set forth in Table 1. The term “transmembrane domain” means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signaling domains. This may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Typically, the transmembrane domain denotes a single transmembrane alpha helix of a transmembrane protein, also known as an integral protein. A chimeric antigen receptor may optionally comprise a “hinge domain” which serves as a linker between the extracellular and transmembrane domains. As used herein the terms “hinge”, “spacer”, or “linker” refers to an amino acid sequence of variable length typically encoded between two or more domains of a polypeptide construct to confer for example flexibility, improved spatial organization and/or proximity. The terms “intracellular domain”, “internal domain”, “cytoplasmic domain” and “intracellular signaling domain” are used interchangeably herein and mean any oligopeptide or polypeptide known to function as a domain that transmits a signal that causes activation or inhibition of a biological process in a cell.
In some embodiments, an immune cell comprising a chimeric antigen receptor described herein (e.g. CAR-T cells, CAR-NK cells) may be particularly useful for immunotherapy, such as including for treating a BTN2A1 -related disorder.
SEQUENCES
The sequences of the Fabs are as follows: Table 1. BTN2A1 Fab CDRs
Figure imgf000036_0001
Figure imgf000037_0001
Fab 2A1.1
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNLYSSSIHWVRQAPGKGLEWVASIYSSSGY TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYYQRGYIYSGFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 34) Fab 2A1.2
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQASDWPITFGQGTKVEIKRTVAAPSVFIFPPS
DSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSYSSIHWVRQAPGKGLEWVASIYSYYGS
TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYYARGYVYAFDYWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ
ID NO: 36)
Fab 2A1.3
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQASDWPITFGQGTKVEIKRTVAAPSVFIFPPS
DSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 37)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNIYSSSIHWVRQAPGKGLEWVASIYSYSGY
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYYYWGGVGDALDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
(SEQ ID NO: 38) Fab 2A1.4
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASIYSSSGY TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARWQSYRGYPWPGFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 40)
Fab 2A1.5
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASISSSSGST
SYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGHLYWEYVQWGFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 42) Fab 2A1.6
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD
SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 43)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASISSSSGST
SYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGHYYYWYYPGYGFDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 44)
Fab 2A1.7
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD
SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 45)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVAYIYPSSGS
TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYYYRGYIDAMDYWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 46) Fab 2A1.8
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD
SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 47)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSYIHWVRQAPGKGLEWVASIYSYYGS
TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARETVYKGYVPALDYWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 48)
Fab 2A1.9
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD
SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 49)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNLYSSSIHWVRQAPGKGLEWVAYIYPSSG
YTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYYYTRGYPDGMDYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 50) Fab 2A1.10
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 51)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASISSSSGST SYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARWFYERGYVWAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ
ID NO: 52)
Fab 2A1.11
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 53)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASISSSSGST SYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGHTYWMYYSGWGMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 54) Fab 2A1.12
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 55)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASIYSSSGY TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARIEYGRGYWDAFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ
ID NO: 56)
Fab 2A1.13
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSQGELITFGQGTKVEIKRTVAAPSVFIFPPS DSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 57)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASISSSSGST
SYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGHYYYYYYSGWGFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 58) Fab 2A1.14
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSKYMLITFGQGTKVEIKRTVAAPSVFIFPPS DSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 59)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSYSSIHWVRQAPGKGLEWVASISPYSSS TSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGHVYYHYYPGYGMDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH T (SEQ ID NO: 60)
Fab 2A1.15
Light chain:
SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSSSLITFGQGTKVEIKRTVAAPSVFIFPPSD SQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 61)
Heavy chain:
EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASISSSSGST SYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGHQYYYYYGGWGFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
(SEQ ID NO: 62) EXAMPLES
Example 1
Target cells trigger Vy9V52 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vy9V52 T cell receptor (TCR). However, an incomplete understanding of the molecular dynamics in this signaling complex hampers Vy9V82 T cell immunotherapeutic efficacy. Herein, a panel of engineered a-BTN3Al and a-BTN2Al antibody (mAb) reagents was used to probe the roles of BTN3A1 and BTN2A1 in pAg signaling. Modified a-BTN3Al mAbs with increased inter-Fab distances establish that tight clustering of BTN3A1 is not necessary to stimulate Vy9V82 T cell activation, and that antagonism may occur through occlusion of a binding interaction between BTN3A1 and a yet unknown co-receptor. A panel of additional a- BTN2A1 antagonists utilize different biophysical mechanisms to compete with Vy9V62 TCRs for BTN2A1 binding. The complex structures of BTN2A1 ectodomain and Fabs from three antagonist antibodies provide molecular insights into BTN2A1 epitopes involved in pAg- signaling.
Methods
Protein Expression and Purification
Fabs
U-BTN2A1 Fabs with a human-IgGl Herceptin scaffold were cloned into the RH2.2 vector. Fab Heavy and Light chains were on the same plasmid. Fab overexpression was induced in Escherichia coli BL21 Gold (DE3) cells in 2xYT media using ImM isopropyl p-d-1- thiogalactopyranoside (IPTG) for 4 hr at 37C. Bacterial cells were centrifuged, resuspended and lysed by homogenization and sonication. Bacterial lysate was spun at 20,000 x g for 45’. Fabs were purified from the periplasmic fraction with Protein Gal or G-F resin (Kossiakoff Lab, U.Chicago), eluted with 0.1 M Glycine pH 2.3 and neutralized with a 1:5 volume ratio of 1 M Tris Buffer pH 8.0. Fabs were then dialyzed into Phosphate Buffered Saline (PBS). 0.-BTN3A1 Fab 20.1 and 103.2 Heavy and Light chains with a hybrid murine-IgGl Fab IgV and human-IgGl Herceptin Fab IgC scaffold were cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site, acid- and basic-zipper, respectively, and hexa-histidine tag. Baculovirus was generated by transfection of plasmid and linearized baculovirus DNA into Sf9 insect cells using Cellfectin transfection reagent. Baculovirus was then added to High-Five insect cells and proteins were expressed for 60-68 hrs at 27°C. Supernatant was isolated by centrifugation at 1700 rpm for 15’ and filtered through glass-fibre. Proteins were purified using Protein-G resin, eluted with 0.1 M Glycine pH 2.3 and neutralized with a 1:5 volume ratio of 1 M Tris Buffer pH 8.0. Fabs were then dialyzed into Phosphate Buffered Saline (PBS). mAbs a-BTN2Al mAb sequences with a hybrid human-IgGl Herceptin Fab IgV and murine- IgGl IgC-Hinge-Fc scaffold were cloned into the AbVec vector. Heavy and Light chains were on separate plasmids. U-BTN3A1 mAbs with a murine-IgGl scaffold were cloned into the AbVec vector. MAbs were expressed in Expi293 cells at 37C using the ExpiFectamine™ transfection system with 0.5 pg each of Heavy and Light chain plasmids per 1 mL of culture. 7 days after transfection, supernatant was isolated by centrifugation at 3000 x g for 10’. mAbs were then purified using Protein G or Protein Gal resin, eluted with 0.1 M Glycine pH 2.3 and neutralized with a 1:5 volume ratio of 1 M Tris Buffer pH 8.0. MAbs were then dialyzed into PBS.
BTN2A1 ectodomain, BTN2A1 ectodomain C219S
BTN2A1 ectodomain (residues 1-219) and BTN2Al ectodomain C219S (residues 1 -219) were cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site and hexa-histidine tag. Proteins were expressed in High-Five insect cells with the baculovirus expression system. Proteins were purified using Ni-NTA resin and eluted with Hanks Buffered Saline (HBS) + 200 mM and 500 mM imidazole. Proteins were de-glycosylated for 2 hrs at 37C using Endo-F3 in HBS + 15mM imidazole at a concentration of ~1 mg/mL. Proteins were repurified using Ni-NTA resin and HBS + 200 mM imidazole elution to remove Endo-F3 and incubated with 3C protease overnight in HBS + 75 mM imidazole for the removal of the hexa-histidine tag. Proteins were purified further using anion-exchange chromatography over the MonoQ column in 20 mM Tris pH 8.0 with a 20 mL gradient of 30-700 mM NaCl. Proteins were subsequently utilized for structural determination.
Biotinylated BTN2A1 ectodomain + avitag and BTN3A1 ectodomain + avitag
BTN2A1 ectodomain (residues 1-217) and BTN3Al ectodomain (residues 1-217) were cloned into the pAcGP67a Vector containing a C-terminal BirA biotinylation sequence, human rhinovirus 3C protease cleavage site and hexa-histidine tag. Proteins were expressed in High- Five insect cells with the baculovirus expression system. Proteins were purified using Ni-NTA resin. Proteins were then incubated with 3C protease overnight in HBS + 75mM Imidazole for the removal of the hexa-histidine tag. Proteins were buffer-exchanged to HBS + 15 mM Imidazole with 0.05 M bicine buffer pH 8.3, 10 mM ATP, 10 mM MgOAc and 50 pM d-biotin and biotinylated overnight using the BirA protein. Proteins were then purified using sizeexclusion chromatography (SEC) over the S200 column in HBS and verified for biotinylation by size-shift on an SDS-PAGE gel following coincubation with Traptavidin.
G115 and DP 10.7 TCRs
The y chain of the G115 or DP-alpha constant-region TCR was cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site, acidic- zipper and hexa-histidine tag. The 8 chain of the G115 or DP-beta constant-region TCR was cloned into the pAcGP67a Vector containing a C-terminal BirA biotinylation sequence, human rhinovirus 3C protease cleavage site, basic-zipper and hexa-histidine tag. TCRs were expressed in High-Five insect cells with the baculovirus expression system. TCRs were purified using Ni- NTA resin and eluted. TCRs were then incubated with 3C protease overnight in HBS and 75 mM Imidazole for the removal of the hexa-histidine tag. For TCR being used for tetramerization, TCR was buffer exchanged to HBS + 15 mM Imidazole HBS + 15 mM Imidazole with 0.05 M bicine buffer pH 8.3, 10 mM ATP, 10 mM MgOAc and 50 pM d-biotin and biotinylated overnight using the BirA protein. Unbiotinylated and biotinylated TCR were then purified using SEC over the S200 column in HBS and verified for biotinylation by size-shift on an SDS-PAGE gel following coincubation with Traptavidin, if applicable. Biotinylated TCR was tetramerized by co-incubation with Streptavidin-PE at a ratio of 1 : 1.1 streptavidin monomer to biotinylated TCR monomer.
Nanobody
Histidine-tagged anti-Fab nanobody (Ereno-Orbea, J., et al.. J Mol Biol, 2018. 430(3): p. 322-336) was expressed in E. coli BL21 (DE3) cells overnight at 20°C post induction with ImM IPTG at OD600 = 0.6-0.8. The cells were harvested by sonication in 25 mM TRIS, pH 8.0, 300 mM NaCl and 10% glycerol. After centrifugation, the supernatant was passed over a lOmL HisTrap HP column and eluted with 25 mM TRIS, pH 8.0, 300 mM NaCl, 150 mM imidazole and 10% glycerol. The protein was further purified by SEC in 20 mM HEPES, pH 7.4, and 200 mM NaCl.
Phage Display Selection
To obtain high-affinity binders, five rounds of selection were performed using phage display selection. Biotinylated BTN2A1 was immobilized onto streptavidin-coated paramagnetic beads for five rounds of phage selection. In the first round, 1 pM of BTN2A1 was immobilized on 200 pl SA magnetic beads and was incubated with 1 mL phage library (1010 CFU) for 1 hour at room temperature with gentle shaking. The beads were washed three times to remove nonspecific phage, added to log phase E. coli XL-1 blue cells and incubated for 20 minutes at room temperature. Then, media containing 100 pg/mL ampicillin and 10 9 p.f.u./mL of M13K07 helper phage was added for overnight phage amplification at 37°C. The amplified phage was precipitated in 20% PEG/2.5 M NaCl for 20 minutes on ice for subsequent rounds. Before each round, the phage pool was negatively selected against empty paramagnetic beads for 30‘ with shaking to eliminate nonspecific binders. The final antigen concentration was dropped systematically from 1 pM to 10 nM from the first to the fifth round (2nd round: 200 nM, third round: 50 nM, fourth round 20 nM, and fifth-round 10 nM). After phage binding, the beads were subjected to five washing rounds with 0.5% BSA/PBST. The bound phages were eluted using 0.1 M Glycine, pH 2.6, and neutralized with TRIS-HC1, pH 8. Then, the phage eluate was used for E. coli infection and phage amplification, as described above. Additional selection pressure using 1 pM of not biotinylated BTN3A1 in all washes was applied to ensure specificity to BTN2A1. After the fourth and fifth rounds the infected cells were plated on ampicillin agar and 192 colonies were picked to produce phage clones for single-point phage ELISA assay. The promising clones demonstrating high specificity were sequenced and reformatted into a RH2.2 expressing vector.
Affinity Analyses
Cellular Affinity
For determination of 0.-BTN2A1 mAb cellular affinity, Daudi cells were resuspended in PBS + 2% FBS and incubated with increasing concentrations of U-BTN2A1 mAb. ct-BTN2Al mAbs were stained with 1:200 fluorescently-tagged secondary mAb and staining was assessed by flow-cytometry.
Enzyme-Linked Immunosorbent Assays (ELISA)
Single-point phage ELISA was used to test specificity of O-BTN2A1 Fabs for BTN2A1 binding. 50 nM of BTN2A1 or BTN3A1 protein was directly immobilized on high-binding experimental wells for 30', followed by extensive blocking with 2% BSA for 1 hour. After 15' of incubation with phage, the wells were extensively washed three times with 0.5% BSA/PBST and incubated with Protein L-HRP (1:5000 dilution in HBST) for 20’. The plates were again washed and developed with TMB substrate and quenched with 10% H3PO4, followed by the absorbance at A450 determination.
BLI
For 20.1 and 103.2 engineered variants relative affinity determination, biotinylated BTN3A1 ectodomain in HBS was immobilized on Streptavidin biosensor tips until Binding reached between 3 and 4 nm. Tips were blocked with 1 pM biotin for 30”. After baseline was established in PBS for 30”, the biosensor tip was exposed to 1.3 pM mAb for 90”. Dissociation in PBS was then assessed for 120”.
For a-BTN2Al Fab competition with the Vy9V82 TCR experiments, biotinylated BTN2A1 ectodomain in HBS was immobilized for 150” on Streptavidin biosensor tips. Tips were blocked with 1 pM biotin in HBS for 30s followed by 2 mg/mL BSA for 180”. The biosensor tip was exposed to 66 pM a-BTN2Al Fab concentration for 120”. Tips were immediately placed into 66 pM U-BTN2A I Fab + 80 pM Vy9V62 TCR for 120”, in HBS. Dissociation in HBS was then assessed for 120”.
Surface Plasmon Resonance Analysis
All Surface plasmon resonance (SPR) analyses were performed on a MASS-1. BTN2A1 was immobilized via a 6x His-tag to a Ni-NTA sensor chip. Fabs in two-fold dilutions were run as analytes at 30 pl/min flow rate at 20°C. Sensograms were corrected through double referencing, and a 1 : 1 binding model was fit using Sierra Analyzer.
Cell-Lines
The y and 8 chain of the G115 Vy9V82 TCR clone were cloned into the pMSC-V vector with puromycin and zeocin selection genes, respectively. Jurkat JRT3.3 cells were transfected with these plasmids and selected first for y chain expression for 7-14 days using 1 mg/mL puromycin. Cells were then selected for ~4 weeks with 200 mg/mL zeocin. Cells were stained with a-y9 and a-82 antibodies and sorted on TCR expression at both 1 and 4 weeks. Daudi-Cas9 AAAVS1, ABTN2A1, ABTN3A1 cell lines were generated as described (Mamedov, M.R., et al., Nature, 2023. 621(7977): p. 188-195). Daudi-Cas9 AAAVS1 were generated with a CRISPR guide RNA targeting AAVS1, a safe harbor site that acted as a CRISPR cutting control. JRT3.3 Jurkat cells expressing the G115 TCR, DaudiAAAVS1, DaudiA2A1, and DaudiA3A1 were cultured in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 10 U/mL Penicillin/Streptomycin (R10). Cells were split to 0.3E6 cells/mL daily and split 24 hours prior to the start of an activation assay.
Isolation and Expansion oj V 9Vd2 T cells from Peripheral Blood
Peripheral blood was isolated from healthy donors through the IRB 13-0666 protocol and subjected to density gradient centrifugation using Ficoll. Lymphocytes were isolated and brought to 1E6 cells/mL in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 0.1% P-me, 0.5X non-essential amino acids (Coming), 1 mM sodium pyruvate and 10 U/mL Penicillin/Streptomycin (R10+). Cells were pulsed with 5 pM Zoledronate and 100 U/mL IL-2 and incubated at 37°C. 100 U/mL IL-2 was supplemented into the media every 2-3 days and the culture volume was doubled on day 6 with fresh R10+ media supplemented with 100 U/mL IL-2. One day prior to use in activation assays, 100 U/mL IL-2 was pulsed into the media. Vy9V82 T cells were utilized on Day 9-10 for activation assays and assayed via flow-cytometry for expansion at such time.
Agonism/ Antagonism of Vy9V32 T cells
In 96-well round-bottom plates with edge-wells containing 200 pL PBS, 0.05E6 Daudi target cells were incubated with PBS, mAb, or Fab +/- HMBPP, Pamidronate or MOPC isotype mAb in RIO for 2 hours in 100 pL. Target cells were washed with 200 pL warm PBS and centrifuged at 300 x g for 5' 3 X and co-incubated with 0.1E6 Jurkat or Primary Vy9V52 T cells per condition overnight in 200 pL R10 or R10+, respectively. 1.6 pg/mL PHA was added to wells with T cells alone prior to overnight incubation as a positive control for activation. T Cell activation was assessed via flow-cytometry.
Cellular mAb :TCR Competition for BTN2A1 Binding Assay
Full-length BTN2A1 was cloned into the pAcGP67a Vector containing a C-terminal human rhinovirus 3C protease cleavage site and hexa-histidine tag. BTN2A1 was expressed in High-Five insect cells with the baculovirus expression system. BTN2A1 -expressing Insect cells were seeded at 1.5E5 cells/well in 96-well round-bottom plates with edge- wells containing 200 pL PBS and incubated with 150nM a-BTN2Al mAb for 30’. Insect cells were washed with 200 pL PBS and centrifuged at 300 x g for 5’. Insect cells were then sequentially stained with DAPI followed by additional washes and stained with 120 nM G115 tetramer labeled with the PE- fluorophore. MAb:tetramer competition was assessed via flow-cytometry.
Flow Cytometry
For Jurkat T cell activation assays, target and T cells from each condition were resuspended in PBS + 2% FBS. Cells were stained with Live-Dead fluorescent dye, ct-CD19 to gate target-cells, ct-Vy9 to gate T cells and a-CD69 fluorophore-conjugated mAbs as a marker for T cell activation. For Primary T cell activation assays, target and T cells from each condition were stained with 1:1500 dilution Live/dead Fixable Near-IR Dead Cell Stain, 1:50 dilution of a- Vy9, a-V52 to gate T cells, and 1:50 dilution of a-CD25, a-CD107a and a-CD69 fluorophore- conjugated mAbs as markers for activation. For mAb and TCR-tetramer competition, cells were resuspended in PBS + 2% FBS and stained with DAPI and fluorescently-labeled G115 TCR tetramers. Data were collected on either the Aurora or AttuneNxt.
X-Ray Crystallography
Sample Preparation
De-glycosylated and purified BTN2A1 ectodomain C219S was mixed with purified Fab at a 1:1 ratio. Complexes were concentrated and purified over SEC using the S200 column in HBS. Complexes were then concentrated to 6.9mg/mL and tested for crystallization using the Morpheus I protein crystallization screen in 3-well sitting drop plates. Crystallization conditions from well G9 were optimized to 0.1 M Buffer System 3, pH 8.3 with 0.1 M Carboxylic Acids and 20% Precipitant mix 1. Optimized crystals were cryoprotected with 20% Ethylene Glycol in 0.1 M Buffer System 3, pH 8.3 with 0.1 M Carboxylic Acids and 25% Precipitant mix 1 and flash-frozen in Liquid Nitrogen.
Data Collection and Processing
X-ray datasets were collected at the Stanford SSRL beamline 14-1 on a Dectris Pilatus 6M at 100K. A complete dataset was collected at a wavelength of 1.1950E-10 m. XDS, truncate, pointless, freeR, and aimless were used for data reduction and scaling. An initial molecular replacement solution was obtained using PHASER through Phenix (Liebschner, D., et al., Section D, Structural biology, 2019. 75: p. 861-877) with PDB ID code 8DFW (Fulford, T.S., et al., bioRxiv, 2023: p. 2023.08.30.555639) Chain B and 4RRP (Kuzin, A., et al., Northeast Structural Genomics Consortium (NESG) Target PdR16. 2014): Chains D,J with CDR loop atoms deleted (D:25-33, 49-61, 90-96. J:28-34, 50-58, 95-100). The initial model was improved using iterative rounds of manual building with Coot followed by refinement with Phenix. A second round of phasing was performed using Chains C,D and J from the refined BTN2A1-Fab complex structure and followed by subsequent rounds of refinement.
Cryo-Electron Microscopy
Sample Preparation Deglycosylated and purified BTN2A1 ectodomain was mixed with purified Fab and Nanobody at a 1:1.2:1.44 ratio. Complexes were concentrated and purified over SEC using the S200 column in HBS. Purified complexes were concentrated to ~2.3mg/mL and diluted to 0.85 mg/mL in HBS containing CHAPSO detergent at 0.25 x CMC. Complexes were frozen on Quantifoil Au 200 grids using the FEI Vitrobot.
Data Collection and Processing
Data was collected on the Thermo Scientific Titan Krios G3i at the University of Chicago Advanced Electron Microscopy Core. Relion was used for motion correction, CTF finding (Max Res<6A), particle-picking (laplacian, mask: 130-280), particle-extraction (Box: 420A), 2D classification, 3D-classification (Initial-model) and 3D-refinement (Zivanov, J., et al. eLife, 2022. 11: p. e83724).
Data Analysis
General
Flow-Jo was utilized to analyze flow -cytometry data. All results from flow-cytometry, and affinity-determination experiments were plotted and statistically analyzed in GraphPad Prism. Adobe Illustrator was used to design figures.
Analysis of protein complex structures
CCP4-Contact was used to determine van der Waals interactions with a distance cut-off of 4.001. PDB-ePISA was used to find salt-bridge and hydrogen-bond interactions between Fab and BTN2Al.
Statistics
All statistical analyses were performed in Graphpad Prism. All data sets were representative of at least 3 replicates with means displayed as points and standard deviations (SD) displayed as error bars. Data from activation assays involving titration of a reagent were displayed on an XY graph and, in specific cases, analyzed using non-linear regression (variableslope, 4 parameters). Data from specific concentrations within a titration were assessed for statistical significance using multiple t-tests with the Dunnett’s method. Activation assays involving use of a reagent at a single concentration and the TCR competition insect-cell assay were analyzed using multiple t-tests with the Dunnett’s method. Crystallography statistical analyses were carried out in Phenix.
Results
Rational engineering ofa-BTN3A mAb 20.1 provides insights into the role ofBTN3A clustering in pAg-signaling
It was hypothesized that the 20.1 antibody (mAb) multimerizes BTN3A dimers when binding BTN3A bivalently due to its lateral binding orientation (Fig. 1A). This model suggests that 20.1 mimics a mechanism by which pAg binding to the intracellular B30.2 domain of BTN3A1 may drive tight clustering of BTN3A, seeding early immunological synapses that recruit BTN2A1 and potentially other binding partners to engage with the Vy9V52 TCR and stimulate activation.
To test the role of pAg-induced BTN3A1 clustering in pAg signaling, the 20.1 agonist mAb was engineered with increasing hinge region lengths (Fig. IB). It was hypothesized that these 20.1 mAb hinge variants would increase inter-BTN3A membrane-mobility and clustering radius within BTN3A multimers (Fig. 1A) thereby affecting the potency of Vy9V82 agonism. The wildtype (WT) 20.1 mAb is a murine IgGl antibody in which 1 glycine separates the heavychain cysteine residues forming the final disulfide bond with the Fab light chain and the first of 3 inter-heavy-chain disulfide bonds. Glycine-serine (GS) linkers ranging from 4 to 29 amino acids were added to this glycine in the 20.1 heavy-chain hinge region to generate 20.1 mAb hinge variants (20.15, 20.110, 20.115, 20.130) (Fig. IB). Each additional hinge-linker amino acid added -3.5A to the radius of diffusion between two 20.1 Fab:BTN3A complexes. This incrementally increased the radius of diffusion in multimerized BTN3A, from -155 A with the WT 20.1 mAb to -360A with the 20.130 mAb hinge variant, by a maximum factor of -2.3X (Fig. IB). GS linkers in these engineered variants did not affect protein stability nor affinity for biotinylated BTN3A1 ectodomain as assessed by SDS-PAGE gel electrophoresis and Bio-Layer Interferometry (BLI), respectively. Increasing concentrations of the WT 20.1 mAb, the 20.1 mAh hinge variants or the 20.1 Fab were added to Daudi-Cas9- AAVS1 (DaudiAAAVS1), a B-cell lymphoma target-cell line that readily activates Vy9V82 T cells, or Daudi-Cas9-ABTN3A1 (DaudiA3A1) . Jurkat JRT3.3 T cells expressing the G115 Vy9V82 TCR (G115 Jurkats) were then co-incubated with these mAb- pulsed target-cells overnight and assayed for activation by analysis of CD69 expression via flow cytometry. There was no obvious trend between the 20.1 mAb hinge length and Vy9V82 activation (Fig 1C) though increasing the 20.1 mAb hinge length had minor effects on Vy9V82 activation at different concentrations, and on the EC50 of Vy9V82 agonism (Fig. ID). This indicated that tight clustering and multimerization of BTN3A is likely not the driving factor in 20.1 mAb agonism of Vy9V82 activation. The 20.1 Fab has an infinite radius of diffusion and retains the ability to activate Vy9V82 T cells with reduced potency (Fig. 1C-D). The WT 20.1 mAb, 20.1 mAb hinge variants and the 20.1 Fab were mildly agonistic when incubated with DaudiA3A1 target cells likely due to interactions with BTN3A2 and BTN3A3 expression of which was retained in this cell line. These data suggest that 20.1 agonism of Vy9V82 activation is driven by the molecular or steric effects of 20.1 contact with BTN3A. Indeed, BTN3A1 residues bound by both the 20.1 agonist mAb and 0.-BTN3A agonist mAb CTX2026 are directly adjacent to a recently reported pAg-signaling hotspot. a-BTN3A mAb 103.2 inhibits Vy9V32 activation through bi-valent engagement ofBTN3A ectodomains
The engineering of the 20.1 mAb provided insights into the role of cellular reorganization of BTN3A during pAg signaling. Next, the role of BTN3A conformational change in pAg signaling was investigated by similarly engineering the antagonist 103.2 mAb. Original analysis of the complex structure between the 103.2 scFv and BTN3A (PDB ID: 4F9P) used a model derived from IgGs that had long, flexible linkers between the Fab and Fc domains (Palakodeti, A., et al., Journal of Biological Chemistry, 2012. 287(39): p. 32780-32790). Based on this analysis, it was concluded that the 103.2 mAb would be able to bind one BTN3A dimer, with each Fab engaging with one BTN3A monomer in an overall 1 :1 stoichiometry (mAb to BTN3A dimer). This complex was re-analyzed in the context of the functional 103.2 IgG isotype (muIgGl), which has a much more restricted length and flexibility of 7 A in the hinge region between the Fabs connected by the Fc domain (Fig. 2A). To do this, the 103.2 scFv was aligned with 5 full-length murine IgGl Fabs. The distance between the heavy-chain cysteines that form the final inter-chain disulfide bond of the Fab are between 79-113A apart (Fig. 2A), a distance that the hinge region must bridge in a bi- valent mAb. Therefore, there is either significant flexibility in the BTN3A monomers between their IgV and IgC domains, or the 103.2 mAb engages the BTN3A dimer with a 1:2 stoichiometry (one 103.2 mAb to 2 BTN3A dimers). In the first scenario, while the IgC domains of BTN3A have negligible structural flexibility when dimerized, the IgV domains can rotate up to ~20 A at the point of the linker between the IgV and IgC domains (Fig. 2A). Including the general flexibility of ~20A between Fab IgV and IgC domains (Fig. 2A), this model positions the 103.2 mAb just within the calculated threshold of monovalent binding to BTN3A with a limited overall flexibility of -8.5A. If monovalent (1:1) binding between 103.2 and BTN3A occurs in physiological contexts, substantial torsional force on the BTN3A IgV domains would be required, potentially locking them in an inactive conformation. Alternatively, bivalent 103.2 binding to BTN3A may also influence the conformational flexibility of BTN3A dimers, preventing the propagation of intracellular pAg- binding information through JMs to BTN3A extracellular domains, ultimately influencing BTN3A interactions between heterodimers or with other proteins such as BTN2A1.
To test these models, 103.2 variants with longer hinge regions (103.25, 1O3.210, 103.215, 1O3.230) were generated to reduce conformational torsion on BTN3A extracellular domains (Fig. 2B), potentially altering the potency of 103.2 antagonism of Vy9V82 T cell activation. As with the 20.1 mAb hinge variants, GS linkers in the 103.2 mAb hinge valiants did not affect protein stability as assessed by SDS-PAGE gel electrophoresis although a minor increase in their relative affinity for biotinylated BTN3A1 ectodomain was noted as assessed by BLI.
Increasing concentrations of the 103.2 mAb hinge valiants or the 103.2 Fab were added to DaudiAAAVS1 or DaudiA3A1 in the presence of 5pM HMBPP. While DaudiAAAVS1 are capable of activating Vy9V52s without exogenous pAg, addition of exogenous pAg boosts the potency with which DaudiAAAVS1 stimulate V/9V82 activation thus increasing the dynamic range of inhibition assays. G 115 Jurkats were then co-incubated with mAb + pAg-pulsed target-cells overnight and assayed for activation by analysis of CD69 expression via flow cytometry. Increasing the 103.2 hinge-length had no significant effect on the maximum (Fig. 2C) or potency (Fig. 2D) of Vy9V82 activation. Minor effects on Vy9V52 inhibition at different concentrations were observed. The 103.2 Fab has an infinite radius of diffusion and did not antagonize Vy9V82 activation (Fig. 2C,D). DaudiA3A1 cells did not stimulate Vy9V82 activation in the presence of the 103.2 reagents tested. This data suggest that the mechanism of 103.2 antagonism does not involve releasing conformational constraint on BTN3A ectodomains or multimerizing BTN3A. It was therefore hypothesized that the importance of the 103.2 mAb bivalent engagement of BTN3A in Vy9V82 antagonism may be due to its higher avidity or the added steric bulk of a mAb over Fab or scFv reagents. Higher avidity or steric hindrance may enable the 103.2 mAb to successfully compete with a yet unknown ligand of BTN3A. a-BTN2Al mAbs antagonize Vy9Vd2 activation with varying potency
The 20.1 and 103.2 mAbs provide valuable insight into the cellular and molecular changes in BTN3A during pAg- signaling. To better map immunogenic and inhibitory hotspots for Vy9V82 activation on BTN2A1, a panel of 15 O-BTN2A1 Fabs were generated using phage display selection with the BTN2A1 ectodomain as the target antigen. Negative selection was carried out using the BTN3A1 ectodomain as the target antigen and Fab selectivity was verified using phage-enzyme linked immunosorbent assay (ELISA) (FIG. 6). U-BTN2A1 Fabs bound the biotinylated, monomeric BTN2A1 ectodomain with varying biophysical affinities in the pico- nanomolar range as assessed by Surface Plasmon Resonance (SPR) (FIG. 7). Full-length mAb constructs bound endogenous BTN2A1 on DaudiAAAVS1 with a range of cellular affinities measured by ECso of binding and had negligible background staining on Daudi-Cas9 cells where BTN2A1 expression had been knocked out (DaudiA2A1) (FIG. 8).
To test the effect of these O.-BTN2A1 mAbs on Vy9V82 activation, O.-BTN2A1 mAbs were incubated with DaudiAAAVS1 or DaudiA2A1 at a concentration of 6.8nM (Ipg/mL) in the presence and absence of 5pM HMBPP. G115 Jurkats were then co-incubated with these mAb- pulsed target-cells overnight and assessed for CD69 expression via flow-cytometry. CD69 expression was assessed relative to a no mAb control group. Both in the presence and absence of exogenous pAg, O.-BTN2A 1 mAbs antagonized Vy9V62 activation with varying strength (Fig. 3A), whereas no mAb had agonist properties (Fig. 3A). DaudiA3A1 cells co-incubated with u- BTN2A1 mAbs did not stimulate Jurkat Vy9V82 T cell activation. 2A1.9 was as strong an antagonist of Vy9V82 activation as the O-BTN3A1 mAb 103.2 (Fig. 3A) whereas other mAbs like 2A1.4 and 2A1.12 were weak antagonists, and some including 2A1.11 had negligible effects on Vy9V82 activation (Fig. 3A).
The potency of antagonism for selected mAbs was tested using primary Vy9V82 T cells expanded from Peripheral Blood Mononuclear Cells (PBMCs) derived from healthy donors, (Fig. 3B) in activation assays (FIG. 9). 2A1.9 was the strongest antagonist of primary Vy9V82 T cell activation, exceeding even 103.2, reducing CD107a, CD25 and CD69 expression below baseline expression levels (Fig. 3B). 2A1.11 and 2A1.12 had no effect on primary Vy9V82 T cell activation (Fig. 3B). 2A1.12 also had greater variation in antagonism potency than other tested mAbs, suggestive of biochemical differences in its molecular interactions with BTN2A1. DaudiA2A1 cells co-incubated with O.-BTN2A1 mAbs did not stimulate primary Vy9V82 T cell activation (FIG. 9). a-BTN2Al mAb antagonists block Vy9Vd2 TCR engagement with BTN2A1 ectodomain
Linking 0.-BTN2A1 Fab epitopes on the BTN2A1 ectodomain to mAb effects on Vy9V82 activation provides potential insights into the molecular architecture of the mature pAg- signaling complex. BTN2A1 is a binding partner of the Vy9V82 T cell Receptor (TCR) and binding of its CFG-IgV face to the HV4 germline-encoded region of the Vy9V82 TCR y-chain is essential but not sufficient for pAg- signaling. To explore if antagonistic O-BTN2A1 mAbs block pAg- signaling by competing for the Vy9V82 TCR epitope on BTN2A1 , mAb-TCR competition was assessed by using High-Five insect cells expressing full-length BTN2A1 on their cell surface (FIG. 10). These cells were incubated with 150nM saturating concentrations of O-BTN2A 1 mAbs and were then stained with 120nM fluorescently tagged tetramers of the G115 Vy9V82 TCR clone. TCR tetramer staining intensity was assessed relative to a no mAb, full TCR staining control group. G115 tetramer staining of BTN2A1 was significantly reduced to varying degrees in the presence of every O-BTN2A1 mAb (Fig. 3C). Addition of certain mAbs (2A1.9, 2A1.14, 2A1.8 and 2A1.7) had a greater effect on TCR tetramer staining of BTN2A1 (Fig. 3C), potentially indicative of a larger overlapping mAb:TCR epitope on BTN2A1. The G115 tetramer had no background staining on High-five insect cells expressing the control transmembrane protein ADLRG3 (Fig. 10). To understand the relationship between Vy9V82 antagonism and TCR competition potency, mAbs based on their potency of Vy9V82 antagonism and TCR competition (Fig. 3D). A class of strongly antagonistic mAbs that dramatically reduce TCR:BTN2A1 staining emerged (Fig. 3D), represented by the 2A1.9 mAb. Another class of mAbs substantially reduced TCR staining of BTN2A1 but did not reduce Vy9V82 activation (Fig. 3D) represented by the 2A1.12 mAb. The third class of mAbs weakly blocked TCR binding to BTN2A1 but had neutral effects on Vy9V82 activation (Fig. 3D) represented by the 2A1.11 mAb. Although no mAbs had strong antagonistic properties without blocking the TCR (Fig. 3D), the 2A1.4 mAb significantly reduced Vy9V82 activation and was the weakest TCR blocker (Fig. 3D) suggesting that it may have a mechanism of antagonism different from direct TCR competition for BTN2A1 binding.
The mAb:TCR competition for BTN2A1 binding was validated for representative Fabs using BLI (Fig. 3E, Fig. 10). The BTN2A1 ectodomain was immobilized and exposed first (1) to 0.-BTN2A1 Fab at 66pM for 150 seconds (s) followed by (2) 66pM Fab mixed with 80pM monomeric G115 Vy9V82 TCR clone for 150s. Fabs were included in the TCR association step (2) to minimize Fab dissociation. The 20.1 Fab, as control, was used to determine maximum TCR binding (FIG. 10). The results from this biophysical epitope competition tracked with that assessed in High-Five insect cells: 2A1.9 completely blocked Vy9V82 TCR association with BTN2A1 ectodomain (Fig. 3E, Fig. 10), 2A1.12 and 2A1.13 also dramatically reduced Vy9V82 TCR association with BTN2A1 ectodomain (Fig. 3E, Fig. 10D), and 2A1.4 and 2A1.11 did not substantially reduce Vy9V82 TCR association with BTN2A1 ectodomain (Fig. 3E, Fig. 10). The largely nonlinear relationship between Vy9V32 antagonism and TCR competition (Fig. 3D) prompted us to pursue structural determination of Fab:BTN2Al complexes to assess how mAb epitopes contributed to Vy9V82 antagonism.
Complex structure shows 2A1.9 Fab sequestering critical residues in the Vy9V32 TCR epitope on BTN2A1
To better understand the molecular underpinnings of mAb antagonism of Vy9V82 TCR, a 2.8A structure of 2A1.9 Fab in complex with monomeric BTN2A1 ectodomain C219S (BTN2A1 ectodomain) was solved by X-ray crystallography (Fig. 4A). 2A1.9 Fab bound to the IgV-like domain of BTN2A1 ectodomain utilizing all CDR loops except CDRL2 to make molecular contacts with residues located throughout BTN2A1 C”, C’, C, F, and G P strands (CFG-face) (Fig. 4B). The total Buried Surface Area (BSA) on the BTN2A1 ectodomain was 964.6 A, with O 0 the Fab heavy and light chains contributing 658.6 A and 306.0 A, respectively. The 2A1.9 Fab formed 15 Hydrogen-bonds (HB) and salt-bridges (SB) with BTN2A1 ectodomain, 7 of which involved residues in the CDRH3 loop. These stable contacts were strengthened through 154 calculated van der Waals (vdW) interactions between both sidechain and main-chain atoms of 2A1.9Fab CDR loop and BTN2A1 ectodomain CFG-face residues. Due to the abundance of vdW interactions, representative contacts were chosen for each residue pair when multiple atoms formed vdW interactions for visual clarity.
The 2A1.9 binding footprint also included critical residues for pAg-signaling (Fig. 4C) identified through rational mutagenesis based on structural models of the BTN2A1:TCR complex. The binding footprints of the 2A1.9 Fab and the Vy9V62 TCR (PDB ID: 8DFW) were then mapped on the BTN2A1 ectodomain (Fig. 4D). Consistent with cellular and biophysical results, the epitopes of the 2A1.9 Fab and the Vy9V52 TCR overlapped substantially, with the 2A1.9 Fab sequestering all but 3 residues in the interface between the Vy9V82 TCR and the BTN2A1 ectodomain (Fig. 4D). To compare the structural and molecular differences in BTN2A1 when bound to either the 2A1.9 Fab or the Vy9V52 TCR, the BTN2A1 IgV domain in the complex structure was aligned with that in the structure of the BTN2A1 ectodomain in complex with the V 9V32 TCR (PDB: 8DFW). The angle between the BTN2A1 ectodomain IgV and IgC domains was altered 7.3° when bound to the 2A1.9 Fab versus the Vy9V82 TCR (Fig. 4D). Critical BTN2A1 residue side chain orientations and molecular contacts with the 2A1.9 Fab were then assessed. Residues that formed electrostatic interactions with the Vy9V82 TCR or had been shown by mutagenesis to be critical for pAg-signaling were highlighted. The 2A1.9 Fab residue R103 forms a network of HB and vdW interactions with BTN2A1 residues R96 and E107 and alters the position of E107 (Fig. 4E), likely contributing to the high affinity of the 2A1.9 Fab for BTN2A1 as well as its antagonism potency. The 2A1.9 Fab contacts BTN2A1 Y98, Q100, and Y105 residues through G104 and Y105 in its CDRH3 loop via main and sidechain HB and vdW interactions (Fig. 4F). The 2A1.9 Fab uses side- and main-chain atoms in CDRH3 and CDRH1 residues to sequester BTN2A1 residues F43 and S44 through extensive vdW interactions (Fig. 4G). Altogether these data establish that the mechanism by which 2A1.9 antagonizes Vy9V52 activation is by direct competition with the Vy9V82 TCR for BTN2A1 binding.
Preliminary Complex Structures ofBTN2Al with 2A1.4 and 2A1.11 Fabs reveal insight into molecular features governing BTN2A1 ’s role in pAg-signaling.
Having verified the importance of the BTN2A1 epitope shared between 2A1.9 and the Vy9V62 TCR in Vy9V82 activation, it was next investigated whether other epitopes on BTN2A1 were critical for pAg signaling. The 2A1.4 mAb was prioritized, as it is the poorest TCR blocker but significantly inhibits V,y9V62 activation more potently than stronger TCR blockers and the TCR blocking mAb 2A1.11 has insignificant effects on Vy9V82 activation (Fig. 3D). Structural determination of the 2A1.9, 2A1.4, and 2A1.11 Fabs in complex with the BTN2A1 ectodomain dimer (BTN2A1 dimer) and an anti-Fab nanobody was pursued via single-particle Cryo-electron microscopy. Maps of these complexes were refined to 7.4, 7.5 and 6.5A approximate resolutions, respectively (Fig. 5A, B), and models were generated by fitting the structures of the BTN2A1 ectodomain (PDB ID: 8DFW, Chain A-B) and a mulgGl Fab complexed with a nanobody (PDB ID: 7PIJ) into the low-resolution maps (Fig. 5A). At these resolutions the overall conformation of binding to BTN2A1 and putative Fab docking angles can be evaluated (Fig. 5C). 2A1.11 and 2A1 .4 Fabs dock to BTN2A1 in a similar location and orientation to that of the 2A1 .9 Fab (Fig. 5B,C) at approximate angles of 106.4°, 91.1° and 97.1°, respectively (Fig. 5C). These data suggest that the epitopes of 2A1.4 and 2A1.11 on BTN2A1 likely overlap with that of 2A1.9 and subsequently the Vy9V82 TCR. However, the different abilities of 2A1.4, 2A1.11 and 2A1.9 mAbs to antagonize Vy9V82 activation and block TCR recognition of BTN2A1 cannot be explained by the overall docking location of Fabs on BTN2A1.
Molecular intricacies in Fab:BTN2Al interface may drive the potency of mAb Vy9V32 antagonism.
It was next investigated why various mAbs had the ability to sequester the Vy9V52 TCR epitope on BTN2A1 with variable effects on Vy9V52 activation ranging from strong inhibition to neutral. To probe whether the physiological properties of mAbs could be characterized by differences in biophysical or cellular affinity, Fab KD, kA, ko, and mAb EC50 were plotted as a function of Vy9V82 TCR blocking potency or antagonism. It was observed that 2A 1.11 and 2A1.4 have 10.7X and 1.7X faster respective ko properties than 2A1.9 and the strongest antagonists tended to have biophysical affinities (KD) below 1 nM. However, there was no overall correlation between mAb affinity for BTN2A1 and Vy9V82 antagonism or TCR blocking. The CDRH3 loop sequences of O-BTN2A1 mAbs were aligned (Fig. 5E) and it was observed that the R103 and GY motif (Fig. 4E,F) used by the 2A1.9 Fab to sequester critical BTN2A1 residues from the Vy9V82 TCR were highly conserved in the panel (Fig. 5E). Thus, subtle molecular differences in the interactions between Fab CDR loops and BTN2A1 may play a role in mAb antagonism potency, controlling whether Fab contact with BTN2A1 persists on the time scale necessary to compete with Vy9V82 TCR binding in a physiological context. Overall, the data presented herein highlight the complicated mechanisms by which antibodies bind to and influence the behaviors of their targets with important implications in the development of antibody -based therapeutics and research tools.
Discussion
Upon binding to a pAg metabolite, BTN3A1 is responsible for initiating a series of cellular and molecular events on the target cell membrane involving BTN2A1 to signal distress to Vy9V62 T cells. Herein, antibodies against these key players in pAg signaling were developed and rationally modified to resolve long-standing questions in the understanding of the Vy9V82 activation mechanism. The 20.1 mAb or pAg binding to BTN3A1 causes BTN3A immobilization, the formation of BTN3A puncta on the cell membrane and BTN3A localization to the target cell - T cell interface, potentially creating BTN3A clusters that seed early immunological synapses. While the 20.1 mAb may indeed cluster and immobilize BTN3A on the cell membrane, contact between 20.1 and the BTN3A ectodomain alone appears to drive 20.1 agonism. This may suggest an allosteric mechanism whereby 20.1 contact with BTN3A generates an epitope for binding to BTN2A1 or a yet unknown ligand or disrupts a distal inhibitory interaction. Alternatively, a steric mechanism by which the 20. 1 mAb occludes an inhibitory epitope on BTN3A is possible as 20.1 agonism potency decreases as the bulk of the 20.1 reagent decreases from a mAb to a Fab and then to an scFv. Indeed, an antigenic hotspot on BTN3A1 involved in pAg signaling to the MOP clone of the Vy9V82 TCR lies directly adjacent to the 20.1 epitope on BTN3A1. Binding of the 20.1 agonist to BTN3A1 may alter or occlude this antigenic hotspot enabling the recruitment of a Vy9V82 TCR ligand or binding to the Vy9V82 TCR itself.
In contrast to the 20.1 mAb, the 103.2 mAb loses antagonistic function when modified into a Fab or scFv, both of which do not inhibit Vy9V82 activation. The bivalent 103.2 mAb may conformationally constraint BTN3A cctodomain dimers to inhibit Vy9V82 activation. It was predicted that mAb hinge lengths greater than 10 amino acids would alleviate torsional force of monovalent 103.2 mAb binding to BTN3A dimers, potentially rescuing Vy9V82 activation. The data presented herein shows that the 103.2 mAb likely does not antagonize Vy9V82 activation by locking BTN3A ectodomains in an inactive conformation. The 103.2 mAb may instead sterically occlude access to epitopes on the rest of the BTN3A ectodomain as it binds to the top of the IgV domain of BTN3A dimers. While the bulkiness of the Fc domain in the 103.2 mAb may be necessary for steric hindrance, valency may be key to 103.2 antagonism due to its overall low binding affinity for BTN2A1 of 15 nM.
The putative interactions between the BTN3A1 and BTN2A1 ectodomains, the established interaction between BTN2A1 and the Vy9V82 TCR, and yet unknown BTN2A1 binding partners were explored herein by developing mAbs against BTN2A1. BTN2A1 antibodies either antagonized or had no effect on Vy9V82 antagonism. This suggests that the primary role BTN2A1 serves is to coordinate the formation of the pAg signaling complex as a central protein covered in critical epitopes for pAg signaling, one of which binds the Vy9V82 TCR (Fig. 4). Cellular and structural data suggest that many of the U-BTN2A I mAbs bind the Vy9V82 TCR epitope on BTN2A1 with pM-nM range affinities. However, some do not inhibit Vy9V82 T cell activation although the affinity between BTN2A1 and the HV4 loop of the Vy9V62 TCR is ~40 pM. Though the biophysical principles underlying TCR-signaling are still poorly understood, it is possible that antibodies with fast off-rates provide a sufficient opportunity for the Vy9V82 TCR to bind to BTN2A1 and initiate TCR- mediated signaling. None of the tested antibody reagents bound non-overlapping epitopes with the Vy9V82 TCR, therefore the roles of the BTN2A1 IgC domain as well as the IgV domain membrane distal and ABE faces remain undetermined. It is possible that additional epitopes exist to coordinate binding with BTN3A or another TCR CDR loop ligand. The reagents developed in this work improve understanding of the molecular events driving pAg signaling. These reagents may be used to aid in the improvement of Vy9V82 immunotherapies, and may also be directly useful in a clinical setting. The 20.1 mAb is already being explored as an immunotherapy adjuvant. Herein, an improvement on its agonist properties was shown with the addition of 4 amino acids to its hinge region (Fig. 1C,D). Therapeutic development surrounding BTN3A has focused on reagents with agonistic properties. An intriguing idea is the use of antagonist antibodies in clinical contexts where the presence of Vy9V82 T cells or the expression of BTNs is a poor prognostic marker. Pro-inflammatory subsets of Vy9V82 T cells have been implicated in autoimmune disorders including psoriasis, inflammatory bowel disease, celiac disease, and multiple sclerosis. Inhibiting the activity of Vy9V82 T cells in such contexts may alleviate symptoms through the management of excessive inflammation. In the context of cancer, data surrounding the association between BTN expression in tumors and clinical outcomes are complicated and tumor dependent. High expression of BTN3A1 in ovarian and pancreatic cancers and BTN2A1 in metastatic renal cell carcinoma has been associated with reduced patient survival. Indeed, BTN3A1 has been shown to suppress c/.p T cell TCR signaling in tumors by preventing the segregation of CD45 from the immune synapse. Thus, the role of tumor-infiltrating Vy9V82 T cells, as well as how BTN3A and BTN2A1 operate outside of pAg signaling, is poorly understood. In contexts where Vy9V32 T cells or BTNs play immunosuppressive roles, targeting BTN2A1 with antagonistic antibodies may sensitize tumors to immunotherapy.

Claims

1. An antibody that binds specifically to a-Butyrophilin2Al (BTN2A1), the antibody comprising a light chain comprising a light chain variable region and a heavy chain comprising a heavy chain variable region, wherein: a) the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 set forth in Tabic 1; and b) the light chain variable region comprises a CDR-L3 set forth in Table 1.
2. The antibody of claim 1, wherein: a) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 5 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 9 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; e) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; f) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17, respectively; g) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; h) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 20, SEQ ID NO: 7, and SEQ ID NO: 21, respectively; i) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 2, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; j) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 24, respectively; k) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 25, respectively; l) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 3, and SEQ ID NO: 26, respectively; m) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 27 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 28, respectively; n) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 29 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 6, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or o) the light chain variable region comprises a CDR-L3 of SEQ ID NO: 1 and the heavy chain variable region comprises a CDR-H1, a CDR-H2, and a CDR-H3 of SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 32, respectively.
3. The antibody of claim 1 or claim 2, wherein: a) the light chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61; and b) the heavy chain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
4. The antibody of claim 3, wherein: a) the light chain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61; and b) the heavy chain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:
50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
5. The antibody of claim 3, wherein: a) the light chain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61; and b) the heavy chain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:
50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.
6. The antibody of claim 3, wherein the antibody comprises: a) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 33 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 34; b) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36; c) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 37 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 38; or d) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 39 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 40; e) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 41 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42; f) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 43 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 44; g) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 46; h) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 48; i) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 49 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 50; j) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 51 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52; k) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 53 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 54; l) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 56; m) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 58; n) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 59 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 60; or o) a light chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 61 and a heavy chain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62.
7. The antibody of claim 4, wherein the antibody comprises: a) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 33 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 34; b) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36; c) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 38; or d) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 39 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40; e) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 41 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42; f) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 43 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 44; g) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 46; h) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 48; i) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 50; j) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 51 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 52; k) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 53 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 54; l) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 56; m) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 58; n) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 59 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 60; or o) a light chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 61 and a heavy chain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 62.
8. The antibody of claim 5, wherein the antibody comprises: a) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 33 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 34; b) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 36; c) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 37 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 38; or d) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 39 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 40; e) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 41 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 42; f) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 43 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 44; g) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 46; h) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 47 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 48; i) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 49 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 50; j) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 51 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 52; k) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 53 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 54; l) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 56; m) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 58; n) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 59 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 60; or o) a light chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61 and a heavy chain comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62.
9. The antibody of any one of the preceding claims, wherein the antibody comprises an antigen binding fragment (Fab).
10. The antibody of any one of the preceding claims, further comprising a detectable label.
11. A kit comprising the antibody of any one of claims 1-10.
12. The antibody of any one of claims 1-10 or the kit of claim 11, for use in a method of detecting the presence and/or amount of BTN2A1 in a sample, inhibiting BTN2A1 activity in a sample or a subject, inhibiting Vy9V52 T-cell activation in a sample or a subject, diagnosing a BTN2A1 -related disorder in a subject, or treating a BTN2A1- related disorder in a subject.
13. A method comprising: a) contacting a sample with the antibody of claim 10; and b) detecting a signal from the detectable label, wherein detection of a signal is indicative of the presence and/or amount of BTN2Alin the sample.
14. A method of inhibiting Vy9V52 T-cell activation in a subject, comprising providing to the subject the antibody of any one of claims 1-10.
15. The method of claim 14, wherein the subject has or is at risk of having a BTN2Al-related disorder.
16. A method of diagnosing a BTN2A1 -related disorder in a subject, the method comprising contacting a sample obtained from the subject with the antibody of any one of claims 1- 10.
17. A method of treating a BTN2A1 -related disorder in a subject, the method comprising providing to the subject the antibody of any one of claims 1-10 or an antibody-drug conjugate comprising the antibody of any one of claims 1-10.
18. The method of any one of claims 15-17, wherein the BTN2A1 -related disorder comprises an autoimmune disorder, an inflammatory disorder, or transplant rejection.
19. The method of any one of claims 14-18, wherein the subject is a human.
PCT/US2024/034693 2023-06-20 2024-06-20 α-BUTYROPHILIN2A1 ANTIBODIES Pending WO2024263692A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016061504A2 (en) * 2014-10-17 2016-04-21 The University Of Chicago Recombinant antibodies that recongnize the c-terminal domains of ebola virus nucleoprotein
US20200283519A1 (en) * 2017-09-21 2020-09-10 Imcheck Therapeutics Sas Antibodies having specificity for btn2 and uses thereof
US20220162305A1 (en) * 2019-03-20 2022-05-26 Imcheck Therapeutics Sas Antibodies having specificity for btn2 and uses thereof
WO2023060211A1 (en) * 2021-10-06 2023-04-13 The University Of Chicago Borealin targeting polypeptides for detection and treatment of cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016061504A2 (en) * 2014-10-17 2016-04-21 The University Of Chicago Recombinant antibodies that recongnize the c-terminal domains of ebola virus nucleoprotein
US20200283519A1 (en) * 2017-09-21 2020-09-10 Imcheck Therapeutics Sas Antibodies having specificity for btn2 and uses thereof
US20220162305A1 (en) * 2019-03-20 2022-05-26 Imcheck Therapeutics Sas Antibodies having specificity for btn2 and uses thereof
WO2023060211A1 (en) * 2021-10-06 2023-04-13 The University Of Chicago Borealin targeting polypeptides for detection and treatment of cancer

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