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WO2024263692A1 - ANTICORPS α-BUTYROPHILINE 2A1 - Google Patents

ANTICORPS α-BUTYROPHILINE 2A1 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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seq
amino acid
acid sequence
cdr
sequence identity
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Erin J. ADAMS
Anthony A. KOSSIAKOFF
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University of Chicago
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University of Chicago
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    • 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

L'invention concerne des anticorps qui se lient à l'α-Butyrophiline 2A1 (BTN2A1), et des procédés thérapeutiques, de diagnostic et de recherche d'utilisation de ceux-ci. En particulier, l'invention concerne des anticorps BTN2A1 n'ayant sensiblement aucune réactivité croisée avec l'α-Butyrophiline 3A1 (BTN3A1), et dans certains modes de réalisation présentant un antagonisme vis-à-vis de BTN2A1 et/ou une inhibition de l'activation des lymphocytes T Vγ9Vδ2.
PCT/US2024/034693 2023-06-20 2024-06-20 ANTICORPS α-BUTYROPHILINE 2A1 Pending WO2024263692A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016061504A2 (fr) * 2014-10-17 2016-04-21 The University Of Chicago Anticorps de recombinaison qui reconnaissent les domaines c-terminal de la nucléoprotéine du virus ebola
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 (fr) * 2021-10-06 2023-04-13 The University Of Chicago Polypeptides ciblant la boréaline pour la détection et le traitement du cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016061504A2 (fr) * 2014-10-17 2016-04-21 The University Of Chicago Anticorps de recombinaison qui reconnaissent les domaines c-terminal de la nucléoprotéine du virus ebola
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 (fr) * 2021-10-06 2023-04-13 The University Of Chicago Polypeptides ciblant la boréaline pour la détection et le traitement du cancer

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