EP3253796A1 - Anti-garp-protein und verwendungen davon - Google Patents

Anti-garp-protein und verwendungen davon

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Publication number
EP3253796A1
EP3253796A1 EP16710325.8A EP16710325A EP3253796A1 EP 3253796 A1 EP3253796 A1 EP 3253796A1 EP 16710325 A EP16710325 A EP 16710325A EP 3253796 A1 EP3253796 A1 EP 3253796A1
Authority
EP
European Patent Office
Prior art keywords
tgf
seq
antibody
garp
human
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP16710325.8A
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English (en)
French (fr)
Inventor
Michael Saunders
Pierre Coulie
Sophie Lucas
Gitte DE BOECK
Sebastian VAN DE WONING
Hans De Haard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Catholique de Louvain UCL
ArgenX BVBA
Original Assignee
Universite Catholique de Louvain UCL
ArgenX SE
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Publication of EP3253796A1 publication Critical patent/EP3253796A1/de
Ceased legal-status Critical Current

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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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • A61P37/02—Immunomodulators
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to human anti-GARP protein that inhibits TGF- ⁇ signaling.
  • the present invention also relates to the treatment of immune disorders and diseases such as cancer. BACKGROUND OF INVENTION
  • Tregs are a subset of CD4+ T lymphocytes specialized in the inhibition of immune responses. Insufficient Treg function results in autoimmune pathology, while excessive Treg function may inhibit anti-tumor immune responses in cancer patients. The exact mechanisms by which Tregs inhibit immune responses are not fully understood. Due to their immunosuppressive functions, Tregs represent potential inhibitors of spontaneous or vaccine-induced anti-tumor immune responses. In murine models, the depletion of Tregs can improve immune responses against experimental tumors (Colombo et al. Nat. Rev. Cancer 2007, 7:880-887). Thus, targeting Tregs in humans could improve the efficacy of immunotherapy against cancer.
  • TGF- ⁇ active TGF- ⁇ is produced by human Tregs, but not other types of human T lymphocytes (Stockis, J. et al. Eur. J. Immunol. 2009, 39:869-882), TGF- ⁇ could be a target of interest.
  • FSGS focal segmental glomerulosclerosis
  • IPF idiopathic pulmonary fibrosis
  • RCC renal cell carcinoma
  • One object of the invention is to provide a new strategy for improving cancer treatment by targeting Tregs via their production of TGF- ⁇ .
  • TGF- ⁇ is tightly regulated by a multi-step process.
  • the precursor pro-TGF- ⁇ 1 homodimerizes prior to cleavage by pro- protein convertase FURI .
  • the resulting product is called latent TGF- ⁇ , in which the C-terminal fragment, or mature TGF-Bl, remains non-covalently bound to the N- terminal fragment known as the Latency Associated Peptide, or LAP.
  • LAP Latency Associated Peptide
  • This latent complex is inactive because LAP prev ents mature TGF-131 from binding to its receptor.
  • the inventors show that latent TGF-B is shown to bind to the surface of Tregs through the transmembrane protein GARP (glycoprotein A repetitions predominant).
  • the present invention therefore provides a new strategy for targeting Treg based on an anti-GARP protein inhibiting TGF-B signaling.
  • One object of the invention is a protein binding to Glycoprotein A repetitions predominant (G ARP) in the presence of TGF-B.
  • G ARP Glycoprotein A repetitions predominant
  • said protein binds to GARP only in the presence of TGF-B.
  • said protein binds to GARP when GARP is complexed to TGF-B.
  • said protein binds to a complex of G ARP and TGF-B.
  • said protein is an antibody molecule selected from the group consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a F(ab)'2, a defucosylated antibody, a bi-specific antibody, a diabody, a triabody, a tetrabody.
  • said protein is an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody.
  • said protein is an antibody mimetic selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, a versabody and a duocalin.
  • Another object of the inv ention is a protein as described here above or a protein binding GARP and inhibiting TGF-B signaling.
  • said protein is an antibody or antigen binding fragment thereof that binds to a conformational epitope comprising one or more amino acids of G ARP or an epitope of G ARP modified as a result of G ARP being complexed with latent TGF-B.
  • said antibody or antigen binding fragment thereof further binds one or more amino acids of latent TGF- ⁇ .
  • said antibody or antigen binding fragment thereof binds an epitope comprising one or more residues from residues 101 to 14 1 of GARP as set forth in SEQ ID NO: 1 .
  • Another object of the invention is a protein having the variable region of the heavy chain comprising at least one of the following CDRs:
  • V H-CDR l GFSLT GYGIN (SEQ ID NO: 2) or GYGI N (SEQ ID NO: 52);
  • VH-CDR3 D NYYDYDGAMDY (SEQ ID NO: 4),
  • variable region of the light chain comprising at least one of the following CDRs:
  • V L-CDRl KASDHIKNWLA SEQ ID NO: 5
  • VL-CDR2 GATSLEA ( SEQ ID NO: 6);
  • VL-CDR3 QQYWSTPWT (SEQ ID NO: 7),
  • variable region of the heavy chain comprises at least one of the following CDRs:
  • V H-CDR l SYYID SEQ ID NO: 13
  • VH-CDR2 RIDPEDGGTKYAQKFQG (SEQ ID NO: 14);
  • V H-CDR3 or NEWETVVVGDLMYEYEY SEQ ID NO: 15
  • variable region of the light chain comprises at least one of the following CDRs:
  • VL-CDRl QASQX, I X 2 S X 3 LA ( SEQ ID NO: 16), wherein X, is S or T, X 2 is S
  • V L-CDR2 X 1 X 2 SX 3 X 4 X 5 T ( SEQ ID NO: 17), wherein X, is G or R; X 2 is A or
  • variable region of the heavy chain comprises at least one of the following CDRs:
  • VH-CDR1 GFSLTGYGIN (SEQ ID NO: 2) or GYGIN (SEQ ID NO: 52);
  • VH-CDR2 MIWSDGSTDYNSVXTS (SEQ ID NO: 3);
  • VH-CDR3 DRNYYDYDGAMDY (SEQ ID NO: 4),
  • variable region of the light chain comprises at least one of the following
  • VL-CDR1 KASDHIKNWLA (SEQ ID NO: 5);
  • VL-CDR2 GATSLEA (SEQ ID NO: 6);
  • VL-CDR3 QQYWSTPWT (SEQ ID NO: 7),
  • variable region of the heavy chain comprises at least one of the following CDRs:
  • VH-CDR2 RIDPEDGGTKYAQKFQG (SEQ ID NO: 14);
  • VH-CDR3 or NEWETVVVGDLMYEYEY (SEQ ID NO: 15);
  • variable region of the light chain comprises at least one of the following
  • V L-CDRl QASQXjI X 2 S X 3 LA (SEQ ID NO: 16), wherein X, is S or T, X 2 is
  • VL-CDR2 XiX 2 SX 3 X 4 X 5 T (SEQ ID NO: 17), wherein X, is G or R; X 2 is A or T; X is R or I; X 4 is L or P; X 5 is Q or K; VL-CDR3: QQYX 1 SX 2 PX 3 T, wherein Xi is D, A, Y or V; X 2 is A, L or V; X 3 is V or P (SEQ ID NO: 18);
  • variable region of the heavy chain comprises the following CDRs: GFSLTGYGIN (SEQ ID NO: 2), MIWSDGSTDYNSVLTS (SEQ ID NO: 3), DRNYYDYDGAMDY (SEQ ID NO: 4) and the variable region of the light chain comprises the following CDRs: KASDHIKNWLA (SEQ ID NO: 5), GATS LEA (SEQ ID NO: 6), QQYWSTPWT (SEQ ID NO: 7) or any CDR having an amino acid sequence that shares at least 60% identity with said SEQ ID NO: 2-7; or the variable region of the heavy chain comprises the following CDRs: GYGIN (SEQ ID NO: 52), MIWSDGSTDYNSVLTS (SEQ ID NO: 3), DRNYYDYDGAMDY (SEQ ID NO: 4) and the variable region of the light chain comprises the following CDRs: KASDHIKNWLA (SEQ ID NO: 2), MIWSDGSTDYNSVLTS (SEQ ID NO: 3), DRNYY
  • amino acid sequence of the heavy chain variable region is SEQ ID NO: 8 or SEQ ID NO: 50 and the amino acid sequence of the light chain variable region is SEQ ID NO: 9 or SEQ ID NO: 51, or the amino acid sequence of the heavy chain variable region is SEQ ID NO: 34 and the amino acid sequence of the light chain variable region is one of SEQ ID NO: 35-39 or any sequence having an amino acid sequence that shares at least 60% identity with said SEQ ID NO: 8-9, 50-51 or 34- 39.
  • Another object of the invention is a protein as defined here above binding to an epitope on the polypeptide having the amino acid sequence SEQ ID NO: 1 recognized by an antibody comprising a heavy chain variable region as set forth in SEQ ID NO: 8 or in SEQ ID NO: 50 and a light chain variable region as set forth in SEQ ID NO: 9 or in SEQ ID NO: 51, or by an antibody comprising a heavy chain variable region as set forth in SEQ ID NO: 34 and one of the light chain variable region as set forth in SEQ ID NO: 35-39.
  • Another object of the invention is an antibody or antigen binding fragment produced by a hybridoma registered under the accession number LMBP 10246CB on May 30, 2013.
  • Another object of the invention is a polynucleotide sequence encoding the antibody or antigen binding fragment as described here above.
  • Another object of the invention is an expression vector comprising the polynucleotide according to claim as described here above.
  • Another object of the invention is a hybridoma cell line producing an antibody against GARP registered under the accession number LMBP I 0246CB on May 30, 2013.
  • Another object of the invention is a pharmaceutical composition comprising the protein as described here above and a pharmaceutically acceptable excipient.
  • Another object of the invention is a pharmaceutical composition as described here above for treating a TGF- ⁇ related disorder in a subject in need thereof.
  • the TGF-13 related disorder is selected from the group consisting of inflammatory diseases, chronic infection, cancer, fibrosis, cardiovascular diseases, cerebrovascular disease (e.g. ischemic stroke), and neurodegenerative diseases.
  • the pharmaceutical composition as described here above is to be administered in combination with another treatment for cancer or another immunotherapeiitic agent such as a tumor vaccine or an immunostimulatory antibody .
  • the pharmaceutical composition as described here above is to be administered as an immunostimulatory antibody for treatment of cancer patients,
  • M HGARP8 inhibits active TGF- ⁇ production by a human Treg clone.
  • Clone Treg Al was stimulated during 24 hours with >CD3/CD28 antibodies, alone or in the presence of the indicated anti-hGARP mAbs (20 iig/ ' ml ).
  • A Cell ly sates were analyzed by WB with anti-pSMAD2 and anti-B-ACTIN antibodies.
  • B Quantification of ECL signals from WB shown in A.
  • FIG. 3 Regions in the hGARP protein required for binding by anti-hGARP antibodies.
  • Murine BW5147 T cells expressing the HA-tagged proteins schematized on the left were stained with anti-hGARP (MHGARP 1 to 9, as indicated on top of the figure) or anti-HA antibodies, and analyzed by flow cytometry. Histograms are gated on live cells. Based on the FACS results, regions required for binding by the various MHGARP mAbs were identified and are indicated by horizontal bars above the representations of the H A-tagged chim eras.
  • the HA-tagged forms of hGARP tested here comprised aa 20-662 of hG ARP (wild type, WT), or aa 20-662 of hGARP in which groups of 3 amino-acids located in region 10 1 - 14 1 were replaced by the amino-acids found in the corresponding region of mGARP (Mut I, Mut II and Mut 111 ).
  • Amino-acid sequences of region 10 1 - 14 1 of hGARP -WT, -Mut I, -Mut II, -Mut III and mGARP are indicated on the left.
  • Amino-acids that differ between human and mouse GARP are highlighted by grey vertical boxes, and amino-acids mutated in Mut I, Mut II and Mut 111 are indicated by black horizontal boxes.
  • MHGARP8 inhibits Treg function in vivo.
  • the indicated groups of NSG mice received i.v. injections of human PBMCs, in combination or not with human Tregs.
  • Mice from groups III and IV were treated with the MHGARP8 antibody, injected i.p. once a week, starting on day - 1.
  • Objective signs of GvHD development in the recipient mice were monitored bi-weekly.
  • a GvHD score was established based on weight loss (0: ⁇ 10%; 1 : 10%-20%; 2: >20%; 3 : >30%), anemia (0: red or pink tail; 1 : white tail), posture (0: normal; 1 : hump), general activity (0: normal; 1 : limited), hair loss (0: no hair loss; 1 : hair loss) and icterus (0: white or red tail; 1 : yellow tail ).
  • Maximum disease severity or death corresponded to a score of 7.
  • A Experiment 1. Values represent mean scores.
  • FIG. 7 New anti-hGARP niAbs.
  • A Schematic representation of the experimental strategies used to derive anti-hGARP mAbs.
  • B Flow cytometry analyses of clone Th A2 (human CD4+ Th cells which do not express hGARP), or Th.A2 cells transduced with hGARP, after staining with biotinylated MHG- 1 to - 14 mAbs and streptavidin coupled to PE (SA-PE), with LHG- 1 to -17 mAbs and a secondary anti-hlgGl antibody coupled to PE, or with a commercially available mouse anti-hGARP m Ab (clone Plato- 1) and a secondary anti-mIgG2b antibody coupled to AF647.
  • SA-PE biotinylated MHG- 1 to - 14 mAbs and streptavidin coupled to PE
  • SA-PE biotinylated MHG- 1 to - 14 mAbs and streptavidin
  • FIG. 1 shows immune responses from immunized llamas.
  • A shows immune responses from DNA immunized llamas.
  • B shows immune responses from llamas immunized with BW cells expressing hGARP/hTGFp.
  • Figure 9 Cross-reactivity to cynomolgus GARP-TG Fp measured on cells by FACS. 293 E cells were transfected with human / cyno GARP and human / cyno TGFB. LHG- 10-D and the affinity optimized variants are cross-reactive with cynomolgus GARP-TGFB. Figure 10. Sequences of LHG- 10 antibodies and its shuffle variants.
  • M HGA RP8 and LHG- 10 inhibit production of active TGF- ⁇ by human Tregs.
  • human CD4+CD25hiCD 1271o cells (Tregs) were re-stimulated with anti-CD3/CD28 coated beads during 24 hours, in the presence or absence of the indicated mAbs (10 , ug/ml).
  • Cells lysates were analyzed by Western Blot with antibodies against phosphorylated SMAD2 (pSMAD2), as a read-out for active TGF-13 production, or B-ACTIN (loading control).
  • pSMAD2 phosphorylated SMAD2
  • FIG. 12 M HGARP8 and LHG-10 inhibit the suppressive activity of human Tregs in vitro.
  • Th Freshly isolated human CD4+CD25-CD127hi cells (Th; 2xl0 4 per microwell) were seeded alone or with clone Treg Al (Stockis, J. et al. Eur. J. Immunol. 2009, 39:869-882) at a 1/1 Treg to Th ratio.
  • Cells were stimulated with coated anti-CD3 and soluble anti-CD28, in the presence or absence of the indicated anti-hGARP mAbs (10 ⁇ «/ ⁇ 1 ).
  • FIG. 13 Forms and regions of GARP bound by anti-GARP mAbs.
  • A Schematic representations of GARP and GARP/TGF- ⁇ complexes. Protein GARP is represented by a thick curved grey line. Numbers indicate amino-acid positions. TGF- ⁇ is represented with the Latency Associated Peptide (LAP) as thick black lines, and the mature TGF- ⁇ 1 peptide as thick straight grey lines. Thin black lines represent interchain disulfide bonds.
  • LAP Latency Associated Peptide
  • B Classification of anti-hGARP mAbs based on their binding requirements.
  • hTGFB 1 was co-transfected with mGARP/hGARP chimeras for the analyses of mAbs that bind liGARP/hTGF-B 1 complexes only (LHG-3, MHGARP8 (MHG-8), LHG- 10).
  • FIG. 16 Inhibition of human Treg function by anti-hGARP in vivo.
  • A shows the protocol on day 0, the indicated groups of NSG mice received i.v. injections of human
  • FIG. 1 shows the results of 4 independent experiments ( 1 to IV), performed with cells from donors A, B or C, with the indicated numbers of mice per group (n).
  • Disease onset is the day when mean disease score becomes >1, and is indicated for 3 experimental groups in which mice were grafted with PBMCs only (group a), PBMCs and Tregs (group b), or PBMCs and Tregs and treated with MHGARP8 (MHG-8) (group c).
  • C Detailed results from experiment IV, showing the evolution of mean disease score (left) and survival curves (right) in the indicated groups of mice.
  • FIG. 17 Anti-hGARP niAbs that block TGF- ⁇ production inhibit suppression by human Tregs in vivo.
  • Top graph shows progression of disease scores (means per group + sem), bottom graph shows survival.
  • NSG mice injected as in Fig. 16, part A were sacrificed 20 days after cell transfer.
  • A Serum levels of human cytokines were measured in a multiplex bead assay.
  • each mutation corresponds to the ratio between the EC 50 of MHG-8 binding to the mutant and the EC 50 of MHG-8 binding to the WT. Mutations with a ratio >2 are considered to decrease the avidity for binding by MHG-8.
  • NE non evaluable (residual binding ⁇ 10%); nt: not tested.
  • Figure 20 Impact of mutations in GARP or TGF-B l on the inhibitory activity of MHG-8 and LHG- 10.
  • the term “immunoglobulin” includes a polypeptide having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity.
  • Antibodies refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g. human GARP).
  • GARP antibodies is used herein to refer to antibodies which exhibit immunological specificity for human GARP protein. As explained elsewhere herein, “specificity” for human GARP does not exclude cross- reaction with species homologues of GARP.
  • Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.
  • the generic term "immunoglobulin" comprises five distinct classes of antibody that can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000 Daltons.
  • the four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y” and continuing through the variable region.
  • the light chains of an antibody are classified as either kappa or lambda ( [K], [ ⁇ ]).
  • Each heavy chain class may be bonded with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the "tail" regions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages w hen the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • heavy chains In the heavy chain, the amino acid sequences run from an Ni-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • heavy chains are classified as gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g. , yl - ⁇ 4). It is the nature of this chain that determines the "class" of the antibody as IgG, igM, IgA IgG, or IgE, respectively.
  • the immunoglobulin subclasses isotypes) e.g.
  • variable region of an antibody allows the antibody to selectively recognize and speci ically bind epitopes on antigens. That is, the VL domain and VI I domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y.
  • the antigen binding site is defined by three complementarity determining regions (CDRs) on each of the ⁇ T 1 and VL chains.
  • CDRs complementarity determining regions
  • the antibody is purified : (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity as shown by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue or, preferably, silver staining.
  • An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody' s natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.
  • affinity variants refers to a variant antibody which exhibits one or more changes in amino acid sequence compared to a reference GARP antibody, wherein the affinity variant exhibits an altered affinity for the human GARP protein or GARP/TGF- ⁇ complex in comparison to the reference antibody.
  • affinity variants will exhibit an improved affinity for human GARP or human G.ARP/TGF- ⁇ complex, as compared to the reference GARP antibody. The improvement may be a lower KD for human GARP, a faster off- rate for human GARP, or an alteration in the pattern of cross-reactivity with non-human GARP homologues.
  • Affinity variants typically exhibit one or more changes in amino acid sequence in the CDRs, as compared to the reference GARP antibody. Such substitutions may result in replacement of the original amino acid present at a given position in the CDRs with a different amino acid residue, which may be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue.
  • the amino acid substitutions may be conservative or non-conservative.
  • Binding Site comprises a region of a polypeptide which is responsible for selectiv ely binding to a target antigen of interest (e.g. human GARP). Binding domains or binding regions comprise at least one binding site. Exemplary binding domains include an antibody variable domain.
  • the antibody molecules of the invention may comprise a single antigen binding site or multiple (e.g., two, three or four) antigen binding sites.
  • Constant amino acid substitution is one in which the amino acid residue is replaced w ith an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid ), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalan
  • a nonessential amino acid residue in an immunoglobulin polypeptide may be replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • a “chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide.
  • a chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • Exemplary chimeric GARP antibodies include fusion proteins comprising camelid-derived VH and VL domains, or humanised variants thereof, fused to the constant domains of a human antibody, e.g. human IgG I , IgG2, IgG3 or IgG4.
  • CDR CDR
  • CDR complementarity determining region
  • CDR means the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 ( 1977) and Kabat et al.. Sequences of protein of immunological interest. (1991), by Chothia et al ., J. Mol. Biol. 196:90 1 -9 1 7 (1987), and by MacCallum et al, J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison.
  • the term “CDR” is a CDR as defined by Kabat based on sequence comparisons.
  • CH2 domain includes the region of a heavy chain molecule that extends, e.g. , from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system, Kabat EA et al. Sequences of Proteins of Immunological Interest. Bethesda, US Department of Health and Human Services, NIH. 1991 ).
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the tw o CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
  • the GARP antibody molecules of the invention comprise framework amino acid sequences and/or CDR amino acid sequences derived from a camelid conventional antibody raised by active immunization of a camelid with GARP antigen.
  • GARP antibodies comprising camelid- derived amino acid sequences may be engineered to comprise framework and/or constant region sequences derived from a human amino acid sequence or other non- camelid mammalian species.
  • a human or non-human primate framework region, heav y chain region, and/or hinge region may be included in the subject GARP antibodies.
  • one or more non-camelid amino acids may be present in the framework region of a "camelid-derived" GARP antibody, e.g. , a camelid framework amino acid sequence may comprise one or more amino acid mutations in which the corresponding human or non-human primate amino acid residue is present.
  • camelid-derived VH and V ' L domains, or humanized variants thereof may be linked to the constant domains of human antibodies to produce a chimeric molecule, as extensively described elsewhere herein.
  • the term "derived from" a designated protein refers to the origin of the polypeptide.
  • the polypeptide or amino acid sequence which is derived from a particular starting polypeptide is a CDR sequence or sequence related thereto.
  • the amino acid sequence hich is deriv ed from a particular starting polypeptide is not contiguous. For example, in an embodiment, one, two, three, four, fiv e, or six CDRs are deriv ed from a starting antibody.
  • the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a region thereof wherein the region consists of at least of at least 3-5 amino acids, 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
  • the one or more CDR sequences derived from the starting antibody are altered to produce variant CDR sequences, e.g. affinity variants, wherein the variant CDR sequences maintain GARP binding activity.
  • Diabodies refers to small antibody fragments prepared by constructing sFv fragments (see sFv paragraph) with short linkers (about 5- 10 residues) between the VI I and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a biv alent fragment, i.e. , fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two "crosso ver” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Holliger et al ., Proc. Natl. Acad. Sci., 90:6444-6448 (1993).
  • engineered includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
  • the antibodies of the invention are engineered, including for example, humanized and/or chimeric antibodies, and antibodies which have been engineered to improve one or more properties, such as antigen binding, stability/half-life or effector function.
  • Epitope refers to a specific arrangement of amino acids located on a peptide or protein or proteins to which an antibody binds. Epitopes often consist of a chemical ly active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear or conformational, i.e. , involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.
  • Framework region includes the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g. , using the Kabat definition of CDRs). Therefore, a variable region framework is between about 100- 120 amino acids in length but includes only those amino acids outside of the CDRs.
  • framework region I corresponds to the domain of the variable region encompassing amino acids 1-30
  • framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49
  • framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94
  • framework region 4 corresponds to the domain of the variable region from amino acids 103 to the end of the variable region.
  • the framework regions for the light chain are similarly separated by each of the light claim variable region CDRs.
  • the framework region boundaries are separated by the respective CDR termini as described above.
  • the CDRs are as defined by Kabat.
  • the six CDRs present on each monomeric antibody are short, non- contiguous sequences of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the heavy and light variable domains show less inter- molecular variability in amino acid sequence and are termed the framework regions.
  • the framework regions largely adopt a [betaj-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the [beta]-sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non- covalent binding of the antibody to the immunoreactive antigen epitope.
  • the position of CDRs can be readily identified by one of ordinary skill in the art.
  • fragment refers to a part or region of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain.
  • antigen-binding fragment refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e. , with the intact antibody from which they were derived) for antigen binding (i.e. , specific binding to human GARP).
  • fragment of an antibody molecule includes antigen-binding fragments of antibodies, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a single domain antibody fragment (DAb), a one-armed (monovalent) antibody, diabodies or any antigen-binding molecule formed by combination, assembly or conjugation of such antigen binding fragments. Fragments can be obtained, e.g. , via chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.
  • Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, ev en a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • heav y chain region includes amino acid sequences derived from the constant domains of an immunoglobulin heav y chain.
  • a polypeptide comprising a heavy chain region comprises at least one of: a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a v ariant or fragment thereof, in an embodiment, a binding molecule of the invention may comprise the Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain).
  • a binding molecule of the invention lacks at least a region of a constant domain (e.g., all or part of a CI 12 domain).
  • at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain.
  • the heavy chain region comprises a fully human hinge domain.
  • the heavy chain region comprising a fully human Fc region (e.g., hinge, CH2 and CH3 domain sequences from a human immunoglobulin ).
  • the constituent constant domains of the heavy chain region are from different immunoglobulin molecules.
  • a heavy chain region of a polypeptide may comprise a CH2 domain deriv ed from an IgGl molecule and a hinge region derived from an IgG3 or IgG4 molecule.
  • the constant domains are chimeric domains comprising regions of different immunoglobulin molecules.
  • a hinge may comprise a first region from an IgGl molecule and a second region from an IgG3 or IgG4 molecule.
  • the constant domains of the heavy chain region may be modified s ch that they vary in amino acid sequence from the naturally occurring (wild-type) immunoglobulin molecule.
  • polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the heavy chain constant domains (CHI, hinge, CH2 or CH3) and/or to the light chain constant domain (CL).
  • exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.
  • Hinge region includes the region of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N -terminal antigen binding regions to move independently. Hinge regions can be subdiv ided into three distinct domains: upper, middle, and lower hinge domains (Roux et al . J. Immunol. 1998 16 1 :4083).
  • hypervariable loop and “complementarity determining region” are not strictly synonymous, since the hypervariable loops (HVs) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined based on sequence variability ( Kabat et al , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Ml)., 1983) and the limits of the HVs and the CDRs may be different in some VH and VL domains.
  • the CDRs of the VL and VH domains can typically be defined as comprising the following amino acids: residues 24-34 (CDRL 1 ), 50-56 (CDRL2) and 89-97 ( CDRL3 ) in the light chain variable domain, and residues 3 1 -35 or 3 1 -3 b (CDRH 1 ), 50-65 (CDRH2) and 95- 102 (CDRH3 ) in the heav y chain variable domain; ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
  • the H Vs may be comprised within the corresponding CDRs and references herein to the "hyperv ariable loops" of VI I and VL domains should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise indicated.
  • the more highly conserved regions of variable domains are called the framework region (FR), as defined below.
  • the v ariable domains of native heavy and light chains each comprise four FRs (FRl, FR2, FR3 and FR4, respectively), largely adopting a [beta]-sheet configuration, connected by the three hypervariable loops.
  • the hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies.
  • Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et ai, J. Mol. Biol. 227: 799-817 (1992)); Tramontane et al., J. Mol. Biol, 215 : 175-182 (1990)).
  • five of the six loops adopt just a small repertoire of main-chain conformations, called "canonical structures " . These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the ability to assume unusual main-chain conformations.
  • “Humanising substitutions” refers to amino acid substitutions in which the amino acid residue present at a particular position in the VH or VL domain antibody GA RP antibody (for example a camelid- derived GARP antibody) is replaced with an amino acid residue which occurs at an equivalent position in a reference human VH or VL domain .
  • the reference human VH or VL domain may be a VH or VL domain encoded by the human germ line, in which case the substituted residues may be referred to as “germlining substitutions " .
  • H uman i si ng/ge m 1 i n i ng substitutions may be made in the framework regions and/or the CDRs of a GARP antibody, defined herein.
  • High human homology An antibody comprising a heavy chain variable domain ( VH ) and a light chain variable domain (VL) will be considered as having high human homology if the VH domains and the VL domains, taken together, exhibit at least 90% amino acid sequence identity to the closest matching human germline VH and VL sequences.
  • Antibodies having high human homology may include antibodies comprising VH and VL domains of native non-human antibodies which exhibit sufficiently high % sequence identity human germline sequences, including for example antibodies comprising VH and VL domains of camelid conventional antibodies, as well as engineered, especially humanized, variants of such antibodies and also "fully human" antibodies.
  • the VH domain of the antibody with high human homology may exhibit an amino acid sequence identity or sequence homology of 80% or greater with one or more human VH domains across the framework regions FR l , FR2, FR3 and FR4.
  • the amino acid sequence identity or sequence homology between the VH domain of the polypeptide of the invention and the closest matching human germline VH domain sequence may be 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%.
  • the VH domain of the antibody with high human homology may contain one or more(e.g.
  • the VL domain of the antibody with high human homology may exhibit a sequence identity or sequence homology of 80% or greater with one or more human VL domains across the framework regions FRl, FR2, FR3 and FR4
  • the amino acid sequence identity or sequence homology between the VL domain of the polypeptide of the invention and the closest matching human germline VL domain sequence may be 85% or greater 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% .
  • the VL domain of the antibody with high human homology may contain one or more (e.g. 1 to 10) amino acid sequence mis-matches across the framework regions FRl, FR2, FR3 and FR4, in comparison to the closest matched human VL sequence.
  • the canonical folds may be determined, w hich allow the identification of the family of human germline segments with the identical combination of canonical folds for H 1 and H2 or L 1 and L2 (and L3).
  • the human germline family member that has the highest degree of sequence homology with the variable region of the antibody of interest is chosen for scoring the sequence homology.
  • the determination of Chothia canonical classes of hypervariable loops L I , L2, L3, I I 1 and H2 can be performed ith the bioinformatics tools publicly available on webpage www . b i o i n f . org . u k/ab s/ch o t h i a . h t m 1. page .
  • the output of the program shows the key residue requirements in a data file. In these data files, the key residue positions are shown with the allowed amino acids at each position.
  • the sequence of the variable region of the antibody of interest is given as input and is first aligned with a consensus antibody sequence to assign the Kabat numbering scheme.
  • the analysis of the canonical folds uses a set of key residue templates derived by an automated method developed by Martin and Thornton (Martin et al., J. Mol. Biol. 263 :800-815 (1996)).
  • Martin and Thornton Martin et al., J. Mol. Biol. 263 :800-815 (1996).
  • the best matching family member in terms of sequence homology can be determined.
  • bioinformatics tools the percentage sequence identity between the VH and VL domain framework amino acid sequences of the antibody of interest and corresponding sequences encoded by the human germline can be determined, but actually manual alignment of the sequences can be applied as well.
  • Human immunoglobulin sequences can be identified from several protein data bases, such as X Base ( http ://vbase. mrc-cpe. cam ac . uk/ ) or the Pluckthun/Honegger database (http:// ' ww i w.bioc.unizh.ch, / antibody/Sequeiices/Germline s).
  • X Base http ://vbase. mrc-cpe. cam ac . uk/
  • Pluckthun/Honegger database http:// ' ww i w.bioc.unizh.ch, / antibody/Sequeiices/Germline s.
  • sequence alignment algorithm such as available via websites like www. expasy.ch/tools/# align can be used, but also manual alignment with the limited set of sequences can be performed.
  • the boundaries of the individual framework regions may be assigned using the I MOT numbering scheme, which is an adaptation of the numbering scheme of Chothia (Lefranc et al., NAR 27: 209-212 (1999); http://im.gt.cines.fr).
  • Antibodies with high human homology may comprise hypervariable loops or CDRs having human or human-like canonical folds, as discussed in detail below.
  • At least one hypervariable loop or CDR in either the VH domain or the VL domain of the antibody with high human homology may be obtained or derived from a VH or VL domain of a non-human antibody, for example a conventional antibody from a species of Camelidae, yet exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.
  • the predicted canonical fold structures for the hypervariable loops of any given VH or VL sequence in an antibody with high human homology can be analysed using algorithms which are publicly available from www.bioinf.org.uk/abs/chothia.html, www .biochem.ucl.ac uk/ - martin/antibodies. html and wwv.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.html. These tools permit query VH or VL sequences to be aligned against human VH or VL domain sequences of known canonical structure, and a prediction of canonical structure made for the hypervariable loops of the query sequence.
  • HI and H2 loops may be scored as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are ful illed:
  • the H I and H2 loops in the antibody of interest may also be scored as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typical ly by +1 or +2 amino acids) but the actual structure of the H I and H2 loops in the antibody of interest matches the structure of a human canonical fold.
  • both HI and H2 in the VH domain of the antibody with high human homology exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.
  • Antibodies with high human homology may comprise a VH domain in which the hypervariable loops H 1 and H2 form a combination of canonical fold structures which is identical to a combination of canonical structures known to occur in at least one human germline VH domain. It has been observed that only certain combinations of canonical fold structures at H I and H2 actually occur in V I 1 domains encoded by the human germline.
  • H I and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g. a Camelidae species, yet form a combination of predicted or actual canonical fold structures hich is identical to a combination of canonical fold structures known to occur in a human germline or somatically mutated VH domain .
  • H 1 and I I 2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g.
  • An antibody with high human homology may contain a V I I domain which exhibits both high sequence identity/sequence homology with human VH, and which contains hypervariable loops exhibiting structural homology with human VI I. It may be advantageous for the canonical folds present at HI and H2 in the VH domain of the antibody with high human homology, and the combination thereof, to be "correct" for the human VH germline sequence which represents the closest match w ith the VH domain of the antibody with high human homology in terms of overall primary amino acid sequence identity.
  • H 1 and H2 may be advantageous for H 1 and H2 to form a combination of canonical folds which also occurs naturally in a human VH3 domain.
  • This may be particularly important in the case of antibodies w ith high human homology which are derived from non-human species, e.g. antibodies containing VH and VL domains which are derived from camelid conventional antibodies, especially antibodies containing humanised camelid VH and VL domains.
  • the VH domain of the GARP antibody w ith high human homology may exhibit a sequence identity or sequence homology of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VH domain across the framework regions FR I , FR2 , FR3 and FR4, and in addition H 1 and 112 in the same antibody are obtained from a non-human VH domain (e.g.
  • LI and L2 in the VL domain of the antibody with high human homology are each obtained from a VL domain of a non-human species (e.g. a camel id-derived VL domain), and each exhibits a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.
  • the hypervariable loops of VL domains of both VLambda and V appa types can adopt a limited number of conformations or canonical structures, determined in part by length and also by the presence of key amino acid residues at certain canonical positions.
  • L 1 , L2 and L3 loops obtained from a VL domain of a non-human species e.g. a Camel idae species, may be scored as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are fulfilled:
  • L 1 , 1.2 or L3 loops derived from the antibody of interest may also be scored as having a canonical fold structure "substantial ly identical" to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typically by +1 or +2 amino acids) but the actual structure of the Camelidae loops matches a human canonical fold.
  • Key amino acid residues found in the human canonical structural classes for the CDRs of human VLambda and V Kappa domains are described by Morea et al.
  • LI and L2 in the VLambda domain of an antibody with high human homology may form one of the following canonical fold combinations: 1 1 -7, 13-7(A,B,C), 14-7( A,B), 1 2- 1 1 , 14- 1 1 and 12- 12 (as defined in Williams et al. J. Mol. Biol. 264:220 -32 (1996) and as shown on http://www.bioc.uzh h/antibody/Sequences/Germlines/VBase_hVL.html).
  • L 1 and L2 in the Vkappa domain may form one of the following canonical fold combinations: 2- 1, 3- 1 , 4- 1 and 6- 1 (as defined in Tomlinson et al. EMBO J . 14:4628-38 ( 1995 ) and as shown on http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase _hVK.html).
  • all three of L 1 , L2 and L3 in the VL domain of an antibody with high human homology may exhibit a substantial ly human structure. It is preferred that the VL domain of the antibody with high human homology exhibit both high sequence identity/sequence homology ith human VL, and also that the hypervariable loops in the VL domain exhibit structural homology with human VL.
  • the VL domain of the GARP antibody with high human homology may exhibit a sequence identity of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VL domain across the framework regions FR I , FR2 , FR3 and FR4, and in addition hypervariable loop L 1 and hypervariable loop L2 may form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VL domain.
  • VH domains exhibiting high sequence identity/sequence homology with human VH, and also structural homology w ith hyperv ariable loops of human VH will be combined with VL domains exhibiting high sequence identity/sequence homology with human VL, and also structural homology with hypervariable loops of human VL to provide antibodies with high human homology containing VH/VL pairings (e.g. camel id-derived VH/VL pairings) with maximal sequence and structural homology to human-encoded VH/VL pairings.
  • VH/VL pairings e.g. camel id-derived VH/VL pairings
  • an antibody is said to be “immunospecific”, “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with the antigen, preferably ith an affinity constant, Ka, of greater than or equal to about 10 4 M _1 , or greater than or equal to about l O 5 M “1 , greater than or equal to about 10 6 M “1 , greater than or equal to about 10 7 M “1 , or greater than or equal to 10 8 M “1 , or greater than or equal to 10 9 M “1 , or greater than or equal to 10 1 ⁇ M "1 .
  • Ka affinity constant
  • Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant Kd, and in certain embodiments, an antibody specifically binds to antigen if it binds with a Kd of less than or equal to 10 "4 M, less than or equal to about 10 "5 M, less than or equal to about 10 "6 M, less than or equal to 10 " ' M, or less than or equal to 10 "8 M, or less than or equal to 5. 10 "9 M, or less than or equal to 10 “9 M, or less than or equal to 5.10 "10 M, or less than or equal to 10 "10 M. Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard G et al.
  • IHC immuno-histochemistry
  • FACS fluorescence-activated cell sorting
  • the term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course. this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
  • polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product.
  • polypeptide Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen.
  • An "isolated polypeptide" is one that has been identified and separated and/or recovered from a component of its natural environment.
  • the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining.
  • Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • identity refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.
  • Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. ⁇ 2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA ( Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al, supra). The well-known Smith Waterman algorithm may also be used to determine identity.
  • NCBI National Center for Biotechnology Information
  • Modified antibody includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain regions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules joined to scFv molecules and the like. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
  • modified antibody includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen).
  • a modified antibody of the invention is a fusion protein comprising at least one heavy chain region lacking a CH2 domain and comprising a binding domain of a polypeptide comprising the binding region of one member of a receptor ligand pair.
  • mammal refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
  • “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by K oh let " et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g. , U. S. Pat. No 4,8 16,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222: 58 1 -597 (1991), for example.
  • “Native sequence” refers to a polynucleotide is one that has the same nucleotide sequence as a polynucleotide derived from nature.
  • a “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g. , antibody) derived from nature (e.g., from any species).
  • Such native sequence polynucleotides and polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
  • a polynucleotide "variant" is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide a s described herein and/or using any of a number of techniques well known in the art.
  • a polypeptide "variant" is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art. Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivativ e polypeptide with desirable characteristics.
  • amino acid sequence of a polypeptide When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, v ariant or region of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells.
  • polypeptides e.g., antigens
  • a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nat ure of the polypeptide to be substantially unchanged.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take several of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
  • “Pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Said excipient does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.
  • Specificity refers to the ability to specifically bind (e.g., immunoreact with) a given target, e.g., GARP.
  • a polypeptide may be monospecific and contain one or more binding sites which specifically bind a target, or a polypeptide may be multi specific and contain two or more binding sites which specifically bind the same or different targets.
  • an antibody of the invention is specific for more than one target.
  • a multispecific binding molecule of the invention binds to GARP and a second molecule expressed on a tumor cell.
  • Exemplary antibodies which comprise antigen binding sites that bind to antigens expressed on tumor cells are known in the art and one or more CDRs from such antibodies can be included in an antibody of the in vention.
  • Synthetic with respect to polypeptides includes polypeptides which comprise an amino acid sequence that is not naturally occurring.
  • non-naturally occurring polypeptides are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion) or polypeptides which comprise a first amino acid sequence (which may or may not be naturally occurring) that is linked in a linear sequence of amino acids to a second amino acid sequence (which may or may not be naturally occurring) to which it is not naturally linked in nature.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” -
  • the terms “Single-chain Fv”, “sFv” or “scFv” are antibody fragments that comprise the VFI and VL antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the VFI and VL domains that enables the sFv to form the desired structure for antigen binding.
  • sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Ver lag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
  • variable region or “variable domain” -
  • variable refers to the fact that certain regions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called “hypervariable loops" in each of the VL domain and the VFI domain which form part of the antigen binding site.
  • the first, second and third hypervariable loops of the V Lambda light chain domain are referred to herein as LI ( ⁇ ), L2 ( ⁇ ) and L3 ( ⁇ ) and may be defined as comprising residues 24-33 (L1( ), consisting of 9, 10 or 11 amino acid residues), 49-53 L2 ( ⁇ ), consisting of 3 residues) and 90-96 (L3 k), consisting of 6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)).
  • the first, second and third hypervariable loops of the VKappa light chain domain are referred to herein as L1(K), L2(K) and L3(K) and may be defined as comprising residues 25-33 (L1(K), consisting of 6, 7, 8, 1 1, 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et al. , Methods 20:267-279 (2000)).
  • the first, second and third hypervariable loops of the VH domain are referred to herein as HI, H2 and H3 and may be defined as comprising residues 25-33 (HI, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et al, Methods 20:267-279 (2000)).
  • L I , L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both Vkappa and V lambda isotypes.
  • HI, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including [gamma], [epsilon], [delta], a or [mu].
  • the hypervariable loops L , L2, L3, HI, H2 and H3 may each comprise part of a "complementarity determining region" or "CDR", as defined below.
  • valency refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes on the same antigen).
  • the subject binding molecules preferably have at least one binding site specific for a human GARP molecule.
  • the GARP antibodies provided herein may be at least bivalent.
  • Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully "treated” for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, of one or more of the symptoms associated with the specific disease or condition, reduced morbidity and mortality, and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • TGF- ⁇ refers to the three isoforms named TGF- ⁇ , TGF-P2 and ⁇ - ⁇ 3.
  • the peptide structures of the TGF- ⁇ isoforms are highly similar (homologies on the order of 70-80 %). They are all encoded as large protein precursors, TGF- ⁇ 1 (Gen Bank Access No: NM_000660 contains 390 amino acids and ⁇ : - ⁇ 2 (GenBank Access No: NM 001 135599 and NM 003238) and TGF ⁇ 33 (GenBank Access No: XM_005268028) each contain 4 12 amino acids.
  • TGF- ⁇ 1 has the following amino acid sequence:
  • LAP has the following amino acid sequence:
  • TGF- ⁇ ⁇ has the following amino acid sequence:
  • One object of the invention is a protein binding to GARP in the presence of TGF-13.
  • Another object of the invention is a protein comprising an antigen binding domain, wherein the antigen binding domain binds specifically to GARP in the presence of
  • said protein binds to GARP only in the presence of TGF- ⁇ .
  • GARP is also called Leucin Rich Repeat Containing 32 ( LRRC32) and belongs to the Leucin Rich Repeat family.
  • SEQ I D NO: 1 GenBank Accession NJV1 001 128922
  • the protein of the invention binds to GARP when GARP is complexed to T GF-13.
  • the protein of the invention binds to GARP when GARP is complexed to latent TGF-13. In another embodiment, the protein of the invention binds to a complex of G ARP and TGF-13. In an embodiment, the protein of the invention binds to a complex of GARP and TGF- B l ; TGF-B2, isoform 1 ; TGF-B2, isoform 2; TGF-B3. Preferably, the protein of the invention binds to a complex of GARP and TGF-B 1 .
  • the protein of the invention binds to a complex of G ARP and latent TGF-B.
  • lymphothelial TGF-B as used herein comprises a complex whose C-terminal fragment, or mature TGF-B 1 , remains non-covalently bound to the N-terminal fragment known as LAP.
  • the protein of the invention binds to a complex of G ARP and latent TGF-B at a KD (the equilibrium dissociation constant between the antibody and its antigen) of less than 10 "! 0 M.
  • said protein is an antibody molecule selected from the group consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a F(ab)'2, a defucosylated antibody, a bi- specific antibody, a diabody, a triabody, a tetrabody.
  • said protein is an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody.
  • said protein is an antibody mimetic selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, a versabody and a duocalin.
  • a domain antibody is well known in the art and refers to the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies.
  • a nanobody is ell known in the art and refers to an antibody-derived therapeutic protein that contains the unique structural and functional properties of naturally- occurring heavy chain antibodies. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3).
  • VHH single variable domain
  • CH2 and CH3 constant domains
  • a unibody is well known in the art and refers to an antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies.
  • An affibody is well known in the art and refers to affinity proteins based on a 58 amino acid residue protein domain, derived from one of the IgG binding domain of staphylococcal protein A.
  • DARPins Designed Ankyrin Repeat Proteins
  • DRP designed repeat protein
  • Anticalins are well known in the art and refer to another antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins.
  • Avimers are ell known in the art and refer to another antibody mimetic technology. Versabodies are well known in the art and refer to another antibody mimetic technology. They are small proteins of 3-5 kDa with >15% cysteines, which form a high disulfide density scaffold, replacing the hydrophobic core the typical proteins have.
  • said protein is an immunoconjugate comprising an antibody or fragment thereof conjugated to a therapeutic agent.
  • said protein is a conjugate comprising the protein of the invention conjugated to an imaging agent. Said protein could be used for example for imaging applications.
  • Another object of the invention is a protein that binds to G ARP and inhibits TGF- ⁇ signaling.
  • said protein binds to GARP when GARP is complexed to TGF- ⁇ .
  • said protein binds to GARP when GARP is complexed to latent TGF- ⁇ .
  • said protein binds to a complex of GARP and TGF-13. In another embodiment, said protein binds to a complex of GARP and latent TGF-B.
  • said protein is an antibody molecule selected from the group consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a F(ab)'2, a defucosylated antibody, a bi- specific antibody, a diabody, a triabody, a tetrabody.
  • said protein is an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody.
  • said protein is an antibody mimetic selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, a versabody and a duocalin.
  • said protein is an anti-hGARP (anti human GARP) antibody or antigen binding fragment thereof that inhibits TGF-B signaling.
  • anti-hGARP anti human GARP
  • said protein prevents or inhibits active TGF-B to be released or inhibits the release of mature TGF-B from GARP/ TGF-B. In an embodiment, said protein prevents or inhibits the release of active TGF-B from membrane-bound GARP/TGF-B.
  • said protein prevents or inhibits active TGF-B to be released or inhibits the release of mature TGF-B from Tregs.
  • said protein inhibits or prevents mature TGF-B to bind to TGF- B receptors.
  • said protein inhibits TGF-B activity and/or the activation of molecules from the TGF-B receptor signaling pathway.
  • the term “inhibit” means that the protein is capable of blocking, reducing, preventing or neutralizing TGF-B signaling or the release of mature TGF-B from Tregs or the binding of mature TGF-B to TGF-B receptors or TGF-B activity and/or the activation of molecules from the TGF-B receptor signaling pathway.
  • said protein is a monoclonal antibody. In another embodiment, said protein is a polyclonal antibody. In an embodiment, said protein binds to a conformational epitope.
  • said conformational epitope comprises one or more amino acids of hGARP.
  • said conformational epitope comprises an epitope of GARP modified as a result of GARP being complexed with latent TGF- ⁇ .
  • said conformational epitope comprises amino acids of hGARP and amino acids of latent TGF-B.
  • said conformational epitope is a mixed conformational epitope and comprises amino acids from both GARP and TGF-B.
  • said conformational epitope is a binding-induced conformational epitope and comprises amino acids from GARP only, but that adopts a different conformation in the presence of TGF-B.
  • said epitope comprises one or more residues from 101 to 141 residues of hG ARP amino acid sequence (SEQ ID NO: 1 ).
  • said epitope comprises the residues 137, 138 and 139: YSG of hGARP amino acid sequence ( SEQ ID NO: 1). In another embodiment of the invention, said epitope comprises the residues 137, 138 and 139: YSG of hGARP amino acid sequence ( SEQ ID NO: 1) and requires the presence of TGF-B.
  • said epitope comprises the residues 137, 138 and 139: YSG of hGARP amino acid sequence (SEQ ID NO: 1) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20 contiguous residues in N-terminal and/or C- terminal of the residues 137, 138 and 139: YSG of SEQ ID NO: 1 .
  • said epitope comprises the residues 137, 138 and 139: YSG of hG ARP amino acid sequence (SEQ ID NO: 1) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 contiguous residues in N-terminal and/or C- terminal of the residues 137, 138 and 139: YSG of SEQ ID NO: 1, and requires the presence of TGF- ⁇ .
  • the protein of the invention binds to epitopes preferably within the region 10 1 - 14 1 of hGARP and inhibits the release of latent TGF-13 from GARP.
  • One skilled in the art can determine the ability of a protein to inhibit TGF-13 signaling by measuring for example activation of molecules from the TGF- ⁇ receptor signaling pathway.
  • One example of such test is the measurement of the phosphorylation of SMAD2 (as shown in Example 2 of the present invention).
  • Another object of the invention is a protein binding to an epitope of a complex formed by human GARP and TGF-13, said epitope comprising at least one of the residues 137, 138, or 139 of GARP (SEQ I D NO: 1) and at least one residue of TGF-13 (SEQ ID NO: 53).
  • said protein is an antibody or an antigen binding fragment thereof.
  • said antibody or antigen binding fragment thereof is selected from the group consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a F(ab)'2, a defucosylated antibody, a bi-specific antibody, a diabody, a triabody, a tetrabody; or an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody; or an antibody mimetic selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, a versabody and a duocalin.
  • the epitope comprises one, two or three of the residues 137, 138, and 139 of GARP (SEQ I D NO: 1). In another embodiment, the epitope comprises at least one of the residues 137, 138, or 139 of G ARP (SEQ ID NO: 1) and at least one residue from the Latency associated peptide ( LAP ) of TGF-B (SEQ ID NO: 54) and at least one residue from mature TGF-B
  • the epitope comprises at least one of the residues 137, 138, or 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 1 8, 19, or 20 residue(s) from the Latency associated peptide (LAP) ( SEQ ID NO: 54) and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from mature TGF-B ( SEQ ID NO: 55).
  • LAP Latency associated peptide
  • the epitope comprises one, two or three of the residues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from mature TGF-B (SEQ ID NO: 55).
  • LAP Latency associated peptide
  • the epitope comprises one, two or three of the residues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least I, 2, 3, 4, 5, 6, 7, or 8 residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) selected from the group of residues 58, 100, 146, 269, 270, 271, 272, 273 of TGF-B (SEQ ID NO: 53) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from mature TGF-B (SEQ ID NO: 55).
  • LAP Latency associated peptide
  • the epitope comprises one, two or three of the residues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least I, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) and at least 1, 2, 3, 4, 5, or 6 residue* s) from mature TGF-B (SEQ ID NO: 55) selected from the group of residues 284, 336, 337, 338, 341, and 345 of TGF B (SEQ ID NO: 53).
  • LAP Latency associated peptide
  • SEQ ID NO: 55 selected from the group of residues 284, 336, 337, 338, 341, and 345 of TGF B (SEQ ID NO: 53).
  • the epitope comprises one, two or three of the residues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, or 8 residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) selected from the group of residues 58, 100, 146, 269, 270, 271, 272, and 273 of TGF- ⁇ (SEQ ID NO: 53) and at least 1, 2, 3, 4, 5, or 6 residue(s) from mature TGF-B (SEQ ID NO: 55) selected from the group of residues 284, 336, 337, 338, 34 1 , and 345 of TGF 13 (SEQ ID NO: 53 ).
  • LAP Latency associated peptide
  • the epitope comprises one, two or three of the residues 137, 138, and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 1 3, or 14 residue(s) selected from the group of residues 58, 100, 146, 269, 270, 27 1 , 272, 273, 284, 336, 337, 338, 341, and 345 of TGF-B (SEQ ID NO: 53).
  • the epitope comprises at least one, two or three of the residues 137, 138 and 139 of GARP (SEQ ID NO: 1) and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, or 19 residue(s) selected from the group of residues 1 13, 1 14, 1 16, 1 17, 1 18, 1 19, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 1 89 of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 1 8, 19, or 20 residue(s) from mature TGF-B ( SEQ ID NO: 55).
  • LAP Latency associated peptide
  • the epitope comprises at least one, two or three of the residues 137, 138 and 139 of GARP (SEQ ID NO: 1 ) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, or 19 residue(s) selected from the group of residues 1 13, 1 14, 1 16, 1 17, 1 18, 1 19, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 189 of GARP ( SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, or 8 residue(s) from the Latency associated peptide (LAP) selected from the group of residues 58, 100, 146, 269, 270, 271, 272, and 273 of TGF-B and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from mature TGF-B (SEQ ID NO: 55).
  • LAP Latency associated peptide
  • the epitope comprises at least one, two or three of the residues 137, 138 and 139 of GARP (SEQ ID NO: 1) and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, or 19 residue(s) selected from the group of residues 1 13, 1 14, 1 16, 1 17, 1 18, 1 19, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 1 89 of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residue(s) from the Latency associated peptide (LAP) (SEQ ID NO: 54) and at least 1, 2, 3, 4, 5, or 6 residue(s) from mature TGF- ⁇ (SEQ ID NO: 55) selected from the group of residues 284, 336, 337, 338, 341 , and 345 of TGF 13 (SEQ ID NO: 53).
  • LAP Latency associated peptide
  • the epitope comprises at least one, two or three of the residues 137, 138 and 139 of GARP (SEQ ID NO: 1) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 1 2, 13, 14, 15, 1 6, 1 7, 1 8, or 19 residue(s) selected from the group of residues I 13, 1 14, 1 16, 1 17, 1 18, 1 19, 140, 142, 143, 144, 145, 146, 162, 163, 165, 166, 167, 170 and 189 of GARP (SEQ ID NO: 1 ) and at least 1, 2, 3, 4, 5, 6, 7, or 8 residue(s) from the Latency associated peptide (L AP) selected from the group of residues 58, 100, 146, 269, 270, 271, 272, and 273 of TGF- ⁇ and at least 1 , 2, 3, 4, 5, and 6 residue(s) from mature TGF- ⁇ ( SEQ ID NO: 55 ) selected from the group of residues 284, 336, 337, 338, 34 1 , and 3
  • An object of the invention is an antibody against human GARP or antigen binding fragment thereof wherein the variable region of the heavy chain comprises at least one of the fallowings CDRs:
  • VH-CDR1 GFSLTGYGIN (SEQ ID NO: 2) or GYGIN (SEQ ID NO: 52);
  • VH-CDR2 MIWSDGSTDYNSVLTS (SEQ ID NO: 3);
  • VH-CDR3 DRNYYDYDGAMDY (SEQ ID NO: 4).
  • Another object of the invention is an anti-hGARP antibody or antigen binding fragment thereof wherein the variable region of the light chain comprises at least one of the follow ings CDRs:
  • VL-CDRl KASDHIKNWLA (SEQ I D NO: 5);
  • VL-CDR2 GATSLEA ( SEQ ID NO: 6);
  • VL-CDR3 QQYWSTPWT (SEQ ID NO: 7).
  • Another object of the inv ention is an antibody against human G ARP or antigen binding fragment thereof wherein the variable region of the heavy chain comprises at least one of the followings CDRs:
  • VH-CDR1 SYYID ( SEQ ID NO: 13);
  • VH-CDR2 RTOPEDGGTKYAQKFQG (SEQ ID NO: 14); and VH-CDR3: NEWETVWGDLMYEYEY (SEQ ID NO: 15).
  • Another object of the invention is an anti-hGARP antibody or antigen binding fragment thereof wherein the variable region of the light chain comprises at least one of the fallowings CDRs:
  • VL-CDR1 QASQXil X 2 S X 3 LA (SEQ ID NO: 16), wherein Xi is S or T, X 2 is S or V, X 3 is Y or F;
  • VL-CDR2 1 1 SX 5X X5T (SEQ ID NO: 17), wherein X, is G or R; X 2 is A or
  • X 3 is R or I
  • X 4 is L or P
  • X 5 is Q or K
  • VL-CDR3 QQYX] SX 2 PX 3 T, wherein Xj is D, A, Y or V; X 2 is A, L or V; X 3 is V or P (SEQ ID NO: 18).
  • variable region of the heavy chain comprises the VH-CDR l of SEQ ID NO: 13, VH-CDR2 of SEQ ID NO: 14 and VH-CDR3 of SEQ ID NO: 15 and the variable region of the light chain comprises at least one of VL-CDR l.
  • SEQ ID NO: 19 as set forth in SEQ ID NO: 19; SEQ ID NO: 22; SEQ ID NO: 25; SEQ ID NO: 28; or SEQ ID NO: 31; at least one of VL-CDR2 as set forth in SEQ ID NO: 20; SEQ ID NO: 23; SEQ ID NO: 26; SEQ ID NO: 29; or SEQ ID NO: 32 and at least one of VL-CDR3 as set forth in SEQ ID NO: 21 ; SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 30; or SEQ ID NO: 33.
  • Another object of the invention is an anti-hGARP antibody or antigen binding fragment thereof wherein the variable region of the light chain comprises at least one of the followings CDRs:
  • VL-CDR2 GASRLQT (SEQ ID NO: 20);
  • VL-CDR3 QQYDSLPVT (SEQ ID NO: 2 1 ).
  • Another object of the invention is an anti-hGARP antibody or antigen binding fragment thereof wherein the variable region of the light chain comprises at least one of the followings CDRs:
  • VL-CDRl QASQSIVSYLA (SEQ ID NO: 22); VL-CDR2: GASRLQT (SEQ ID NO: 23); and
  • VL-CDR3 QQYASAPVT (SEQ ID NO: 24).
  • Another object of the invention is an anti-hGARP antibody or antigen binding fragment thereof wherein the variable region of the light chain comprises at least one of the followings CDRs:
  • VL-CDRl QASQSISSYLA (SEQ ID NO: 25);
  • VL-CDR2 GTSRLKT (SEQ ID NO: 26);
  • VL-CDR3 QQYYSAPVT (SEQ ID NO: 27).
  • Another object of the invention is an anti-hGARP antibody or antigen binding fragment thereof wherein the variable region of the light chain comprises at least one of the followings CDRs:
  • VL-CDRl QASQTISSFLA (SEQ ID NO: 28);
  • VL-CDR2 RASIPQT (SEQ ID NO: 29);
  • VL-CDR3 QQYVSAPPT (SEQ ID NO: 30).
  • Another object of the invention is an anti-hGARP antibody or antigen binding fragment thereof wherein the variable region of the light chain comprises at least one of the followings CDRs:
  • VL-CDRl QASQSISSYLA (SEQ ID NO: 31);
  • VL-CDR2 GASRLKT (SEQ ID NO: 32);
  • VL-CDR3 QQYASVPVT (SEQ ID NO: 33).
  • the anti-hGARP antibody or antigen binding fragment thereof may comprise the CHI domain, hinge region, CH2 domain and CH3 domain of a human antibody, in particular IgGl, IgG2, IgG3 or IgG4.
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its heavy chain the following CDRs: VH-CDR 1 GFSLTGYGIN (SEQ ID NO: 2), VH-CDR2 MIWSDGSTDYNSVLTS (SEQ ID NO: 3) and VH-CDR3 DRNYYDYDGAMDY (SEQ ID NO: 4).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its heavy chain the following CDRs: VH-CDR 1 GYGIN (SEQ ID NO: 52), VH-CDR2 MIWSDGS ' l DYNSVLTS (SEQ ID NO: 3) and VH- CDR3 DRNYYDYDGAMDY (SEQ ID NO: 4).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its light chain the following CDRs: VL-CDR 1 KASDHIKNWLA (SEQ ID NO: 5), VL-CDR2 GATSLEA (SEQ ID NO: 6) and VL-CDR3 QQYWSTPWT (SEQ ID NO: 7).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its heavy chain the following CDRs: VH-CDR1 SYYID (SEQ ID NO: 13), VH-CDR2 RIDPEDGGTKYAQKFQG (SEQ I D NO: 14) and VH-CDR3 NEWETVVVGDLMYEYEY ( SEQ ID NO: 15).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its light chain the following CDRs: VL-CDR I QASQXil X 2 SX 3 LA (SEQ ID NO: 16), wherein Xi is S or T, X 2 is S or V, X 3 is Y or F; VL-CDR2 XiX 2 SX 3 X 4 X 5 T ( SEQ ID NO: 17), wherein X] is G or R; X 2 is A or T; X 3 is R or I; X 4 is L or P; X 5 is Q or K; and VL-CDR3 QQYX 1 SX 2 PX 3 T, wherein Xi is D, A, Y or V; X 2 is A, L or V; X 3 is V or P (SEQ ID NO: 18).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its light chain the follow ing CDRs: VL-CDR 1 QASQSISSYLA ( SEQ ID NO: 19), VL-CDR2 GASRLQT ( SEQ ID NO: 20), and VL-CDR3 QQYDSLPVT ( SEQ ID NO: 2 1 ).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its light chain the following CDRs: VL-CDR 1 QASQSIVSYLA (SEQ I D NO: 22); VL-CDR2 GASRLQT (SEQ ID NO: 23); and VL-CDR3 : QQYASAPVT (SEQ ID NO: 24).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its light chain the following CDRs: VL-CDR 1 QASQSISSYLA (SEQ ID NO: 25); VL-CDR2 GTSRLKT (SEQ ID NO: 26); and VL-CDR3 QQYYSAPVT (SEQ ID NO: 27).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its light chain the following CDRs: VL-CDR1 QASQTISSFLA (SEQ ID NO: 28); VL-CDR2 RASIPQT (SEQ ID NO: 29); and VL-CDR3 QQYVSAPPT (SEQ ID NO: 30).
  • the anti-hGARP antibody or antigen binding fragment thereof comprises in its light chain the following CDRs: VL-CDR I QASQSISSYLA (SEQ ID NO: 31); VL-CDR2 GASRLKT (SEQ ID NO: 32); and VL-CDR3 QQY ASVPVT (SEQ ID NO: 33).
  • any of the CDRs 1, 2 and 3 of the heavy and light chains may be characterized as having an amino acid sequence that shares at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NO.
  • the anti-hGARP antibody or antigen binding fragment thereof is selected from the group consisting of an antibody having:
  • the anti-hGARP antibody or antigen binding fragment thereof is selected from the group consisting of an antibody having: (i) the heavy chain CDR 1 , 2 and 3 (VH-CDR1 , VH-CDR2, VH-CDR3 ) amino acid sequences as shown in SEQ I D NO: 52, 3 and 4; and
  • the anti-hGARP antibody or antigen binding fragment thereof is selected from the group consisting of an antibody having:
  • the anti-hGARP antibody or antigen binding fragment thereof comprises:
  • the anti-hGARP antibody or antigen binding fragment thereof comprises:
  • the anti-hGARP antibody or antigen binding fragment thereof comprises: (i) the heavy chain CDR 1, 2 and 3 (VH-CDR I , VH-CDR2, VH-CDR3) amino acid sequences as shown in SEQ ID NO: 13, 14 and 15; and
  • the anti-hGARP antibody or antigen binding fragment thereof comprises:
  • the anti-hGARP antibody or antigen binding fragment thereof comprises:
  • the anti-hGARP antibody or antigen binding fragment thereof comprises a variable heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 4 ( DRN Y YD YDG AMD Y ), or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence.
  • the anti-hGARP antibody or antigen binding fragment thereof comprises a variable heavy chain CDR3 comprising an amino acid sequence of SEQ I D NO: 15, or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence.
  • Another object of the invention is the anti-hGARP antibody MHGARP8 or antigen binding fragment thereof comprising a heavy chain variable region of sequence SEQ ID NO: 8 and a light chain variable region of sequence SEQ ID NO: 9.
  • Another object of the invention is the anti-hGARP antibody MHGARP8 or antigen binding fragment thereof comprising a heavy chain variable region of sequence SEQ ID NO: 50 and a light chain variable region of sequence SEQ ID NO: 51, wherein SEQ ID NO: 50 and SEQ ID NO: 51 correspond, respectively, to SEQ ID NO: 8 and SEQ ID NO: 9 wherein the signal peptide sequences were removed.
  • Another object of the invention is the anti-hG ARP antibody LHG I 0 or antigen binding fragment thereof comprising a heavy chain variable region of sequence SEQ ID NO: 34 and a light chain variable region of sequence SEQ ID NO: 35.
  • VGDLMYEYEYWGQGTQVTVSS (SEQ ID NO: 34).
  • DIQMTQSPTSLSASLGDRVTITCQASQSISSYLAWYQQKPGQAPKLLIYGASRLQ TG SRPSGSGSGTSFTLTISGLEAEDAGTY ⁇ YCQQY ' DSLPVTFGQGTKVELK (SEQ ID NO: 35).
  • Another object of the invention is the anti-hGARP antibody LHG I 0.3 or antigen binding fragment thereof comprising a heavy chain v ariable region of sequence SEQ ID NO: 34 and a light chain variable region of sequence SEQ ID NO: 36.
  • Another object of the inv ention is the anti-hGARP antibody LHG I O.4 or antigen binding fragment thereof comprising a heav y chain v ariable region of sequence SEQ ID NO: 34 and a light chain variable region of sequence SEQ ID NO: 37.
  • Another object of the inv ention is the anti-hGARP antibody LHG I 0.5 or antigen binding fragment thereof comprising a heav y chain v ariable region of sequence SEQ ID NO: 34 and a light chain variable region of sequence SEQ ID NO: 38.
  • Another object of the invention is the anti-hGARP antibody LHG10.6 thereof comprising a heavy chain variable region of sequence SEQ ID NO: 34 and a light chain variable region of sequence SEQ ID NO: 39.
  • amino acids of the heavy chain or light chain variable regions as described here above may be substituted by a different amino acid.
  • an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the MHGARP8 antibody described herein, wherein the antibodies retain the desired functional properties of the protein of the invention.
  • sequence of the heavy chain variable region of an anti-hGARP antibody of the invention encompasses sequences that have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 8 or with
  • sequence of light chain variable region of an anti-hGARP antibody of the invention encompasses sequences that have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 9 or with SEQ ID NO: 51.
  • an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the LUG 10 antibody described herein, and wherein the antibodies retain the desired functional properties of the protein of the invention.
  • sequence of the heavy chain variable region of an anti-hG ARP antibody of the invention encompasses sequences that have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 34.
  • the sequence of light chain variable region of an anti-hGARP antibody of the invention encompasses sequences that have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 35; 36; 37; 38 or 3 .
  • the specified variable region and CDR sequences may comprise conservative sequence modifications.
  • Conservative sequence modifications refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are typically those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties.
  • Specified variable region and CDR sequences may comprise one, two, three, four or more amino acid insertions, deletions or substitutions. Where substitutions are made, preferred substitutions ill be conservative modifications. Families of amino acid residues having simi lar side chains have been defined in the art. These fami lies include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g.
  • aspartic acid, glutamic acid uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine).
  • uncharged polar side chains e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methi
  • anti-hGARP antibodies may also be CDR-grafted antibodies in which the CDRs are derived from a camelid antibody, for example a camelid anti-hGARP antibody raised by active immunization with liGARP.
  • the invention provides an antibody that binds essentially the same epitope as the MI IGARP8 or LHG10 antibody.
  • anti-hGARP antibodies comprising VH and VL domains, or CDRs thereof may comprise CHI domains and/or CL domains, the amino acid sequence of which is fully or substantially human.
  • the antigen binding polypeptide of the invention is an antibody intended for human therapeutic use, it is typical for the entire constant region of the antibody, or at least a part thereof, to have a fully or substantially human amino acid sequence. Therefore, one or more or any combination of the CHI domain, hinge region, CH2 domain, CHS domain and CL domain (and CH4 domain if present) may be fully or substantially human with respect to its amino acid sequence.
  • the CHI domain, hinge region, CH2 domain, CHS domain and CL domain may all have a fully or substantially human amino acid sequence.
  • substantially human refers to an amino acid sequence identity of at least 90%, or at least 95%, or at least 97%, or at least 99% with a human constant region.
  • human amino acid sequence in this context refers to an amino acid sequence which is encoded by a human immunoglobulin gene, which includes germline, rearranged and somatically mutated genes.
  • the invention also contemplates polypeptides comprising constant domains of ' " human " sequence which have been altered, by one or more amino acid additions, deletions or substitutions with respect to the human sequence, excepting those embodiments where the presence of a "fully human " hinge region is expressly required.
  • the presence of a ""fully human " hinge region in the anti-hGARP antibodies of the invention may be beneficial both to minimize immunogenicity and to optimize stability of the antibody. It is considered that one or more amino acid substitutions, insertions or deletions may be made within the constant region of the heavy and/or the light chain, particularly within the Fc region. Amino acid substitutions may result in replacement of the substituted amino acid with a different naturally occurring amino acid, or with a non-natural or modified amino acid.
  • a GARP antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3 :219-230 (1989).
  • the invention also contemplates immunoconjugates comprising an antibody as described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Fc regions may also be engineered for half-life extension, as described by Chan and Carter, 2010 Nature Reviews: Immunology, 10:301 -316, incorporated herein by reference.
  • Variant anti- hGARP antibodies in which the Fc region is modified by protein engineering, as described herein may also exhibit an improvement in efficacy (e.g. in therapeutics/diagnostics), as compared to an equivalent antibody (i.e. equivalent antigen-binding properties) without the Fc modification.
  • the Fc region is modified to increase the abilitv of the antibody to mediate antibody dependent cellular cytotoxicity ( ADCC ) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids.
  • the glycosylation of an antibody is modified.
  • an aglycoslated antibody can be made (i.e. , the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for the GARP target antigen.
  • Such carbohydrate modifications can be accomplished by; for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • variant anti-hGARP antibodies having an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or a non-fucosylated antibody (as described by Natsume et al., 2009 Drug Design Development and Therapy, 3 : 7- 16) or an antibody having increased bisecting GlcNac structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ADCC activity of antibodies, producing typically 10-fold enhancement of ADCC relative to an equivalent antibody comprising a "native " human Fc domain.
  • Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation enzymatic machinery (as described by Yamane-Ohnuki and Satoh, 2009 mAbs l(3):230-236).
  • the anti-hGARP antibody comprises an Fc region having the sequence SEQ ID NO: 47.
  • the anti-hGARP antibody comprises the heavy chain constant domain region having the sequence SEQ ID NO: 48, wherein X is N or is mutated into Q to inhibit ADCC.
  • the residue 297 of SEQ ID NO: 48 is aglycosylated.
  • the N residue at the position 297 of SEQ ID NO: 48 is mutated into Q.
  • the anti-hG ARP antibody comprises the light chain constant domain region having the sequence SEQ ID NO: 49.
  • anti-hGARP antibodies may be lacking effector function, either because the Fc region of the antibody is of an isotype which naturally lacks effector function, or which exhibits significantly less potent effector function than human IgGl, for example human IgG2 or human IgG4, or because the Fc region of the antibody has been engineered to reduce or substantially eliminate effector function, as described in Armour KL, et al., Eur. J. Immunol , 1999, 29:2613-2624.
  • the Fc region of the anti-hG ARP antibody may be engineered to facilitate the preferential formation of bispecific antibodies, in which two antibody heavy chains comprising different variable domains pair to form the Fc region of the bispecific antibody.
  • the anti-hGARP antibody of the invention may exhibit one or more effector functions selected from antibody-dependent cel l-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP) against cells expressing human GARP protein on the cell surface.
  • the antibody may exhibit ADCC against GARP-related dysfunctional cells.
  • the antibody may exhibit enhanced ADCC function in comparison to a reference antibody w hich is an equivalent antibody comprising a native human Fc domain.
  • the ADCC function may be at least lOx enhanced in comparison to the reference antibody comprising a native human Fc domain.
  • the antibody with enhanced ADCC function displays substantially identical antigen-binding specificity and/or shares identical amino acid sequence with the reference antibody, except for any modifications made (relative to native human Fc) for the purposes of enhancing ADCC.
  • the antibody may contain the hinge region, CHI domain, CH2 domain and C B domain of a human IgG, most preferably human IgGl .
  • the antibody may include modifications in the Fc region, such as for example substitutions, deletions or insertion or other structural modifications to enhance or reduce Fc-dependent functionalities.
  • One object of this invention relates to anti-hGARP antibodies or antigen binding fragment thereof which inhibit TGF- ⁇ signaling, and that may be particularly suitable for therapeutic applications which benefit from antibody effector function, i.e. ADCC, CDC, ADCP, and in particular enhanced effector function.
  • the GARP antibodies described herein which exhibit effector function (or enhanced effector function) and which inhibit TGF- ⁇ may be particularly advantageous for certain therapeutic applications, e.g. cancer, chronic infection, and fibrosis treatments which benefit from antibody effector function.
  • Another object of the invention is an isolated polynucleotide sequence encoding the heavy chain variable region of sequence SEQ ID NO: 8 or of SEQ ID NO: 50.
  • said nucleic sequence is SEQ ID NO: 10:
  • Another object of the invention is an isolated polynucleotide sequence encoding the light chain variable region of sequence SEQ ID NO: 9 or of SEQ ID NO: 51.
  • said nucleic sequence is SEQ ID NO: 11 :
  • Another object of the invention is an expression vector comprising the nucleic sequences encoding the anti-hGARP antibody of the invention, in an embodiment, the expression vector of the invention comprises at least one of SEQ ID NO: 10 and SEQ ID NO: 11 or any sequence having a nucleic acid sequence that shares at least: 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with said SEQ ID NO : 10 and SEQ ID NO : 1 1.
  • host cells may be prokaryotic, yeast, or eukaryotic cells, and are preferably mammalian cells, such as, for example: monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et a!., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHQ, Urlaub et al., Proc. Natl.
  • COS-7 monkey kidney CVl line transformed by SV40
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et a!., J. Gen. Virol. 36:59 (1977)
  • baby hamster kidney cells BHK, ATCC CCL 10
  • Chinese hamster ovary cells/-DHFR CHQ, Urlaub et al., Proc. Natl.
  • mice Sertoli cells TM4, Mather, Biol. Reprod. 23 :243-25 I (1980)
  • mouse myeloma cells SP2/0-AG14 ATCC CRL 1581 ; ATCC CRL 8287
  • NSO HP A culture collections no.
  • monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat fiver cells (BRL 3A, ATCC CRL 1442); human lung ceils (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 5 1 ); TRI cells (Mather et al, Annals N.Y. Acad. Sci.
  • host cell generally refers to a cultured cell line. Whole human beings into which an expression vector encoding an antigen binding polypeptide according to the invention has been introduced are explicitly excluded from the definition of a "host cell”.
  • Another object of the invention is a method of producing an anti-hGARP antibody or antigen binding fragment thereof which comprises culturing host cells containing the isolated polynucleotide sequence encoding the anti-hGARP antibody under conditions suitable for expression of the anti-hGARP antibody, and recovering the expressed anti- hGARP antibody.
  • This recombinant process can be used for large scale production of GARP antibodies according to the invention, including antibodies monoclonal antibodies intended for in vitro, ex vivo, in vivo therapeutic, diagnostic uses. These processes are available in the art and will be known by the skilled person.
  • Another object of the invention is a hybridoma cell line which can be used to produce said antibody of the invention.
  • a preferred hybridoma cell line according to the invention was deposited with the BCCM/LMBP Plasmid Collection, Department of Biomedical Molecular Biology, Ghent University, Tiers-Schell-Van Montagu' building, Technologiepark 927, B-9052 Gent - Zwijnaarde BELGIUM (Table 2):
  • Fragments and derivatives of antibodies of this invention (which are encompassed by the term “antibody “ or “antibodies “ as used in thi s application, unless otherwise stated or clearly contradicted by context), preferably a MHGARP8-like antibody, can be produced by techniques that are known in the art. "Fragments " comprise a region of the intact antibody, generally the antigen binding site or variable region.
  • antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment” or “single chain polypeptide"), including without limitation (1) single-chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi-specific antibodies formed from antibody fragments.
  • single-chain antibody fragment single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety
  • Fragments of the present antibodies can be obtained using standard methods. For instance, Fab or F(ab')2 fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques. It will be appreciated that immune-reactive fragments can be modified using known methods, for example to slow clearance in vivo and obtain a more desirable pharmacokinetic profile the fragment may be modified with polyethylene glycol (PEG). Methods for coupling and site-specifically conjugating PEG to a Fab " fragment are described in, for example, Leong et al. Cytokines 16 (3): 106- 1 19 (2001) and Delgado et al, Br J. Cancer 73 (2): 1 75- 1 82 (1 996), the disclosures of which are incorporated herein by reference.
  • PEG polyethylene glycol
  • the DNA of a hybridoma producing an antibody of the invention may be modified so as to encode a fragment of the invention.
  • the modified DNA is then inserted into an expression vector and used to transform or transfect an appropriate cell, which then expresses the desired fragment.
  • the DNA of a hybridoma producing an antibody of this invention can be modified prior to insertion into an expression vector, for example, by substituting the coding sequence for human heavy- and light- chain constant domains in place of the homologous non- human sequences ⁇ e.g. , Morrison et al., PNAS pp. 6851 ( 1 84 )), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • “chimeric " or "hybrid " antibodies may be prepared that have the binding specificity of the original antibody.
  • such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody of the invention.
  • the antibody of this invention preferably a MHGARP8 or LUG 1 0-like antibody
  • "Humanized” forms of antibodies according to this invention are speci ic chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab ' )2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from the murine immunoglobulin .
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of the original antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody.
  • CDR complementary-determining region
  • Fv framework ( FR) residues of the human immunoglobulin may be replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in either the recipient antibody or in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantial ly all of the CDR regions correspond to those of the original antibody and al l or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a region of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • variable domains both light and heavy
  • sequence of the variable domain of an antibody of this invention is screened against the entire library of known human variable-domain sequences.
  • the human sequence that is closed to the mouse sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol. 151 , pp. 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196, pp. 901).
  • Another method uses a particular framework from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly avai lable and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional structures of selected candidate immunoglobulin sequences.
  • a XenoMouse is a murine host according to this invention that has had its immunoglobulin genes replaced by functional human immunoglobulin genes.
  • antibodies produced by this mouse or in hybridomas made from the B cells of this mouse are already humanized.
  • the XenoMouse is described in United States Patent No 6, 162,963, which is herein incorporated in its entirety by reference.
  • Human antibodies may also be produced according to various other techniques, such as by using, for immunization, other transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al. Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods.
  • Camehdae hypervariable loops may be obtained by active immunization of a species in the familv Camehdae with a desired target antigen.
  • Camehdae either the native animal or a transgenic animal engineered to express the immunoglobulin repertoire of a camelid species
  • B cells producing (conventional Camehdae) antibodies having specificity for the desired antigen can be identified and polynucleotide encoding the VH and VL domains of such antibodies can be isolated using known techniques.
  • the invention provides a recombinant antigen binding polypeptide immunoreactive with a target antigen, the polypeptide comprising a VH domain and a VL domain, wherein at least one hypervariable loop or complementarity determining region in the VH domain or the VL domain is obtained from a VH or VL domain of a species in the family Camehdae, which antigen binding polypeptide is obtainable by a process comprising the steps of:
  • substitutions, insertions or deletions may be present in the framework regions of the VH domain and/or the VL domain.
  • the purpose of such changes in primary amino acid sequence may be to reduce presumably unfavourable properties (e.g. immunogenicity in a human host (so-called humanization), sites of potential product heterogeneity and or instability (glycosylation, deamidation, isomerization, etc. ) or to enhance some other favourable property of the molecule (e.g. solubi lity, stability, bioavailability, etc. ).
  • changes in primary amino acid sequence can be engineered in one or more of the hypervariable loops (or CDRs) of a Camelidae VH and/or VL domain obtained by active immunization.
  • Such changes may be introduced in order to enhance antigen binding affinity and/or specificity, or to reduce presumably unfavourable properties, e.g. immunogenicity in a human host (so-called humanization), sites of potential product heterogeneity and or instability, glycosylation, deamidation, isomerization, etc., or to enhance some other favourable property of the molecule, e.g. solubility, stability, bioavailabi lity, etc.
  • the antibodies of the present invention may also be derivatized to "chimeric" antibodies (immunoglobulins) in which a region of the heavy/light chain(s) is identical with or homologous to corresponding sequences in the original antibody, while the remainder of the chain( s) is identical w ith or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity and binding specificity (Cabilly et al., supra; Morrison et al., Proc. Natl. Acad. Sci., pp. 685 (1984)).
  • An object of the invention is a composition comprising at least one of the protein of the invention as described here above.
  • Another object of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of the protein of the invention as described here above and a pharmaceutically acceptable excipient.
  • compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial g
  • Another object of the invention is the protein of the invention for inhibiting TGF-B activity in a subject in need thereof.
  • Another object of the invention is a method for inhibiting TGF-B activity in a subject in need thereof, comprising administering to the subject an effective amount of the protein of the invention.
  • Another object of the invention is the protein of the invention or the pharmaceutical composition as defined here above for treating a TGF-B-related disorder in a subject in need thereof.
  • Another object of the invention is a method for treating a TGF-B-related disorder in a subject in need thereof, comprising administering to the subject an effective amount of the protein of the invention.
  • TGF-B-related disorders include, but are not limited to, inflammatory diseases, chronic infection, cancer, fibrosis, cardiovascular diseases, cerebrovascular disease (e.g. ischemic stroke), and neurodegenerative diseases.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectal ly, nasally, buccally, vaginally or via an implanted reservoir.
  • administration used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water. Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils such as olive oil or castor oil
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as T weens.
  • Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 inL) or 500 mg (50 inL) single-use vials.
  • the product is formulated for intravenous (IV) administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 in g/niL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5.
  • Another object of the invention is a method for reducing immunosuppression in the tumor environment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the protein of the invention.
  • Another object of the invention is a method for boosting the immune system in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the protein of the invention.
  • Another object of the invention is a method for inhibiting the immune suppressive function of human Tregs in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the protein of the invention.
  • Another object of the invention is a method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the protein of the invention.
  • Another object of the invention is a method for treating cancer in a subject in need thereof, wherein the pharmaceutical composition of the invention is to be administered as an immunostimulatory antibody for treatment of cancer patients.
  • Another object of the invention is a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the protein of the invention in combination with another treatment for cancer or an immunotherapeutic agent.
  • Another object of the invention is a combination of the protein of the invention and another treatment for cancer or another immunotherapeutic agent for treating or for use in treating cancer.
  • said immunotherapeutic agent is a tumor vaccine.
  • said i m m u not heraplast i c agent is an i m m u no st i m u 1 at ory antibody.
  • Various cancers can be treated by the present invention such as for an adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor, glioma, breast carcinoma, carcinoid tumor, cervical cancer, colon carcinoma, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing' s tumor, extracranial germ cell tumor, eye cancer, gall bladder cancer, gastric cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, merkel cell carcinoma, metastatic squamous head and neck cancer, myeloma, n
  • Suitable tumor antigens for use as a tumor vaccine known in the art include for example : (a) cancer-testis antigens such as NY-ESO- 1 , SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE- 1 , GAGE-2, MAGE- 1 , MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE- 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors), (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p2 1 /Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CD 4 (associated with, e.g., melanoma), MUM 1 (associated with,
  • MUC-1 may be coupled to LH); (ii) lipopolypeptides (e.g., MUC-1 linked to a lipid moiety); (iii) polysaccharides (e.g., Globo H synthetic hexasaccharide), which may be coupled to a carrier proteins (e.g., to KLH), (iv) gangliosides such as GM2, GM 12, GD2, GD3 (associated with, e.g., brain, lung cancer, melanoma), which also may be coupled to carrier proteins (e.g. , KLH).
  • LH lipopolypeptides
  • polysaccharides e.g., Globo H synthetic hexasaccharide
  • gangliosides such as GM2, GM 12, GD2, GD3 (associated with, e.g., brain, lung cancer, melanoma), which also may be coupled to carrier proteins (e.g. , KLH).
  • tumor antigens include pi 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH- IGK, MYL- RAR, Epstein Barr vims antigens, EBNA, human papillomavirus (IIPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP- 180, pl85erbB2, pl80erbB-3, c-met, mn-23H 1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1 , NuMa, K-ras, p 16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29 ⁇ BCAA), CA 195, CA 242, CA-50, CAM43, CD68VKP1, CO-029, FGF-5, Ga
  • Suitable immunostimulatory antibodies include, but are not limited to: anti-CTLA-4, anti-PD l , anti-PDL 1 and anti- IR antibodies.
  • the method for treating cancer in a subject in need thereof comprises administering to the subject the protein of the invention prior to, concurrent to and/or posterior to another anti-cancer agent or cancer treatment, such as chemotherapy treatment.
  • Another object of the present invention is a method to prevent infectious diseases such as HIV, malaria, or Ebola, or improve vaccination against these infections, comprising administering to the subject a therapeutically effective amount of the protein of the invention.
  • the protein of the invention may be used /// vitro or in vivo to identify samples, tissues, organs or cells that express GARP.
  • assays in which the protein of the invention may be used include, but are not limited to, ELISA, sandwich ELISA, RIA, FACS, tissue immunohistochemistry, Western-blot, and immunoprecipitation.
  • the sample is a biological sample.
  • biological samples include, but are not limited to, bodily fluids, preferably blood, more preferably blood serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascitic fluids, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, and alveolar macrophages, tissue lysates and extracts prepared from diseased tissues.
  • bodily fluids preferably blood, more preferably blood serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascitic fluids, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, and alveolar macrophages, tissue lysates and extracts prepared from diseased tissues.
  • sample is intended to mean a sample taken from an individual prior to any analysis.
  • the protein of the invention may be labeled for diagnostic or detection purposes.
  • labeled herein is meant that a compound has at least one element, isotope or chemical compounds attached to enable the detection of the compound.
  • labels include, but are not limited to, isotopic labels such as radioactiv e or heavy isotopes; magnetic, electric or thermal labels and colored or luminescent dyes.
  • lanthanide complexes For example: lanthanide complexes, quantum dots, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, malachite green, stilbene, Lucifer yellow, cascade blue, texas red, alexa dyes, cy dyes.
  • One object of the invention is a method for identifying activated Tregs in a sample ba sed on the use of the protein of the in vention.
  • Another object of the inv ention is a method for identifying soluble or complexed latent TGF- ⁇ based on the use of the protein of the invention.
  • Another object of the inv ention is a kit comprising at least one protein of the invention.
  • kit any manufacture (e.g., a package or a container) comprising at least one reagent, i.e. for example an antibody, for specifically detecting the expression of GARP.
  • the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • any or all of the kit reagents may be provided within containers that protect them from the external env ironment, such as in sealed containers.
  • the kits may also contain a package insert describing the kit and methods for its use.
  • Kits for performing the sandwich EL IS A generally comprise a capture antibody, optionally immobilized on a solid support (e.g., a microtiter plate), and a revelation antibody coupled with a detectable substance, such as, for example HRP, a fluorescent label, a radioisotope, beta-galactosidase, and alkaline phosphatase.
  • a detectable substance such as, for example HRP, a fluorescent label, a radioisotope, beta-galactosidase, and alkaline phosphatase.
  • Example 1 New monoclonal antibodies directed against human GARP (anti-hGARP monoclonals)
  • DBA/2 or Balb/c mice were immunized with murine PIHTR cells transfected with human GARP.
  • Sera from immunized mice were tested for the presence of anti-hGARP antibodies, by screening for binding to hGARP-expressing BW cells by FACS.
  • Splenocytes from mice with high titers of anti-hGARP antibodies were fused to SP2/neo cells.
  • Hybridomas were selected in HA T medium and cloned under limiting dilution.
  • Supernatants of +/- 1600 hybridoma clones were screened by FACS for the presence of antibodies binding to hGARP-expressing BW cells. Thirty-eight clones producing anti-hGARP monoclonal antibodies were identified in this screening.
  • Nine clones were selected and amplified for large scale-production and purification of nine new anti-hGARP monoclonals (MHGARP 1 to 9).
  • MHGARP 1 to 9 bind to murine BW5 147 cells transfected with hGARP, but not to untransfected cells. MHGARP 1 to 9 also bind 293T cells transfected with hGARP and two human T cells lines (clone Th A 2 and Jurkat) transduced w ith a hGARP-encoding lentivirus, but not the corresponding parental cells (not shown). This recognition pattern is identical to that of a commercially avai lable anti-hGARP mAb (clone Plato- 1 ) used here as a positive control. These results show that MHGARP 1 to 9 recognize hGARP on cell surfaces.
  • MHGARP antibodies MUG- 1 to -14 on the figure
  • ThA2 human CD4+ T helper cells, which do not express hGARP
  • ThA2 transduced with hGARP
  • Example 2 MHGARP8. but none of 12 other anti-hGARP monoclonal s. inhibits active TGF- ⁇ production by human Treg ceils
  • a human Treg clone (1E+06 cells/ml) was stimulated in serum-free medium with coated anti-CD3 (1 iig/ml) and soluble anti-CD28 (1 iig/ ' ml) antibodies, in the presence or absence of 20 Lig/ml of an anti-hGARP monoclonal antibody.
  • Thirteen anti-hGARP monoclonals were tested in this assay: the above mentioned nine new monoclonal s (MHGARP 1 to 9), and commercially available antibody clones Plato- 1 (Enzo Life Sciences, catalog No. ALX-804-867), 272G6 (Synaptic Systems, catalog No. 221 1 1 1), 50G 10 (Synaptic Systems, catalog No.
  • hGARP is required for active TGF-B production by TCR- stimulated Treg cells
  • TCR T cell receptor
  • Active TGF-B produced by stimulated Tregs triggers an autocrine signal, which leads to the phosphorylation and activation of SMAD2 and SMAD3 transcription factors.
  • pSMAD2 phosphorylated SMAD2
  • WB Western Blot
  • Example 3 MHGARP8. but not other anti-hGARP mAbs. recognizes a conformational epitope that requires the presence of TGF-13 Mapping the regions recognized by anti-hGARP monoclonals
  • Murine BW5147 T cells were electroporated with plasmids encoding the HA-tagged proteins schematized in Figure 3, part A, corresponding to hGARP, mGARP or mGARP/hGARP chimeras.
  • Stable clones selected in neomycin were stained with biotinylated anti-hGARP antibodies (anti-hGARP 1 to 9) and streptavidin-PE, with the commercial anti-hGARP antibody (clone Plato- 1 ) and a secondary a n t i - m 1 gG 2 b- A F488 , or with an anti-HA antibody and secondary anti-mouse IgG I -AF488. Histograms are gated on live cells.
  • Black histograms show signals on untransfected BW cells, white histograms show signals on BW cells expressing the HA-tagged hGARP, and grey histograms show signals on BW cells expressing HA-tagged mGARP or mGARP/hGARP chimeras.
  • MHGARP8 may recognize an epitope in hGARP that is distinct from the epitopes recognized by the other anti-hGARP mAbs.
  • MHGARP-1 the MHGARP mAbs of the instant application do not recognize murine GARP (mGARP). Plasmids were therefore constructed encoding HA-tagged hGARP, mGARP or hGARP/mGARP chimeras to map the hGARP regions recognized by the mAbs of the instant application.
  • Murine BW cells were transfected and stable clones were derived expressing the HA-tagged proteins (schematically represented in Figure 3). All clones expressed similar levels of HA-tagged protein on the surface, as indicated by similar fluorescence intensities after staining with an anti- HA mAb ( Figure 3, part A).
  • mAbs in the second group (MHGARP-2, -3 and -8) bound to only 1 of the 5 chimeras, and thus recognize an epitope in region 1 0 1 - 14 1 .
  • a third group comprises MHGARP-5, which bound to 2 of the chimeras and therefore recognizes region 14 1 -207.
  • This group probably al o contains MHGARP- 1 , w hich is cross-reactive but bound these 2 chimeras more efficiently than it bound mGARP or the 3 other chimeras.
  • mAbs in the fourth group (MHG.ARP-4 and Plato- 1 ) bound 4 of the 5 chimeras, and thus recognize region 265-333.
  • the anti-hGARP mAbs were grouped into four families of antibodies that recognize four distinct regions of the hGARP protein.
  • MHGARP-8 which displays neutralizing activity, binds to region 10 1 - 14 1 .
  • This region is also recognized by MHGARP-2 and -3, which are not neutralizing. Therefore, the ability to bind region 10 1 - 14 1 is not sufficient to confer neutralizing activity .
  • the epitope recognized by MHGARP8 appears only when hGARP is bound to murine (m) or human (h) TGF-B 1 . This could be due to one of two mechanisms: either the epitope comprises amino-acids from both hGARP and TGF-Bl (mixed conformational epitope), or it comprises amino- acids from hGARP only, but that adopt a different conformation in the presence of TGF- ⁇ 1 (binding-induced conformational epitope).
  • BW cells express murine TGF-Bl, and murine TGF-Bl binds to hGARP ( Figure 3, part B). Therefore, binding of M HGARP8 to BW + hGARP (in the absence of transfected h ' TGF-B 1 ) could be due to recognition of hGARP/niTGF-B 1 complexes.
  • MHGARP-1, -2, -3, -4 and -5 immunoprecipitated GARP as efficiently as the other anti-GARP mAbs, but they did not co-immunoprecipitate TGF-B ( Figure 3, part C, bottom panels).
  • MHGARP- 1 , -2, -3, -4 and -5 recognize free GARP, but not GARP that is bound to TGF-B.
  • MHGARP-2 and -3 which require the GARPioi-141 region for binding, recognize only free GARP, whereas neutralizing MHGAR 8, which also requires GARPnn-m , recognizes GAR bound to TGF-B.
  • Binding of MHGARP-6, -7, and -9 was not modified by co-transfection of hTGFBl, indicating that these niAbs bind hGARP whether or not it is bound to TGF-B 1 (i.e. they bind both free GARP and GARP bound to TGF-B 1).
  • binding of MHGARP8 increased dose-dependently when hTGFBl was co-transfected with hGARP. This suggests again that in contrast to all other antibodies, MHGARP8 does not bind free GARP, but only GARP bound to TGF-B 1.
  • siRNAs were used to silence the expression of TGFBl in Jurkat cells transduced with hGARP ( Figure 3, part E).
  • the siRNA against TGFBl mRNA efficiently reduced expression of TGF-B 1, as illustrated by the decrease in surface LAP detected on Jurkat ; hGARP cells ( Figure 3, part E, right panel).
  • Reduced expression of TGF-B 1 in Jurkat+hGARP decreased the binding of the MHGARP8 antibody, but it did not modify the binding of the other anti-GARP antibodies ( Figure 3, part E, foreground histograms). This confirms that in contrast to the other anti-GARP antibodies, MHGARP8, does not bind free GARP, but only binds GARP in the presence of TGF-B I .
  • Transfected cells were analyzed by FACS to measure binding of the MHGARP8 antibody, and presentation of hTGF-Bl on the cell surface with an anti- liLAP antibody (Figure 4).
  • transfection of hGARP, mGARP or hGARP/mGARP constructs alone induced low levels of surface L AP, due to low levels of endogenous hTGFB l expression ( Figure 4, part A, left).
  • Surface LAP levels dramatically increased upon transfection of hTGFB l in cells transfected with liGARP, mGARP, or any liG A RP/mG A RP contruct ( Figure 4, part B, left histogram).
  • hTGF-Bl is presented on the cell surface by hGARP, by mGARP and by all the hGARP/mGARP chimeras.
  • M1IGARP8 bound only to the surface of cells transfected with hGARP, or with the hGARP/mGARP constructs encoding amino-acids 101 to 14 1 of hG ARP ( Figure 4, part A B, right).
  • MHGARP8 does not bind mGARP. Its binding to hGARP requires a region comprising amino-acids 101 to 141.
  • region 10 1 - 14 1 in human and murine GARP were compared. In this region, only 13 amino-acids differ between hGARP and mGARP ( Figure 5, amino-acids highlighted by grey boxes).
  • Three HA-tagged mutant forms of hGARP were constructed. In each mutant (Mut I, Mut II and Mut III), three consecutive amino-acids were replaced by the corresponding amino-acids of the mG ARP protein ( Figure 5, black boxes).
  • Stable clones of BW cells transfected with these HA-tagged forms of wild type (WT) or mutant hGARP were derived. All clones expressed similar levels of HA-tagged protein on the surface, as demonstrated by staining with an anti- HA antibody ( Figure 5, histograms on the right). The clones were then analyzed after staining with MHGARP-2, -3 and -8, i.e. , antibodies which require region 101 -141 of hGARP for binding. The three antibodies bound to cells expressing WT, Mut I and Mut II forms of hGARP.
  • MHGARP8 is the only available anti-GARP antibody that inhibits active TGF-Bl production by human Tregs.
  • binding of MHGARP8 requires both region 101-141 of hGARP and the presence of hTGF-B, whereas binding of non-neutralizing antibodies require other regions of hGARP (for MHGARP- 1 , -4, -5, -6, -7 and -9), or occurs only in the absence of TGF-Bl (for MHGARP-2 and -3).
  • region hGARP 101-141 amino- acids 137 to 139 are required for the binding of MHGARP-2, -3 and -8.
  • the affinity of the MHGARP8 antibody to immobilized shGARP-TGFp was measured by BIACOR analysis.
  • the Kd of said antibody is 0.2 ii .
  • Example 4 MHGARP8 inhibits human Treg cell function in vivo
  • MHGARP8 also inhibits human Tregs /// vivo
  • a model of xenogeneic GvHD induced by transfer of human PBMCs (Peripheral Blood Mononuclear Cells) into immuno-compromised NO D-S i - 1 L 2 Rg " " (NSG) mice was used.
  • SG mice lack functional T, B and NK cells. This allows efficient engraftment of human hematopoietic stem cells (FISCs), which proliferate and generate a functional human immune system in recipient mice.
  • FISCs human hematopoietic stem cells
  • GvHD xenogeneic Graft-versus-Host Disease
  • PBMCs Human PBMCs were isolated from total blood of a hemochromatosis donor by centrifugation on density gradients ( Ly mphoprepTM ), and frozen for later use.
  • Autologous Tregs were generated as follows: CD4+ T cells were isolated from the blood of the same donor using the RosetteSepTM Human CD4+ T Cell Enrichment Cocktail (StemCell Technologies) and stained with anti-CD4, anti-CD25 and anti- CD 127 antibodies coupled to fluorochronies.
  • CD4+CD25hiCD1271o cells were sorted by flow cytometry (>99% purity) then stimulated with anti-CD3/CD28 coated beads (Dynabeads® Human T-Activator CD3/CD28 for T-Cell Expansion and Activation, Life Technologies) in the presence of IL-2 (120 lU/ml) during 14 days. These expanded Treg cells were frozen for later use. NSG mice were irradiated (2.5 Gy) on day -1, then injected in the tail vein with human PBMCs (2.7x106 per mouse) alone, or mixed with expanded human Tregs (1.4x106 per mouse) on day 0. Mice also received weekly i.p.
  • MHGARP8 antibody 400 ,ug on day -1 (day minus 1), 200 iig at later time points), or control PBS. Mice were monitored bi-weekly for GvHD development as indicated in the text. Human PBMCs ith or without Tregs were transferred into NSG mice, and the mice were treated with i.p. injections of MHGARP8 antibody or control PBS. The large number of human Treg ceils required for the transfers were obtained through short in vitro amplification of CD4+CD25+CD1271o cells sorted from human PBMCs by flow cytometry. Objective signs of GvHD dev elopment in the recipient mice were monitored bi-weekly. We performed two independent experiments, which yielded similar results.
  • Example 5 New anti-hGARP monoclonal antibodies (mAbs) using Immunization of llamas approach Production of recombinant soluble GARP-TG Fp i complex
  • Human and murine GARP-TGFpl complex was produced as a soluble complex using a truncated GARP expression construct.
  • the human GARP protein sequence was truncated after Leucine 628, followed by a cleavable TEV-3x strep tag (EAAENLYFQGAAWSHPQFEKGAAWSHPQFEKGAAWSHPQFEKGAA*)
  • Murine GARP protein sequence was tmncated after leucine 629, followed by the same cleavable TEV-3x strep tag.
  • the GARP-TGFf] 1 complexes were produced by co-expression of the truncated GARP and the ⁇ ⁇ in HEK293E cells, followed by purification via the Strep-Tag.
  • RNA yields of 450 ⁇ g were achieved, which was used for random cDNA synthesis and PGR amplification of the V-regions of the heavy and the light chains ( ⁇ and VK) for construction of the Fab containing phagemid libraries as described by De Haard H et al., (J Biol Chem. 1999 Jun 25; 274(26): 18218-30), to obtain diverse libraries of good diversity (l -7x l0 8 ).
  • the immune response to the GARP-TGF ⁇ ⁇ complex was investigated by ELISA on coated recombinant soluble GARP-TGF ⁇ ⁇ complex (l ⁇ ig/ml).
  • Five-fold serial dilutions of sera, starting from 10% sera were prepared and 100 ⁇ of diluted sera was added onto the coated wells and incubated for 1 hour at RT. After washing with 3 x PBS/Tween, the plates were blocked with PBS supplemented with 1% casein ( Figure 8). Binding of conventional llama IgG I to its target GARP-TGF was measured in ELISA using a mouse anti llama IgG 1 antibody (clone 27E10, Daley LP, et al. Clin. Diagn Lab Immunol. 1 2, 2005) and a HRP-conjugated donkey anti-mouse antibody (Jackson) for detection.
  • a mouse anti llama IgG 1 antibody clone 27E10, Daley LP
  • Phage expressing Fabs were produced according to standard protocols and selections performed on immobilized recombinant soluble G ARP-TGF
  • hGARP and ⁇ (LAP) counter selections were used to enrich for Fabs binding the hGARP-TGF i complexes.
  • Individual colonies were isolated and periplasmic fractions (peris) in 96- well plates were produced by IPTG induction from all the libraries according to standard protocols. Screening of the hGARP-TGFf] specific Fabs was performed using ELISA.
  • hGARP- TGF i was immobilized on a maxisorb plate. After blocking with 1% casein in PBS for I h. Fab from 20 ⁇ 1 periplasmic extracts were allowed to bind to hGARP-TGFf] 1 .
  • VK chain shuffling was used to improve the affinity of the mAb LHG- 10 ( Figure 10).
  • the heavy chain of the parental clone (VHCH 1 of LHG- 10) was reintroduced in the phagemid-light chain library.
  • the heavy chain was extracted from an expression vector, which lacks the bacteriophage derived gene 3 necessary for display, to further avoid contamination of the parental light chain in the selection procedure.
  • the heavy chain was cloned into the phagemid-light chain library and the ligated DN A was electroporated into E.coli TGI cells to create the light chain shuffled library.
  • the size of libraries was above 10 8 .
  • Affinity selections combined w ith off-rate washes, were performed to select for chain shuffled Tabs with an improved affinity for human GARP-TGF(3 1 .
  • a set-up was chosen where Fab expressing phages were incubated with different concentrations of recombinant soluble human GARP-TGFpi directly coated to the microsorb plate.
  • LHG- 10-D IgGs were checked for cross-reactivity on cyno GARP/cyno TGF- ⁇ 1 in a FACS-based assay using HEK-293E cells transfected with cyno GARP/cyno TGFf] 1 or human GARP/human TGF(31 .
  • MFIGARP8 was also tested in this cross-reactivity assay. All LHG-IO-D and MHG-8 are cross-reactive against cyno GARP/cyno TGFf] 1 ( Figure 9).
  • Example 6 Two anti-hGARP mAbs ( MHGARP8 and LHG- 10) inhibit active TGF-Bl production by human Tregs Stimulated human Tregs produce active TGF-B l close to their cell surfaces. Autocrine and paracrine TGF-Bl activity induces SMAD2 phosphorylation in Tregs themselves, and in Th cells co-cultured with Tregs (Stockis, J. et al. Eur. J. Immunol. 2009, 39:869- 882).
  • phosphorylated SMAD2 was detected in the stimulated Tregs, but not in non-stimulated Tregs, nor in Tregs stimulated in the presence of a neutralizing anti- TGF-Bl antibody ( Figure 1 1).
  • Phosphorylated SMAD2 was greatly reduced in Tregs stimulated in the presence of MHGARP8 (named MHG-8 on Figure 1 1 , part A) or LHG- 10 ( Figure 1 1, part B), indicating that these two anti-hGARP mAbs block active TGF- ⁇ production.
  • the inhibitory activity of MHGARP8 and LHG- 10 shows that GARP is required for active TGF-Bl production by human Tregs.
  • Example 7 MHGARP8 and LHG-10 inhibit the suppressive activity of human Tregs in vitro
  • Tregs suppress other T cells at least in part through production of active TGF- ⁇ I ( Stockis, J. et al. Eur. J. Immunol. 2009, 39:869-882).
  • MHGARP8 MHG-8
  • LHG- 10 also inhibit human Treg function in in vitro suppression assays.
  • a Treg clone was used as a source of Tregs, and freshly isolated CD4 CD25 CD 127 hi cells or a CD4 T cell clone (Th cells) as targets for suppression. Tregs and Th cells were stimulated with >CD3 and >CD28 in the presence or absence of various additional mAbs.
  • clone Treg A 1 inhibited the proliferation of CD4 + CD25 " CD127 hl Th cells by 66 % in the absence of anti-hGARP mAb. Suppression was reduced to 36% and 32% in the presence of MHG- 8 or LHG- 1 0, respectively, but was not reduced in the presence of 6 other anti-hGARP mAbs. Suppression by clone Treg A 1 on another Th target (clone Th A2) was also measured in the presence of MHGARP8, an anti-hTGF- ⁇ 1 mAb or an isotype control.
  • MHGARP8 (MHG-8) inhibited the in vitro suppressive activity of Treg Al in a manner similar to that of the anti-TGF- ⁇ 1 antibody, whereas the isotype control showed no effect (Figure 12).
  • Example 8 Epitopes recognized by inhibitory anti-hGARP mAbs
  • GARP associates with pro- or latent TGF-B 1 to form disulfide-linked GARP/TGF- ⁇ 1 complexes ( Figure 13 and Stockis 2009b Eur. J. Immunol. 2009. 39: 3315-3322 and Gauthy E et al).
  • IP co-immunoprecipitation
  • a first group (left column) comprises the 8 mAbs that did not co-immunoprecipitate pro- or latent TGF- ⁇ 1 : they bound 293 T cells transfected with hGARP alone, but not with hGARP and hTGFBI. This confirms that these mAbs bind free GARP only, as binding to surface GARP is lost in the presence of TGF-B I ( Figure 14, part B, shows 3 representative mAbs of this group).
  • a second group comprises most other mAbs (19 mAbs, middle column of Figure 13, part B): they bound 293 T cells equally well upon transfection with hGARP alone or with hGARP and hl ' Gl-Bl, indicating that they bind both free GARP and GARP/TGF-B 1 complexes ( Figure 14, part B, shows 6 mAbs of this group).
  • a third group of 5 mAbs bound 293 T cells transfected with hGARP and hTGFBl, but not cells transfected with hGARP alone (right column of Figure 13, part B). These mAbs bind GARP/TGF-Bl complexes but not free GARP, and include inhibitory MHGARP8 (MHG-8) and LHG- 10 ( Figure 14, part B, shows 3 mAbs of this group).
  • Binding patterns to mGARP/hGARP chimeras (Figure 15, part A, 10 representative mAbs) allowed to identify the region of hGARP required for binding by each anti-hGARP mAb. This is summarized in Figure 15, part B, where mAbs are distributed in rows corresponding to various regions of hGARP: mAbs in the first row require a region comprising amino- acids 20 to 101 (hGARP 2 o-ioi), mAbs in the second row require hGARPmi-m, those in the third require the fourth, hGARP 2 65-332, and finally, a fifth group requires hGARP 332 ..628.
  • Sequences of mouse and human GARPioi-m differ at 14 amino-acid (aa) positions, comprising three clusters of three contiguous positions ( Figure 15, part B, left panel).
  • aa 14 amino-acid
  • Binding patterns to mutants revealed three types of mAbs ( Figure 15, part B, right panel), which required amino-acids hGARPiii-i ij, hGARP 12 6 -127 , or hGARPi 3 ⁇ 4--i ⁇ ) for binding, respectively.
  • Example 9 Inhibition of human Tregs function by anti-hGARP in vivo
  • GVHD xenogeneic graft-versus-host disease
  • NSG mice have defective cytokine signaling and lack functional T, B and NK cells, allowing very efficient engraftment of human T cells upon i. v. injection of PBMCs.
  • mice dev elop xenogeneic GVHD, due to the activ ity of human cytotoxic T lymphocytes against murine tissues ( Shultz, Nat Rev Immunol. 20 1 2 Nov; 12( 1 1 ):786-98 ).
  • co-transfer of human Tregs w ith human PBMCs attenuates GVHD (Harmon et al. Transfusion. 20 14 Feb ; 54( 2 ) : 353 -63 ), prov iding a model to test the inhibitory activity of anti-hGARP mAbs on human Tregs in vivo.
  • mice We transferred human PBMCs (3xl0 mouse) with or without autologous Tregs (1.5xl0 6 /mouse) in N SG mice ( Figure 16, part A).
  • PBMCs 3xl0 mouse
  • autologous Tregs 1.5xl0 6 /mouse
  • mice were injected with MHGARP8 (named MHG-8 on the figure), anti-TGF-B I , an isotype control or PBS, one day before the graft and weekly thereafter.
  • Objective signs of GVHD were monitored bi-weekly, to establish a disease score based on weight loss, reduced mobility, anemia or icterus, and hair loss.
  • MHGARP8 inhibits the immune-suppressive function of human Tregs in vivo.
  • MHGARP-8 did not aggravate GVHD in mice grafted with PBMCs alone, thus that its effect depended on the co-injection of Tregs (Fig. 17).
  • Fig. 17 We also examined whether abrogation of Treg protection by MHGARP-8 depended on its ability to block TGF- ⁇ production.
  • MHGARP-8 was compared to LHG-10.6, which also blocks TGF- ⁇ production by Tregs, and to LHG-3, which does not.
  • Antibody LHG- 10.6 is a variant of LHG-10 with increased affinity for GARP/ ⁇ GF- ⁇ ⁇ complexes that was selected by phage display from Fabs in which the heavy chain of LHG- 10 was combined to the VK library. Like MHGARP-8, LHG- 10.6 aggravated GVHD, whereas non-blocking antibody LHG-3 had no effect (Fig. 17). This suggested that MHGARP-8 and LHG-10.6 abrogate Treg protection by blocking Treg production of TGF- ⁇ , and not by inducing Treg depletion. To further exclude the latter possibility, a mutated version of LHG-10.6, named LHG-10.6N297Q, was also tested.
  • the N297Q mutation results in loss of Fc glycosylation, thus loss of Fc receptor- and C I q- binding, and consequently loss of ADCC and CDC functions.
  • LHG- I 0.6N297Q aggravated GVHD in mice grafted with PBMCs and Tregs as potently as LHG-10.6, confirming that anti- GARP antibodies do not act by depleting Tregs (Fig. 17).
  • cytokines in the serum of mice 20 days after cell transfer were measured (Fig. 18, part A).
  • Human IL-2 and IFNy were detected at high levels in mice grafted with PBMCs only, indicating a strong xenogeneic activation of human T cells. They were significantly reduced by the cotransfer of Tregs, confirming suppressive activity.
  • MHGARP-8 decreased the suppression by Tregs, but had no effect in mice transferred with PBMCs alone.
  • IL-10 levels were not increased but instead reduced in the presence of Tregs, suggesting that Tregs do not suppress through production of IL-10 in this model (Fig. 18, part A).
  • hCD45+ human hematopoietic cells
  • CD4+ and CD8+ T lymphocytes
  • MHGARP-8 Fig. 18, part B
  • the numbers and proportions of Tregs were not reduced in mice treated with MHGARP- 8.
  • Treg numbers were significantly increased in mice transferred with Tregs and treated with MHGARP-8 as compared to untreated mice (Fig.
  • inhibitory anti-GARP mAbs are capable of inhibiting the immunosuppressive activity of human Tregs in vivo without inducing Treg depletion.
  • Example 10 X-ray crystal structures of the Fab-fragment generated from antibody MHGARP8 in complex with the GARP/TGF-B complex.
  • the Fab fragment of MHG-8 was prepared by papain digestion of MHG-8 and purified using Protein A affinity chromatography and gel filtration chromatography.
  • the MHG- 8 Fab fragment was added to the GARP/T GFP complex to allow binding of the Fab to its antigen.
  • the Fab-fragment purified in this way was mixed with the GARP/ TGFbeta complex and applied on a gel filtration column in 20 mM Tris/HCl pH 8.0, 50 niM NaCl.
  • the Fab/GARP/TGF(3 complex was concentrated on a 50 kD Vivascience ultrafiltration device to a final concentration of 18 mg/mL, as determined by Nanodrop (UV).
  • This MHG-8 Fab/GARP/TGF complex (where ⁇ is comprised of LAP and mature ⁇ ) was purified and used for crystallization.
  • the purified protein was used in crystallisation trials employing a standard screen with approximately 1,200 different conditions. Conditions initially obtained have been optimised using standard strategies, systematically varying parameters critically influencing crystallisation, such as temperature, protein concentration, drop ratio, and others. These conditions were also refined by systematically varying pH or precipitant concentrations.
  • the application of the Free Mounting System (FMS) was necessary to obtain well diffracting crystals.
  • the crystals were coated with oil and transferred to the N2 cryo- stream at 100K. Crystals have been flash- frozen and measured at a temperature of 100 K.
  • the X-ray diffraction data have been collected from complex crystals at the SWISS LIGHT SOURCE (SLS, Villigen, Switzerland) using cryogenic conditions. The crystals belong to space group P 2 1. Data were processed using the programs XDS and XSCALE. Table 5
  • the phase information necessary to determine and analyse the structure was obtained by molecular replacement.
  • the published structures of latent TGFbeta (PDB-ID 3RJR), Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor interacting protein 1 (PDB-ID 40QT) and Fab-fragment (PDB-ID IFNS) were used as a search models.
  • Subsequent model building and refinement was performed according to standard protocols with the software packages CCP4 and COOT.
  • a measure to crossvalidate the correctness of the final model about 0.6 % of measured reflections were excluded from the refinement procedure (see Table 6).
  • VV CllC 0 V/
  • Test-set contains 0.6% of measured reflections
  • the crystal structure of the Fab.GARP/TGF-B complex al lowed the identification of the epitope recognized by the antibody MHGARP8.
  • the Fab-fragment binds a composite three dimensional epitope on the Fab:GARP/TGF-B complex.
  • the interaction surface on the Fab:GARP/TGF-B complex is formed by several continuous and discontinuous sequences from both hGARP (Tables 7a and 7b respectively) and TGF-B (Table 7c), showing interactions between TGF residues and MHG-8 heavy chain residues.
  • Table 7a Interactions between GARP residues (left side) and MHG-8 heavy chain residues (right side)
  • Example 1 1 Impact of mutations in GARP or TGF- ⁇ 1 on the binding and activity of inhibitory anti-GARP antibodies MHG-8 and LHG-10.
  • Geom corresponds to the geometric mean of the fluorescence intensity measured by FACS on 293 T cells transfected with empty plasmid (control) o plasmids encoding the mutant or WT forms of G ARP/TGF-B 1 complexes.
  • Results obtained on all mutants are detailed in Figure 19, parts A-D for binding by inhibitory anti-GARP antibodies MHG-8 and LHG-10, non-inhibitory anti-GARP antibody MHG-6, and anti-LAP antibody, respectively. They are also summarized in Table 8 for MHG-8 and LHG-10.
  • Table 8 Effects of GAEP and TGF
  • part A demonstrates that only one mutation in TGF-Bl results in loss of binding by MHG-8. This mutation corresponds to K338E (25% residual binding).
  • K338E 25% residual binding
  • 8 a a from LAP and 6 a a from mature TGF- ⁇ 1 that are in contact with MHG-8 Fab in the crystal only one aa in mature TGF-Bl (Lys338) is required for binding to the MHG-8 mAb.
  • L140K/E142L does not induce loss of binding to LHG- 10, in contrast to what was observed for MHG-8, confirming that the mode of binding of LHG- 10 to G ARP is different from that of MHG-8;
  • single mutation T162D induces loss of binding by LHG- 1 0 (49% residual binding), whereas it had no effect on binding by MHG-8; single mutation R163E induces loss of binding by LHG- 10 (48% residual binding).
  • Figure 19 part B also shows that one mutation in mature TGF- ⁇ 1 ( 338E, 15% residual binding) induces loss of binding to LHG-10. This mutation also induced loss of binding to MHG-8.
  • One other mutation in mature TGF-I 1 (Y336A, 56% residual binding) induced partial loss of binding to LHG-10, whereas it did not affect binding to MHG-8.
  • Relative staining intensity [Geom with X jig/ml of antibody - Geom in the absence of antibody] / [Geom with 5 jig/ml of antibody - Geom in the absence of antibody]
  • the relative staining intensities were plotted according to the concentration of antibody used for staining, and a non-linear regression analysis was used with the Prism software to determine an EC 50 value for binding of the antibody to the WT and to each of the various mutant GARP/TGF- ⁇ I complexes. If the ratio between the EC 50 measured on the mutant and the EC 50 measured on the WT was > 2, the mutation was considered to induce a reduced avidity for binding by the antibody.
  • the ratio of EC 50 (mutant vs WT) for MHG-8 and LHG-10 are indicated below each mutation on Figure 19, parts A and B, respectively. The data from this experiment is also summarized in Table 8.
  • Transiently transfected cells (293T + ITGB6 or 293T+ITGB6 ; GARP) were incubated during 24 hours with inhibitory anti-GARP antibodies MHG-8 or LHG-10, or with control antibodies corresponding to a neutralizing anti-TGF-B 1 antibody (positive control ), or the non-inhibitory anti-GARP antibody LHG- 14 (negative control ). All antibodies were used at 20 ng/ml. Cells were then lysed and incubated with a luciferase substrate and the luminescent signal was measured in a luminometer. To compare various mutants tested in different experiments, the inhibitory activity of antibodies were experessed as follows:
  • Residual inhibitory activity 100 x
  • Results obtained are detailed in Figure 20, parts A-C for MHG-8, LHG- 10 and anti- T ' GF- ⁇ I , respectively, and summarized in Table 8.
  • mice (DBA/2, Balb/c, and NOD.Cg-Prkdcscid I12rgtm I Wjl/SzJ or NSG from The Jacskon Laboratory) were bred at the animal facility of the Universite Catholique de Louvain, Belgium. Handling of mice and experimental procedures were conducted in accordance with national and institutional guidelines for animal care.
  • Pl .HTR cells a highly transferable variant of the P815 mastocytoma derived from DBA/2 mice, were electroporated with a plasmid encoding the full-length human GARP and selected in puromycin (1.6 ng/ml) under limiting dilution conditions.
  • Two clones expressing high surface hGARP (Pl .HTR+hGARP) were isolated and used to immunize H-2d mice.
  • a stable clone of murine BW5 147.C2 cells expressing high levels of human GARP (BW5147+hGARP) was derived as described (E. Gauthyet al, PLoS One 8, e761 86 (2013)).
  • This clone was electroporated with a plasmid encoding full-length human TGF-b 1 , and selected in neomycin (3 mg/ml) under limiting dilution conditions.
  • a subclone expressing high levels of surface hGARP/h I GF- ⁇ 1 complexes (BW5 147+hGAR ; hTGFB 1 ) was isolated and used to immunize llamas.
  • Human Treg and Th clones were derived and cultured as previously described (J. Stockis, et al. Eur. J. Immunol. 39, 869-882 (2009).).
  • Total human PBMCs were purified from the blood of hemochromatosis donors by centrifugal ion on a Lymphoprep® gradient.
  • Human polyclonal Tregs were obtained by sorting CD4+CD25+CD1271o cells by FACS from total PBMCs, followed by / / vitro stimulation with anti-CD3/CD28 coated beads in the presence of IL-2 during 12-13 days, as described (E. Gauthyet al. PLoS One 8, e76186 (2013).).
  • 293T cells were transiently transfected with hGARP- and hTGF- ⁇ 1 -encoding plasmids using the TransIT-LTl transfection Reagent (Minis Bio).
  • DBA 2 or Balb/c mice were immunized with live Pl .HTR+hGARP cells, following a previously described injection scheme (M. M. Lemaire, et al. J. Immunol. Methods, (201 1)).
  • Lymphocytes from mice with high titers of anti-hGARP antibodies as determined by FACS, were fused to SP2/neo cells in the presence of polyethylene glycol.
  • Hybridomas were selected in HAT medium and cloned under limiting dilution conditions.
  • Supernatants of hybridoma clones were screened by FACS for the presence of antibodies binding to BW5147+liGARP cells. Fourteen positive clones were selected, further subcloned to ensure clonal ity, and amplified for large scale-production and purification of 14 new anti-hGARP mAbs (MHG- 1 to - 14).
  • Blood samples (10 ml) were collected to monitor IgGl responses against hGARP/TGF- ⁇ complexes by EL IS A, using immobilized recombinant GARP/TGF- ⁇ 1 complexes (produced in HEK-293E cells co-transfected with hTGFB 1 and hGARP truncated from the transmembrane-coding region) for capture, followed by a mouse anti-llama IgGl antibody (clone 27E10) and a HRP-conjugated donkey anti-mouse antibody (Jackson) for detection.
  • immobilized recombinant GARP/TGF- ⁇ 1 complexes produced in HEK-293E cells co-transfected with hTGFB 1 and hGARP truncated from the transmembrane-coding region
  • a mouse anti-llama IgGl antibody clone 27E10
  • HRP-conjugated donkey anti-mouse antibody Jackson
  • 450 Lig of RNA were obtained and used for random cDNA synthesis followed by PGR amplification of the immunoglobulin heavy and light chain variable regions (VH, ⁇ and VK).
  • VH, ⁇ and VK immunoglobulin heavy and light chain variable regions
  • Phages expressing Fabs were produced and selected according to standard protocols. Briefly, 2 to 3 rounds of phage selections were performed by binding on immobilized recombinant GARP/TGF- ⁇ 1 , washing and elution with trypsin. In some instances, counter selections with soluble hGARP (hGARP 1-628 fused to a TEV-3xStrepTag produced in 293E cells) and soluble latent TGF- ⁇ 1 were used to enrich for Fabs binding hGARP/TGF- ⁇ 1 complexes only.
  • Fab clones were divided into 1 7 families, based on similarities in the sequences coding for the VH CDR3 region.
  • VH and VL sequences from one representative clone of each family were subcloned in a full human IgGl backbone, and the resulting plasmids were transfected into HEK-293E cells to produce and purify 1 7 new anti-hGARP mAbs (LHG-1 to - 1 7).
  • Proportions of cells w ith a demethylated ⁇ 3 ⁇ 1 in human PBMCs, in human polyclonal Treg populations or in splenocytes from NSG mice grafted with human cells were measured by methyl-specific qPCR as described ( 1. .1. de Vries, et al. Clin. Cancer Res.
  • CCTATAAAATAAAATATCTACCCTC (SEQ ID NO: 58); demethylated FOXP3il alleles: TCTACCCTCTTCTCTTCCTCCA (SEQ ID NO: 59)/ GATTTTTTTGTTATTGATGTTATGGT (SEQ ID NO: 60)/
  • a human Treg clone (10° cells/ml ) was stimulated in serum-free medium with coated anti-CD3 (Orthoclone O I 3; Janssen-Cilag, 1 g/ml) and soluble anti-CD28 (BD Biosciences; I ng-'ml ), in the presence or absence of 10 ug/ml of an anti-hGARP mAb (clones tested: MHG-1 to -14; LHG-1 to -17; Plato- 1 from Enzo Life Sciences; 272G6 and 50G10 from Synaptic Systems; 7B 1 1 from BioLegend) or of an anti-hTGF- ⁇ antibody (clone 1 D 1 I , R&D systems).
  • Intact or permeabilized cells were labeled according to standard protocols, using combinations of the following primary and/or secondary reagents as indicated in the figures.
  • Primary antibodies biotinylated MHG- 1 to 14; LHG-1 to - 1 7; anti-hGARP clone Plato 1 (Enzo Life Sciences); antihCD45-PerCP, anti-hCD3 ⁇ FITC or anti-hCD3- APC, anti-hCD4-FITC or anti-hCD4-APC, antihCD45RA-PE-Cv7 (Biolegend); anti- hCD8a-APC-H7, anti-CD25-PE-Cy7, anti-hCD I 27-PE (BD Biosciences); anti- hFOXP3-PE or anti-hFOXP3-APC (eBiosciences); anti-liLAP-APC (R&D Systems); anti-HA (Eurogentec).
  • Th cells were seeded alone or with the indicated numbers of Tregs, and stimulated with coated anti-CD3 (Orthoclone OKT3, Janssen-Cilag, 1 .ug/ml) and soluble anti-CD28 (BDBiosciences, 1 ⁇ g/ml), in the presence or absence of 10 ⁇ «/ ' ⁇ 1 of an anti-hGARP mAb (MUG or LUG), an anti-TGF-b antibody (clone 1 D1 1, R&D Systems) or an isotype control (mlgGl clone 1 1 7 1 1 , R&D Systems), [methy!- 3H]Thymidine (0,5 mCi/well) was added during the last 16 hours of a 4 day-culture.
  • coated anti-CD3 Orthoclone OKT3, Janssen-Cilag, 1 .ug/ml
  • soluble anti-CD28 BDBiosciences, 1 ⁇ g/ml
  • mice were irradiated (1.5 Gy) one day before tail vein injections of human PBMCs (3xl0 6 per mouse) alone, or mixed with autologous polyclonal Tregs (1.5xl0 6 per mouse).
  • mice received i.p. injections of PBS or 400 tig of MHG-8 (mlgGl), an anti-TGF-bl antibody (mlgGl clone 13A1/A26) or an isotype control (mlgGl anti-TNP clone B840 1 H5.M). Mice were monitored biweekly for the development of GVH D.
  • a global disease score was established by adding up scores attributed in the presence of the following symptoms: weight loss (1 if >10%; 2 if >20%); anemia or icterus (1 if white or yellow ears; 2 if white or yellow ears and tail); humped posture (1); reduced activity (1 if limited activity; 2 if no activity); hair loss (1). Mice were euthanized when reaching a global score >6. Death corresponds to a maximum score of 8. Cytokine concentrations in sera
  • Concentrations of human 1 L-2, IL-10, and IFNg in mouse serum were determined using a Bio-Plex Pro Human Cytokine 17-plex Assay according to the manufacturer's recommendations (Bio-Rad Laboratories). Limits of detection in this assay were: 0.12 pg/ ' ml for IL-2; 1.56 pg/ ' ml for IFNy; 2.48 pg/ml for IL-10.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2016001356A (es) 2013-08-01 2016-10-26 Ludwig Inst For Cancer Res Ltd Proteina anti-garp y sus usos.
IL274151B (en) 2015-05-21 2022-07-01 Harpoon Therapeutics Inc Trispecific binding proteins and methods of use
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US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
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CA3208624A1 (en) * 2021-01-18 2022-07-21 Shanghai Jemincare Pharmaceutical Co., Ltd. Garp protein antibody and application thereof
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Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5892019A (en) 1987-07-15 1999-04-06 The United States Of America, As Represented By The Department Of Health And Human Services Production of a single-gene-encoded immunoglobulin
DE3920358A1 (de) 1989-06-22 1991-01-17 Behringwerke Ag Bispezifische und oligospezifische, mono- und oligovalente antikoerperkonstrukte, ihre herstellung und verwendung
US6673986B1 (en) 1990-01-12 2004-01-06 Abgenix, Inc. Generation of xenogeneic antibodies
ATE297465T1 (de) 1991-11-25 2005-06-15 Enzon Inc Verfahren zur herstellung von multivalenten antigenbindenden proteinen
WO2007113301A1 (en) * 2006-04-03 2007-10-11 Medizinische Hochschule Hannover Pharmaceuticals for influencing the reaction of the human immune system
SG11201508681QA (en) * 2013-05-06 2015-11-27 Scholar Rock Inc Compositions and methods for growth factor modulation
MX2016001356A (es) * 2013-08-01 2016-10-26 Ludwig Inst For Cancer Res Ltd Proteina anti-garp y sus usos.
EP2832747A1 (de) * 2013-08-01 2015-02-04 Université Catholique de Louvain Anti-GARP-Protein und Verwendungen davon

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