WO2014028748A1 - Anticorps d'interleukine 2 et complexes d'anticorps - Google Patents

Anticorps d'interleukine 2 et complexes d'anticorps Download PDF

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WO2014028748A1
WO2014028748A1 PCT/US2013/055157 US2013055157W WO2014028748A1 WO 2014028748 A1 WO2014028748 A1 WO 2014028748A1 US 2013055157 W US2013055157 W US 2013055157W WO 2014028748 A1 WO2014028748 A1 WO 2014028748A1
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seq
sequence
antibody
variable region
chain variable
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PCT/US2013/055157
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Michael DIDONATO
Ann HERMAN
Deborah A. Knee
Fei Wang
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Irm Llc
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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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/246—IL-2
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to antibodies, antibody fragments, and antigen binding molecules that bind to IL-2 and modulate its binding to the IL-2 receptors.
  • High dose IL-2 therapy is used for treatment of cancer via a mechanism in which CD8 + T cells and NK cells that may kill the tumor cells are expanded. Deficiencies in the IL- 2 pathway in humans have also been associated with susceptibility to autoimmune disease. However, dosing of IL-2 in humans is associated with serious adverse events correlated with the expansion of the CD8+ and NK cells that make it unacceptable for the treatment of autoimmune indications.
  • the present invention is based, in part on the discovery of an anti-human IL-2 mAb that can modulate IL-2 results in the expansion of Treg cells and not CD8 or NK cells.
  • the invention thus provides antibodies, pharmaceutical compositions and methods for selectively expanding Treg cells.
  • the invention provides an isolated antibody that binds to the same epitope of human IL-2 as an antibody having a V H region set forth in SEQ ID NO: 12 and a V L region set forth in SEQ ID NO: 13.
  • an antibody of the invention typically binds to an epitope that includes residues Alal, Pro2, Thr3, Ser4, Ser5, Ser6, Thr7, Lys8, Lys9, Glnll, Leul2, Glul5, Hisl6, Leul9 of SEQ ID NO:81.
  • the antibody has a heavy chain V-segment that has at least 95% sequence identity to the V-segment sequence of SEQ ID NO:69 and a light chain V segment shares at least 95% sequence identity to the V- segment sequence of SEQ ID NO:70.
  • the antibody comprises: (a) a heavy chain variable region comprising a human heavy chain V-segment, a heavy chain complementary determining region 3 (CDR3), and a heavy chain framework region 4 (FR4), wherein the heavy chain CDR3 comprises the amino acid sequence EDYN(S/A)DD (SEQ ID NO:73) or ENWEGDN (SEQ ID NO: 17); and (b) a light chain variable region comprising a human light chain V segment, a light chain CDR3, and a light chain FR4, wherein the light chain CDR3 variable region comprises the amino acid sequence GTHFPF (SEQ ID NO: 6).
  • the CDR3 comprises the sequence WQGTHFPFT (SEQ ID NO: 11).
  • the heavy chain CDR3 comprises EDYNSDD (SEQ ID NO:3).
  • the heavy chain FR4 is a human germline FR4.
  • the heavy chain FR4 may, for example, have the sequence WGQGTLVTVSS (SEQ ID NO:71).
  • the light chain FR4 is a human germline FR4.
  • the light chain FR4 may, for example, have the sequence FGSGTKLEIK (SEQ ID NO:72).
  • the heavy chain V region comprises a CDR3 having a sequence EDYN(S/A)DD (SEQ ID NO:73) or ENWEGDN (SEQ ID NO: 17), a CDRl having a sequence DSYMN (SEQ ID NO:7), and a CDR2 having a sequence
  • the CDR3 comprises the sequence EDYN(S/A)DD (SEQ ID NO:73)
  • the CDR2 comprises the sequence DINP(D/K)NA(I/R/Y)TSYN(Q/R)KFRG (SEQ ID NO:75)
  • the CDRl comprises the sequence DSYMN (SEQ ID NO:7).
  • the light chain V region comprises a CDR3 having a sequence WQGTHFPFT (SEQ ID NO: 11), a CDRl sequence (R/K)SSQSL(F/R)DS(D/S)GKTYLN (SEQ ID NO:76) and a CDR2 sequence LVSKLDS (SEQ ID NO: 10).
  • the CDRl sequence comprises RSSQSLRDS(D/S)GKTYLN (SEQ ID NO:77).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPDNGITSYNQKFRG (SEQ ID NO:8), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDRl sequence KSSQSLFDSDGKTYLN (SEQ ID NO:9), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPNNGGTSYNRKFKG (SEQ ID NO: 18), and a CDR3 sequence ENWEGDN (SEQ ID NO: 17); and a light chain variable region having a CDRl sequence KSSQSLFDSDGKTYLN (SEQ ID NO:9), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPDNAITSYNQKFRG (SEQ ID NO:25), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDRl sequence RSSQSLRDSDGKTYLN (SEQ ID NO:26), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPDNAITSYNRKFRG (SEQ ID NO:35), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDRl sequence RSSQSLRDSDGKTYLN (SEQ ID NO:26), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPDN ARTS YNRKFRG (SEQ ID NO:41), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDRl sequence RSSQSLRDSDGKTYLN (SEQ ID NO:26), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPKN ARTS YNRKFRG (SEQ ID NO:47), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDRl sequence RSSQSLRDSDGKTYLN (SEQ ID NO:26), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPKNAYTSYNRKFRG (SEQ ID NO:53), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDRl sequence RSSQSLRDSDGKTYLN (SEQ ID NO:26), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence DSYMN (SEQ ID NO:7), a CDR2 sequence DINPDNAITSYNRKFRG (SEQ ID NO:25), and a CDR3 sequence EDYNADD (SEQ ID NO:58); and a light chain variable region having a CDRl sequence RSSQSLRDSSGKTYLN (SEQ ID NO:60), a CDR2 sequence LVSKLDS (SEQ ID NO: 10), and a CDR3 sequence WQGTHFPFT (SEQ ID NO: 11).
  • an antibody of the invention has a heavy chain variable region comprising a human heavy chain V-segment, a heavy chain complementary determining region 3 (CDR3), and a heavy chain framework region 4 (FR4), wherein the heavy chain CDR3 comprises the amino acid sequence EDYN(A/S)DD (SEQ ID NO:73) or ENWEGDN (SEQ ID NO: 17); and (b) a light chain variable region comprising a human light chain V segment, a light chain CDR3, and a light chain FR4, wherein the light chain CDR3 variable region comprises the amino acid sequence GTHFPF (SEQ ID NO: 6).
  • the antibody has a heavy chain V region that comprises a CDRl having a sequence GYTFTDS (SEQ ID NO: l), and a CDR2 having a sequence
  • the CDR2 comprises the sequence NPDNAI (SEQ ID NO:23), NPDNGI (SEQ ID NO:2), NPNNGG (SEQ ID NO: 16), NPDNAR (SEQ ID NO:40), NPKNAR (SEQ ID NO:46), or NPKNAY (SEQ ID NO:52).
  • the antibody comprises a light chain V region having a CDRl sequence SQSL(F/R)DS(D/S)GKTY (SEQ ID NO:79) and a CDR2 sequence LVS (SEQ ID NO:5).
  • the CDRl sequence has a sequence
  • SQSLFDSDGKTY (SEQ ID NO:4), SQSLRDSDGKTY (SEQ ID NO:24), or
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence GYTFTDS (SEQ ID NO:l), a CDR2 sequence NPDNGI (SEQ ID NO:2), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDRl sequence SQSLFDSDGKTY (SEQ ID NO:4), a CDR2 sequence LVS (SEQ ID NO:5), and a CDR3 sequence GTHFPF (SEQ ID NO:6).
  • an antibody of the invention comprises a heavy chain variable region having a CDRl sequence GYTFTDS (SEQ ID NO:l), a CDR2 sequence NPNNGG (SEQ ID NO: 16), and a CDR3 sequence ENWEGDN (SEQ ID NO: 17); and a light chain variable region having a CDR1 sequence SQSLFDSDGKTY (SEQ ID NO:4), a CDR2 sequence LVS (SEQ ID NO:5), and a CDR3 sequence GTHFPF (SEQ ID NO:6).
  • an antibody of the invention comprises a heavy chain variable region having a CDR1 sequence GYTFTDS (SEQ ID NO:l), a CDR2 sequence NPDNAI (SEQ ID NO:23), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDR1 sequence SQSLRDSDGKTY (SEQ ID NO:24), a CDR2 sequence LVS (SEQ ID NO:5), and a CDR3 sequence GTHFPF (SEQ ID NO:6).
  • an antibody of the invention comprises a heavy chain variable region having a CDR1 sequence GYTFTDS (SEQ ID NO:l), a CDR2 sequence NPDNAR (SEQ ID NO:40), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDR1 sequence SQSLRDSDGKTY (SEQ ID NO:24), a CDR2 sequence LVS (SEQ ID NO:5), and a CDR3 sequence GTHFPF (SEQ ID NO:6).
  • an antibody of the invention comprises a heavy chain variable region having a CDR1 sequence GYTFTDS (SEQ ID NO:l), a CDR2 sequence NPKNAR (SEQ ID NO:46), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDR1 sequence SQSLRDSDGKTY (SEQ ID NO:24), a CDR2 sequence LVS (SEQ ID NO:5), and a CDR3 sequence GTHFPF (SEQ ID NO:6).
  • an antibody of the invention comprises a heavy chain variable region having a CDR1 sequence GYTFTDS (SEQ ID NO:l), a CDR2 sequence NPKNAY (SEQ ID NO:52), and a CDR3 sequence EDYNSDD (SEQ ID NO:3); and a light chain variable region having a CDR1 sequence SQSLRDSDGKTY (SEQ ID NO:24), a CDR2 sequence LVS (SEQ ID NO:5), and a CDR3 sequence GTHFPF (SEQ ID NO:6).
  • an antibody of the invention comprises a heavy chain variable region having a CDR1 sequence GYTFTDS (SEQ ID NO:l), a CDR2 sequence NPDNAI (SEQ ID NO:23), and a CDR3 sequence EDYNADD (SEQ ID NO:58); and a light chain variable region having a CDR1 sequence SQSLRDSSGKTY (SEQ ID NO:59), a CDR2 sequence LVS (SEQ ID NO:5), and a CDR3 sequence GTHFPF (SEQ ID NO:6).
  • an antibody of the invention has a heavy chain variable region that shares at least 90% amino acid sequence identity, or at least 95% sequence identity, to a variable region selected from SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, or SEQ ID NO:61; and a light chain variable region that shares at least 90% amino acid sequence identity to a variable region selected from SEQ ID NO:28 or SEQ ID NO:62.
  • the heavy chain variable region comprises a variable region selected from SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, or SEQ ID NO:61; and the light chain variable region comprises a variable region selected from SEQ ID NO:28 or SEQ ID NO:62.
  • an antibody of the invention as described herein binds to IL- 2 with an equilibrium dissociation constant (3 ⁇ 4) of less than 5 x 10 ⁇ 8 M.
  • An antibody of the invention may be in any format.
  • the antibody is a Fab' fragment.
  • the antibody is an IgG.
  • an antibody of the invention comprises human constant regions.
  • an antibody of the invention that binds IL-2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, or SEQ ID NO:61.
  • an antibody of the invention that binds IL-2 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 13, SEQ ID NO:20, SEQ ID NO:28, or SEQ ID NO:62.
  • an antibody of the invention comprises: (a) a heavy chain variable region comprising SEQ ID NO: 12 or SEQ ID NO: 19 and a light chain variable region comprising SEQ ID NO: 13; (b) a heavy chain variable region comprising SEQ ID NO:27 and a light chain variable region comprising SEQ ID NO:28; (c) a heavy chain variable region comprising SEQ ID NO:36 and a light chain variable region comprising SEQ ID NO:28; (d) a heavy chain variable region comprising SEQ ID NO:42 and a light chain variable region comprising SEQ ID NO:28; (e) a heavy chain variable region comprising SEQ ID NO:48 and a light chain variable region comprising SEQ ID NO:28; (f) a heavy chain variable region comprising SEQ ID NO:54 and a light chain variable region comprising SEQ ID NO:28; or (g) a heavy chain variable region comprising SEQ ID NO:61 and a light chain variable region comprising SEQ ID NO: 62.
  • the invention provides methods of selectively expanding Treg cells in a patient having an autoimmune disease or a transplant patient, the method comprising administering an IL-2 antibody of the invention as described herein.
  • the antibody may be administered to the patient concurrently with IL-2.
  • the antibody and IL-2 are present in the same composition.
  • the IL-2 antibody is administered within 2 hours of administering IL-2 to the patient.
  • the patient may have any autoimmune disease, including, but not limited to, type I diabetes, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, or asthma.
  • the patient is a transplant patient.
  • Figure 1 Binding of antibodies to human IL2.
  • Figures 1 A-C provide illustrative data that hybridoma and fully human IL-2 modulating antibodies binds to human IL-2 as determined by ELISA assays.
  • Figure 2 Binding of antibodies to cyno IL2.
  • Figures 2A-C provide illustrative data that hybridoma and fully human IL-2 modulating antibodies binds to cynomolgus monkey IL-2 as determined by ELISA assays.
  • Figures 3A-C provide illustrative data that hybridoma IL-2 modulating antibodies, specifically bind to IL-2 from human (A) but not from rodent, as determined by ELISA assays.
  • the data from ELISA assays (B and C) also show that the anti-IL-2 antibodies of the invention do not bind to other off-target proteins.
  • Figure 4 Epitope mapping of anti-IL2 mAbs (alanine scanning).
  • Figures 4A-C illustrate epitope mapping of the IL-2 mAbs of the invention. Mutation of residues of human IL-2 to Alanine (A) identified residues with the epitope of MAB1 and 2 (B). All of the alanine scanning mutants were shown to be present in the ELISA assay by detection with an Anti-Fc antibody (C).
  • FIG. 4A SEQ ID NOS:81 and 98-104.
  • Figure 5 Crystal Structure Figures 5A-B illustrate the crystal structure of a Fab from MABlrFab bound to human IL-2 (A) and the position of the Alanine scanning mutations (B).
  • Figure 6 In vitro data.
  • Figures 6A-B illustrate the in vitro ability of MAB 1 to modulate the activity of human IL-2.
  • Mouse splenocytes were incubated with IL-2 complexed with MAB 1 for 72 hours and then analyzed by FACS to determine the change in numbers of CD4+CD25+ T cells (A) and CD8+CD25+ T cells (B).
  • the designation "5355” refers to a commercially available control antibody which non-selelctively expands CD8 cells and Treg cells.
  • Figure 7 In vitro STAT5 phosphorylation data.
  • Figure 7A-B illustrate the in vitro ability of MAB1 and MAB 8 to modulate IL-2 signaling through STAT-5 in cells that express the IL-2R y complex (A) but not cells that express the IL-2 ⁇ complex (B).
  • Figure 8 In vivo data.
  • Figure 8 illustrates the in vivo ability of a MAB 1 / human IL-2 complex to increase T reg frequency in NOD mice.
  • an "antibody” refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen.
  • An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond.
  • immunoglobulin genes include the ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either ⁇ or ⁇ .
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • VL variable light chain
  • VH variable heavy chain
  • an “antibody” encompasses all variations of antibody and fragments thereof that possess a particular binding specificity, e.g. , for IL-2.
  • full length antibodies, chimeric antibodies, and humanized antibodies, and multimeric versions of these fragments e.g. , multispecific (including bispecific) antibodies, multivalent antibodies, tetramers
  • the term "antibody” as used herein also refers to antibody fragments.
  • an “antibody fragment” encompasses all variations of antibody fragments that possess a particular binding specifically, e.g. , for IL-2.
  • single chain antibodies ScFv
  • Fab fragment antigen binding fragment
  • Fab' single chain antibodies
  • multimeric versions of these fragments e.g. , F(ab')2 ,
  • IL-2 antibody or “anti-IL-2 antibody” are used interchangeably to refer to an antibody that specifically binds to IL-2.
  • CDRs complementarity-determining domains
  • VL and VH- The CDRs are the target protein-binding site of the antibody chains that harbors specificity for such target protein.
  • CDRl-3 three CDRs (CDRl-3, numbered sequentially from the N-terminus) in each human VL or VH, constituting about 15-20% of the variable domains.
  • the CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity.
  • the remaining stretches of the VL or VH, the so-called framework regions exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).
  • the positions of the CDRs and framework regions are determined using various well known definitions in the art, e.g. , Kabat, Chothia, international ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.cines.fr/), and AbM (see, e.g. , Johnson et al. , Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al. , Nature, 342:877-883 (1989); Chothia et al. , J. Mol.
  • binding specificity determinant or “BSD” interchangeably refer to the minimum contiguous or non-contiguous amino acid sequence within a complementary determining region necessary for determining the binding specificity of an antibody.
  • a minimum binding specificity determinant can be within one or more CDR sequences. In some embodiments, the minimum binding specificity determinants reside within (i.e. , are determined solely by) a portion or the full-length of the CDR3 sequences of the heavy and light chains of the antibody.
  • an "antibody light chain” or an “antibody heavy chain” as used herein refers to a polypeptide comprising the VL or VH, respectively.
  • the endogenous VL is encoded by the gene segments V (variable) and J (junctional), and the endogenous VH by V, D (diversity), and J.
  • Each of VL or VH includes the CDRs as well as the framework regions.
  • antibody light chains and/or antibody heavy chains may, from time to time, be collectively referred to as "antibody chains.” These terms encompass antibody chains containing mutations that do not disrupt the basic structure of VL or VH, as one skilled in the art will readily recognize.
  • Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab' which itself is a light chain joined to VH-CH1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region. Paul, Fundamental Immunology 3d ed. (1993).
  • antibody While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g. , single chain Fv) or those identified using phage display libraries (see, e.g. , McCafferty et al. , Nature 348:552-554 (1990)).
  • any technique known in the art can be used (see, e.g. , Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al. , Immunology Today 4:72 (1983); Cole et al. , Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985).
  • Techniques for the production of single chain antibodies can be adapted to produce antibodies to polypeptides of this invention.
  • transgenic mice, or other organisms such as other mammals may be used to express humanized antibodies.
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g. , McCafferty et al. , supra; Marks et al. , Biotechnology, 10:779-783, (1992)).
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol.
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some complementary determining region ("CDR") residues and possibly some framework (“FR”) residues are substituted by residues from analogous sites in rodent antibodies.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g. , an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • variable region or "V-region” interchangeably refer to a heavy or light chain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • An endogenous variable region is encoded by immunoglobulin heavy chain V-D-J genes or light chain V-J genes.
  • a V- region can be naturally occurring, recombinant or synthetic.
  • variable segment or “V-segment” interchangeably refers to a subsequence of either a heavy chain or light chain variable region that extends from from FR1 through FR3 (i.e., FR1-CDR1-FR2-CDR2-FR3).
  • An endogenous V-segment is encoded by an immunoglobulin V-gene.
  • a V-segment can be naturally occurring, recombinant or synthetic.
  • J-segment refers to a subsequence of the variable region encoded comprising a C-terminal portion of a CDR3 and the FR4.
  • An endogenous J-segment is encoded by an immunoglobulin J-gene.
  • a J-segment can be naturally occurring, recombinant or synthetic.
  • a "humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with human counterparts. See, e.g. , Morrison et ah , Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et ah , Science, 239: 1534-1536 (1988); Padlan, Molec. Immun. , 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994).
  • corresponding human germline sequence refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences.
  • the corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences.
  • the corresponding human germline sequence can be framework regions only, complementary determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art.
  • the corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 92%, 94%, 96%, 98%, 99% sequence identity with the reference variable region nucleic acid or amino acid sequence.
  • Corresponding human germline sequences can be determined, for example, through the publicly available international ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.cines.fr/) and V-base (on the worldwide web at vbase.mrc- cpe.cam.ac.uk).
  • IMGT international ImMunoGeneTics database
  • V-base on the worldwide web at vbase.mrc- cpe.cam.ac.uk.
  • the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample.
  • Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein.
  • an IL-2 antibody of the invention typically binds to human or primate IL-2, but not to murine IL-2.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid- phase ELISA immunoassays are routinely used to select antibodies specifically
  • a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least than 10 to 100 times over the background.
  • Equilibrium dissociation constant (3 ⁇ 4, M) refers to the dissociation rate constant (k ⁇ j, time "1 ) divided by the association rate constant (k a , time "1 , M “1 ). Equilibrium dissociation constants can be measured using any known method in the art.
  • the antibodies of the present invention generally will have an equilibrium dissociation constant, as determined by surface plasmon resonance analysis performed at 37°C, of less than about 10 "7 or 10 "8 M, for example, less than about 10 "9 M or 10 "10 M, in some embodiments, less than about 10 "11 M, 10 "12 M or 10 "13 M.
  • antigen-binding region refers to a domain of an IL-2- antibody of this invention that is responsible for the specific binding between the antibody and IL-2.
  • An antigen-binding region typically includes at least one antibody heavy chain variable region and at least one antibody light chain variable region.
  • T regulatory cells are T cells that suppress immune responses of other cells.
  • Treg cells are characterized by the expression of CD4 and CD25 (CD4+CD25+ regulatory T cells). Treg cells also express the Foxp3 transcription factor. Treg cells are involved in shutting down immune responses after they have successfully eliminated invading organisms, and also in preventing autoimmunity. Treg cells respond to the presence of 11-2 by rapid proliferation.
  • IL-2 antibody that expands Treg population, but not CD8 or NK cells refers to an antibody that binds to IL-2 and when complexed with IL-2 does not inhibit IL-2- mediated proliferation of Treg cells, but does prevent or inhibit IL-2-induced proliferation of CD8 or NK cells. Accordingly, in the context of the current invention, an IL-2 antibody of the invention when complexed with IL-2 selectively induces proliferation of Treg cells, but not CD8 or NK cells. Proliferation of Treg cells can be assessed using methods known in the art for measuring T cell proliferation, such as an in vitro splenocyte proliferation assay, as illustrated in Example 1.
  • An antibody of the invention increases proliferation of Treg cells when complexed with IL-2 by at least 40%, 50%, or 60%, or more, compared to Treg cells that are treated not treated with the anti-IL-2/IL-2 complex.
  • the antibody when administered with IL-2 also inhibits proliferation of CD8+CD25+ T cells by at least 20%, typically by at least 50%, or greater, compared to CD8+CD25+ T cells treated with IL-2 alone.
  • interleukin-2 or "interleukin-2” interchangeably refer to a cytokine involved in the regulation of immune response cells.
  • IL-2 induces proliferation and activation of T cells, B cells, and NK cells, although T-cells appear to be its major target.
  • IL- 2 plays a role in T cell immunologic memory and in the maturation of regulatory T cells (T- regs).
  • IL-2 is a glycoprotein of 15 kDa that is a member of the four a-helical bundle family of cytokines.
  • the 153 amino acid human IL-2 protein includes a 20-residue signal peptide.
  • the gene encoding human IL-2 is localized to chromosome 4q26-q27.
  • the nucleic acid and amino acid sequences of human IL-2 are known.
  • the human IL-2 protein sequence has been published under GenBank Accession No. NP_000577.2 (IL-2 precursor protein, including signal peptide, GL28178861; PRI 25-MAR-2012).
  • the mRNA sequence is published under GenBank Accession No. NM_000586.3 (GI: 125661059, PRI 25-MAR-2012).
  • an IL-2 polypeptide is functionally an IL-2 that induces Treg proliferation.
  • an IL-2 amino acid sequence shares at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the mature IL-2 protein sequence of GenBank accession numbers NP_000577.2.
  • an IL-2 nucleic acid sequence shares at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the region of the nucleic acid sequence of GenBank accession number NM_000586.3 that encodes the mature IL-2 protein.
  • an "IL-2" protein also includes allelic variants that are encoded by an IL-2 gene that localizes to chromosome 4q26-q27.
  • a "transplant patient” as used in the context of this invention refers to an individual who has received a transplant, is undergoing a transplant procedure, or will undergo a transplant procedure within two weeks, or within one week, e.g., within 72 hours or less, of administration of an IL-2 antibody of the invention.
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • the terms "polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i. e. , gaps) as compared to the reference sequence (e.g. , a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequences.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • polypeptides that are substantially identical to the polypeptides exemplified herein (e.g. , the variable regions exemplified in any one of SEQ ID NOs: 12, 13, 19, 20, 27, 28, 36, 42, 48, 54, 61, 62, 69, or 70).
  • identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence.
  • shorter amino acid sequences e.g. , amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) /. Mol. Biol.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) /. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et ah , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • the terms "subject,” “patient,” and “individual” interchangeably refer to a mammal, for example, a human or a non-human primate mammal.
  • the mammal can be an agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid) or domestic mammal (e.g., canine, feline).
  • a therapeutically acceptable amount or “therapeutically effective dose” interchangeably refer to an amount sufficient to effect the desired result (i.e. , apoptosis of a target cell). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects.
  • a therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.
  • a "prophylactically effective dosage,” and a “therapeutically effective dosage,” of an IL-2 antibody of the invention can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms (e.g., symptoms of disorders associated with T- reg function). Said terms can also promote or increase, respectively, frequency and duration of periods free from disease symptoms.
  • the present invention provides IL-2 antibodies, and pharmaceutical compositions comprising the antibodies, that selectively binds to an epitope at which binding of the antibody results in expansion of Treg without expansion of CD8 + or NK cells.
  • the IL-2 antibodies of the invention find use, either when administered alone or in some embodiments, when administered complexed with IL-2, in reducing, inhibiting and preventing symptoms of conditions and diseases resulting from Treg deficiency. Accordingly, IL-2 antibodies can be used to treat autoimmune diseases, to treat transplant patients, or to treat patients having diabetes.
  • An IL-2 antibody of the invention typically binds to an epitope at the N-terminus of the mature IL-2 that encompasses the unstructured region residues Alal, Pro2, Thr3, Ser4, Ser5, Ser6, Thr7, Lys8, Lys9 of the mature peptide prior to helix A of human IL-2 and several residues on the same face of helix A, residues Glnl l, Leul2, Glul5, Hisl6, and Leul9. Helix A is known to be important in binding of the IL-2R chain.
  • the anti-IL-2 antibody/IL-2 complex expands human Treg cells (CD4+, CD25+, FoxP3+), but does not expand activated human CD8 cells (CD8+, CD25+).
  • the epitope thus includes the region of human IL-2 that contains amino acids 8 and 9 (KK) and includes the residues Alal, Pro2, Thr3, Ser4, Ser5, Ser6, Thr7, Lys8, Lys9, Glnll, Leul2, Glul5, Hisl6, and Leul9.
  • the IL-2 antibodies are administered as a complex with IL-2.
  • the IL-2 antibodies of the present invention bind to an epitope on IL-2 such that treatment with an IL-2 antibody/IL-2 ocmplex results in the explanion of Treg without expanding CD8 or NK cells.
  • Antibodies of the invention can be used in vitro or in vivo to expand Treg cells.
  • an anti-IL-2 antibody is administered to a patient suffering from an autoimmune disease as detailed below or a transplant patient.
  • the antibodies of the present invention specifically bind human IL-2 and when complexed with IL-2, allow for proliferation of Treg cells, but not proliferation of CD8+ or NK cells.
  • the anti-IL-2 antibody can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and for certain desired fragments, enzymatic digestion of antibody tetramers. Full-length monoclonal antibodies can be obtained by, e.g. , hybridoma or recombinant production.
  • an anti-IL-2 antibody of the invention can be any type or subtype, as appropriate, and can be selected to be from the species of the subject to be treated by the present methods (e.g. , human or non-human primate).
  • an antibody of the invention can include a human constant region, such as a human kappa or lambda constant region; and/or a heavy chain constant, e.g., a gamma-1, gamma-2, gamma-3, or gamma-4 constant region.
  • the antibody may be an IgA or IgM.
  • Antibodies may be produced using any number of expression systems, including both prokaryotic and eukaryotic expression systems, such as mammalian expression systems, insect expression systems, yeast expression systems and the like.
  • the expression system is a mammalian cell expression, such as a CHO cell expression system.
  • the VH and VL regions may be expressed using a single vector, e.g., in a discistronic expression unit, or under the control of different promoters. In other embodiments, the VH and VL region may be expressed using separate vectors.
  • antibodies are purified from culture media and host cell using known techniques.
  • antibody chains are expressed with signal sequences and are thus released to the culture media.
  • An antibody of the invention may also be produced in any number of formats involving antigen binding fragments, including as a Fab, a Fab', a F(ab') 2 , a scFv, or a dAB.
  • the anti-IL-2 antibodies can be multimerized and used according to the methods of this invention.
  • Anti-IL-2 antibodies or antigen-binding molecules of the invention also include single domain antigen-binding units which have a camelid scaffold. Animals in the camelid family include camels, llamas, and alpacas. Camelids produce functional antibodies devoid of light chains.
  • the heavy chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit.
  • Camelid antibodies are capable of attaining binding affinities comparable to those of conventional antibodies.
  • Camelid scaffold-based anti-IL-2 molecules with binding specificities of the anti-IL-2 antibodies exemplified herein can be produced using methods well known in the art, e.g., Dumoulin et al., Nature Struct. Biol. 11:500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J Mol Biol. 332:643-55, 2003.
  • the engineered anti-IL-2 antibodies of the invention can be engineered human antibodies with V-region sequences having substantial amino acid sequence identity to human germline V-region sequences while retaining the specificity and affinity of a reference antibody. See, U.S. Patent Publication No. 2005/0255552 and U.S. Patent Publication No. 2006/0134098, both of which are hereby incorporated herein by reference.
  • the process of engineering identifies minimal sequence information required to determine antigen-binding specificity from the variable region of a reference antibody, and transfers that information to a library of human partial V-region gene sequences to generate an epitope-focused library of human antibody V-regions.
  • a microbial-based secretion system can be used to express members of the library as antibody Fab fragments and the library is screened for antigen- binding Fabs, for example, using a colony-lift binding assay. See, e.g. , U.S. Patent
  • Positive clones can be further characterized to identify those with the highest affinity.
  • the resultant engineered human Fabs retain the binding specificity of the parent, reference anti-IL-2 antibody, typically have equivalent or higher affinity for antigen in comparison to the parent antibody, and have V-regions with a high degree of sequence identity compared with human germ- line antibody V-regions.
  • the minimum binding specificity determinant (BSD) required to generate the epitope-focused library is typically represented by a sequence within the heavy chain CDR3 ("CDRH3") and a sequence within the light chain of CDR3 ("CDRL3").
  • the BSD can comprise a portion or the entire length of a CDR3.
  • the BSD can be comprised of contiguous or non-contiguous amino acid residues.
  • the epitope-focused library is constructed from human V-segment sequences linked to the unique CDR3-FR4 region from the reference antibody containing the BSD and human germ-line J-segment sequences (see, Figure 1 and U.S. Patent Publication No. 2005/0255552).
  • the human V-segment sequences linked to the unique CDR3-FR4 region from the reference antibody containing the BSD and human germ-line J-segment sequences (see, Figure 1 and U.S. Patent Publication No. 2005/0255552).
  • V-segment libraries can be generated by sequential cassette replacement in which only part of the reference antibody V-segment is initially replaced by a library of human sequences.
  • the identified human "cassettes" supporting binding in the context of residual reference antibody amino acid sequences are then recombined in a second library screen to generate completely human V-segments (see, U.S. Patent Publication No. 2006/0134098).
  • paired heavy and light chain CDR3 segments, CDR3-FR4 segments, or J-segments, containing specificity determinants from the reference antibody are used to constrain the binding specificity so that antigen-binders obtained from the library retain the epitope-specificity of the reference antibody. Additional maturational changes can be introduced in the CDR3 regions of each chain during the library construction in order to identify antibodies with optimal binding kinetics.
  • the resulting engineered human antibodies have V-segment sequences derived from the human germ-line libraries, retain the short BSD sequence from within the CDR3 regions and have human germ-line framework 4 (FR4) regions.
  • an anti-IL-2 antibody of the invention contains a minimum binding sequence determinant (BSD) within the CDR3 of the heavy and light chains derived from the originating or reference monoclonal antibody.
  • the remaining sequences of the heavy chain and light chain variable regions (CDR and FR), e.g. , V-segment and J-segment, are from corresponding human germline and affinity matured amino acid sequences.
  • the V-segments can be selected from a human V-segment library. Further sequence refinement can be accomplished by affinity maturation.
  • the heavy and light chains of the anti-IL-2 antibodies contain a human V-segment from the corresponding human germline sequence (FR1-CDR1- FR2-CDR2-FR3), e.g. , selected from a human V-segment library, and a CDR3-FR4 sequence segment from the originating monoclonal antibody.
  • the CDR3-FR4 sequence segment can be further refined by replacing sequence segments with corresponding human germline sequences and/or by affinity maturation.
  • the FR4 and/or the CDR3 sequence surrounding the BSD can be replaced with the corresponding human germline sequence, while the BSD from the CDR3 of the originating monoclonal antibody is retained.
  • the corresponding human germline sequence for the heavy chain V-segment is VH1 1-46. In some embodiments, the corresponding human germline sequence for the heavy chain is J-segment is JH4. In some embodiments, the heavy chain J-segment comprises the human germline JH4 partial sequence WGQGTLVTVSS (SEQ ID NO:71). The full-length J-segment from human germline JH4 is YFD YWGQGTLVT VS S (SEQ ID NO: 80).
  • the variable region genes are referenced in accordance with the standard nomenclature for immunoglobulin variable region genes. Current immunoglobulin gene information is available through the worldwide web, for example, on the ImMunoGeneTics (IMGT), V-base and PubMed databases. See also, Lefranc, Exp Clin Immuno genet.
  • the corresponding human germline sequence for the light chain V-segment is VKII-A1. In some embodiments, the corresponding human germline sequence for the light chain J-segment is Jk2. In some embodiments, the light chain
  • J-segment comprises the sequence FGSGTKLEIK (SEQ ID NO:72).
  • the heavy chain V-segment shares at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:69. In some embodiments, the light chain V-segment shares at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:70.
  • the heavy chain V-segment shares at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, or SEQ ID NO:61.
  • the light chain V-segment shares at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence SEQ ID NO:28 or SEQ ID NO:62.
  • the heavy chain CDR3 comprises EDYN(S/A)DD (SEQ ID NO:73) or ENWEGDN (SEQ ID NO: 17);
  • the light chain CDR3 comprises WQGTHFPFT (SEQ ID NO: 11).
  • the antibodies of the invention comprise a heavy chain variable region comprising a CDRl comprising DSYMN (SEQ ID NO:7); a CDR2 comprising DINP(D/N/K)N(G/A)(I/G/R/Y)TSYN(Q/R)KF(R/K)G (SEQ ID NO:74), and a CDR3 comprising EDYN(S/A)DD (SEQ ID NO:73).
  • the antibody comprises a heavy chain variable region that has a CDRl comprising DSYMN (SEQ ID NO:7), a CDR2 comprising DINP(D/K)NA(I/R/Y)TSYN(Q/R)KFRG (SEQ ID NO:75), and a CDR3 comprising EDYN(S/A)DD (SEQ ID NO:73).
  • the CDR3 is EDYNSDD (SEQ ID NO:3).
  • the antibodies of the invention comprise a light chain variable region comprising a CDRl comprising (R/K) S S QS L(F/R)D S(D/ S) GKT YLN, (SEQ ID NO:76), a CDR2 comprising LVSKLDS (SEQ ID NO: 10), and a CDR3 comprising WQGTHFPFT (SEQ ID NO: 11).
  • the antibody comprises a light chain variable region that comprises a CDRl RSSQSLRDS(D/S)GKTYLN (SEQ ID NO:77), a CDR2 comprising LVSKLDS (SEQ ID NO: 10), and a CDR3 comprising WQGTHFPFT (SEQ ID NO: 11).
  • the heavy chain CDR3 comprises EDYN(S/A)DD (SEQ ID NO:73) or ENWEGDN (SEQ ID NO: 17);
  • the light chain CDR3 comprises GTHFPF (SEQ ID NO: 6).
  • the antibodies of the invention comprise a heavy chain variable region comprising a CDRl comprising GYTFTDS (SEQ ID NO: l); a CDR2 comprising NP(N/D/K)N(A/G)(I/G/R/Y) (SEQ ID NO:78) , and a CDR3 comprising
  • the antibody comprises a heavy chain variable region that has a CDRl comprising
  • GYTFTDS (SEQ ID NO: l), a CDR2 comprising NPDNGI (SEQ ID NO:2), NPDNAI (SEQ ID NO:23), NPDNAR (SEQ ID NO:40), NPKNAR (SEQ ID NO:46), or NPKNAY (SEQ ID NO:52) and a CDR3 comprising EDYN(S/A)DD (SEQ ID NO:73).
  • the CDR3 is EDYNSDD (SEQ ID NO:3).
  • the antibody comprises a heavy chain variable region that has a CDRl comprising GYTFTDS (SEQ ID NO: l), a CDR2 comprising NPNNGG (SEQ ID NO: 16), and a CDR3 comprising ENWEGDN (SEQ ID NO: 17).
  • the antibodies of the invention comprise a light chain variable region comprising a CDRl comprising SQSL(F/R)DS(D/S)GKTY (SEQ ID NO:79); a CDR2 comprising LVS (SEQ ID NO:5), and a CDR3 comprising GTHFPF (SEQ ID NO:6).
  • the CDRl sequence comprises SQSLFDSDGKTY (SEQ ID NO:4), SQSLRDSDGKTY (SEQ ID NO:24), or SQSLRDSSGKTY (SEQ ID NO:59).
  • the heavy chain variable region has a FR1 comprising the FR1 amino acid sequence of SEQ ID NO:69, a FR2 comprising the FR2 of SEQ ID NO: 17, and a FR3 comprising the FR3 of SEQ ID NO:69.
  • the FR4 comprises WGQGTLVTVSS (SEQ ID NO:71).
  • the FR1, FR2, and/or FR3 sequences can be determined based on the the CDRs, e.g., the CDRs as defined by Kabat or Chothia. (See also, Table A).
  • the identified amino acid sequences may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.
  • the light chain variable region has a FR1 comprising the FR1 amino acid sequence of SEQ ID NO:70, a FR2 comprising the FR2 of SEQ ID NO: 18, and a FR3 comprising the FR3 of SEQ ID NO:70.
  • the FR4 comprises FGSGTKLEIK (SEQ ID NO:72).
  • the FR1, FR2, and/or FR3 sequences can be determined based on the the CDRs, e.g., the CDRs as defined by Kabat or Chothia. (See also, Table A).
  • the identified amino acid sequences may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.
  • variable regions of the anti-IL-2 antibodies of the invention generally will have an overall variable region (e.g. , FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4) amino acid sequence identity of at least about 90%, for example, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the corresponding human germline variable region amino acid sequence.
  • the heavy chain of the anti-IL-2 antibodies can share at least about 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the human germline variable region VH1 1-46/JH4.
  • the light chain of the anti-IL2 antibodies can share at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the human germline variable region VKII-Al/Jk2. In some embodiments, only amino acids within the framework regions are added, deleted or substituted. In some embodiments, the sequence identity comparison excludes the CDR3.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 12 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 13.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 19 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:20.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:27 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:28.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:36 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:28.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:42 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:28.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:48 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:28.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:54 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:28.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the invention comprise a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:61 and comprise a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:62.
  • the variation in sequence compared to the reference sequences excludes the CDR3s.
  • the anti-IL-2 antibodies of the present invention generally will bind to IL-2 with an equilibrium dissociation constant (3 ⁇ 4) of less than about 10 "8 M or 10 "9 M, for example, less than about 10 "10 M or 10 ⁇ n M, in some embodiments less than about 10 "12 M or 10 "13 M.
  • Anti-Il-2 antibodies that bind to the same epitope and/or compete with Mabl or Mab2 for binding to helix A of IL-2 can be identified using assays well known in the art.
  • the epitope to which the IL-2 antibody binds includes the residues Alal, Pro2, Thr3, Ser4, Ser5, Ser6, Thr7, Lys8, Lys9, Glnl l, Leul2, Glul5, Hisl6, Leu 19 of the mature form of IL-2.
  • an antibody of the invention competes with Mabl, i.e., competes with an antibody that has the ⁇ 1 ⁇ 2 and VL regions of the Mabl antibody (SEQ ID NOS: 1 and 2, respectively) for binding to IL-2.
  • an antibody described herein e.g., a different antibody comprising a VH and VL region combination as described herein, can be used as a reference antibody for assessing whether another antibody competes for binding to IL-2.
  • a test antibody is considered to competitively inhibit binding of a reference antibody, if binding of the reference antibody to the antigen is reduced by at least 30%, usually at least about 40%, 50%, 60% or 75%, and often by at least about 90%, in the presence of the test antibody.
  • Many assays can be employed to assess binding, including ELISA, as well as other assays known in the art, such as immunoblots.
  • an IL-2 antibodyof the invention can be tested using known assays.
  • an antibody can be evaluated for the ability to affect Treg numbers when complexed with IL-2.
  • Such an assay can be performed in vitro or in vivo.
  • an in vitro mouse splenocyte assay can be performed.
  • Human IL-2 induces an increase in the numbers of CD4+ CD25+ T cells and to a greater extent CD8+ CD25+ T cells.
  • the binding of an IL-2 antibody of the invention to human IL-2 does not significantly alter the ability of human IL-2 to expand CD4+CD25+ T cells, but it does significantly decrease the proliferation of CD8+CD25+ T cells.
  • an assay for evaluating activity of an anti-IL-2 antibody typically comprises determining whether the antibody selectively expands Treg cells when the antibody is complexed with IL-2.
  • the ability of an antibody to modulate the function of IL-2 is assessed by determining the ability of an antibody to inhibit IL-2-induced proliferation of CD8+ CD25+ T cells and the influence of the antibody on IL-2 induced CD4+CD25+ T cell proliferation.
  • proliferation may, for example, be measured in splenocytes treated with IL- 2 / Ab complex compared to splenocytes treated with IL-2 alone.
  • An antibody of the invention typically inhibits IL-2 induced CD8+CD25+ T cell proliferation in an in vitro splenocyte assay by at least 20%, typically by at least 50%, or more when compared to splenocytes treated with IL-2 alone, but the proliferation of CD4+CD25+ T cells is not significantly reduced.
  • the proliferation of CD4+CD25+ T cells is reduced by no more than about 30%, preferably by no more than about 20%, or less, compared to proliferation observed of splenocytes treated with IL-2 alone.
  • the ability of an antibody of the invention to stimulate CD4+CD25+ T cell proliferation may be determined by treating splenocytes with the antibody complexed with IL-2 and comparing proliferation to controls that are not treated with IL-2.
  • an antibody of the invention increases proliferation of Treg cells when complexed with IL-2 by at least 40%, 50%, or 60%, or more, compared to Treg cells that are not treated with the anti-IL-2/IL-2 complex.
  • the anti-IL-2 antibodies of the invention can be used to treat or ameliorate any diseases or conditions that benefit from expansion of the Treg T cell population including autoimmune disorders, transplants, and diabetes.
  • the IL-2 antibodies of the invention interact with an epitope on IL-2 and form a complex with IL-2.
  • the IL-2 antibodies are administered with IL-2, e.g., administered in a form where an anti-112 antibody is complexed with IL-2.
  • the IL-2 antibodies of the invention find use to e.g., diagnose, inhibit, prevent, ameliorate the symptoms of, protect against, and treat disorders associated with Treg T cells.
  • disorders that can be treated with IL-2 antibodies include autoimmune diseases such as Type I diabetes, rheumatoid arthritis, asthma, systemic lupus erythematosus, multiple sclerosis, irritable bowel syndrome, Hashimoto's thyroiditis, Crohns disease, inflammatory bowel disease and other autoimmune or inflammatory diseases.
  • the IL-2 antibodies of the invention are additionally suitable for treating transplant patients, or patients who will be receiving a transplant.
  • Transplant patients that can be treated include patients receiving islet, kidney, pancreas, heart, liver, or other transplants.
  • a therapeutically and/or prophylactically effective amount of a second agent effective in treating, diagnosing, preventing, and/or ameliorating autoimmune diseases, or other Treg-associated-associated diseases, disorders or symptoms is administered to the individual in combination with another agent for treating the disease.
  • the invention provides pharmaceutical compositions comprising the anti-IL-2 antibodies formulated together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition additionally comprises IL-2.
  • Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • a pharmaceutical composition of the present invention can be administered by a variety of methods known in the art.
  • the route and/or mode of administration vary depending upon the desired results. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous.
  • the pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, intranasal, inhalational, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • the antibodies can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • the composition is sterile and fluid. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
  • compositions are preferably manufactured under GMP conditions.
  • a therapeutically effective dose or efficacious dose of the anti-IL-2 antibody is employed in the pharmaceutical compositions of the invention.
  • the anti-IL-2 antibodies are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response).
  • a therapeutically or prophylactically effective dose a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular
  • compositions of the present invention employed, or the ester, salt or amide thereof the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • a clinician can start doses of the antibodies of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • effective doses of the compositions of the present invention vary depending upon many different factors, including the specific disease or condition to be treated, means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months.
  • the antibody can be administered, alone or in combination with IL-2, in single or divided doses. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of anti-IL-2 antibody in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml.
  • antibody or antibody/IL-2 complex can be administered as a sustained release formulation, in which case less frequent administration is required.
  • Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies show longer half life than that of chimeric antibodies and nonhuman antibodies.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • an IL-2 antibody or IL-2 antibody/IL-2 complex can be administered after the transplant, during the transplant, or before the transplant. Wherer the IL-2 antibody is administered before the transplant, administration typically takes place less than two weeks, preferably less than one week, and often less than 72, 48, or 24 hours prior to transplantation.
  • IL-2 and the anti-IL-2 antibody can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
  • Concomitant administration of an anti-IL-2 antibody and IL-2 means administration of the two agents at such time that the combination of IL-2 and anti-IL-2 antibody enhances Treg proliferation. Such concomitant administration can involve concurrent (i.e. at the same time), prior, or subsequent administration of either IL-2 or IL-2 antibody, e.g., administration of IL-2 antibody followed by administration of IL-2.
  • the anti-IL-2 antibody can be administered in the same pharmaceutical formulation as the IL-2.
  • an anti-IL-2 antibody prepraration is mixed IL-2 prior to administration
  • An IL-2 antibody of the invention or IL-2 antibody /IL2 complex can be used in combination with other agents known to be beneficial for treating the Treg-associated condition. Accordingly, a patient having an autoimmune disease may receive additional therapeutic agents for treating the disease. For example, a diabetes patient will typically also receive other pharmaceutical agents for treating diabetes. Similarly, a transplant patient will also receive additional compounds to reduce rejection of the transplanted tissue. Active agents can be administered separately or together with an IL-2 antibody or IL-2 antibody-IL- 2 complex. The IL-2 antibody and the other active agent can, but need not, be administered concurrently or can be administered in any order.
  • helix A of human IL-2 (APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATE, SEQ ID NO:81) was cloned from cDNA, expressed as a mouse Fc fusion protein ("mFc-hlL- 2-DM1"), and used as the immunogen.
  • mFc-hIL-2-DMl has the amino acid sequence as shown below:
  • Hybridomas were generated following immunization of mice with the mFc-hIL-2 fusion protein.
  • a primary ELISA screen was carried out using hFc-hIL-2 as the antigen. Positive hybridomas were then subjected to a reconfirmation ELISA screen using hFc-hIL-2 and hFc-hTNFRl to eliminate any clones that bound non-specifically to the hFc tag.
  • Clones were tested in several in vitro assays, including a biochemical assay for binding of IL-2 to IL- 2 ⁇ or IL-2R0C, and a proliferation assay using human IL-2 on mouse splenocytes.
  • mice that showed an ability to modulate IL-2 and block CD8+ CD25+ proliferation were further characterized in vivo.
  • mice were dosed with an IL-2/Ab complex and CD4+CD25+ and CD8+CD25+ T cell numbers were determined by FACS.
  • a 384 well plate was coated with the protein of interest at 50ng per well and incubated overnight at 4°C.
  • the plate was blocked with a solution of 1 BSA in PBS (Blocking Buffer) for one hour at room temperature and then rinsed three times with PBST.
  • the MAB was then diluted to 1 ⁇ g/ml in PBS and 20 ⁇ of this dilution was applied to the plate for 1 hour at room temperature.
  • the plate was then rinsed 3 times with PBST, and Jackson Immunoresearch goat anti-mouse Fc- specific HRP conjugate (#115-035-072) was diluted 1 :5000 in Blocking Buffer.
  • a 96 well plate was coated with anti-CD3 antibody (0.5mg/ml in PBS) overnight at 4°C. The next day 2 x 10 5 cells were added to each well in 50 ⁇ of media.
  • the mAb : human IL-2 complex was made by incubating 50 ⁇ g Ab and 500 ⁇ g of human IL-2 at room temperature for 20 minutes in a volume of 120 ul.
  • the Ab:IL-2 complex and 30 ⁇ of anti-CD28 (0.5 ⁇ g/ml) were then added to the cells in the 96 well plate. The plate was the incubated for 72 hours in a 37°C tissue culture incubator before analyzing by FACS.
  • In vivo model examine effects ofMABl/human IL-2 complex on Treg numbers [0140] The ability of a MAB 1/human IL-2 complex to modulate Treg numbers was assessed in vivo. Prediabetic NOD mice were dosed 6x over 7days with MAB 1/human IL-2 (50 ⁇ g: 1.5 ⁇ g) or human IL-2 alone (1.5 g). On Day 7, the % of Treg cells in blood was determined by FACS. The fold change in Treg frequency was calculated by comparing to mice treated with hIL-2 alone. Each dot represents an independent experiment.
  • Fig. 4A In order to identify the epitope on human IL-2 that is bound by the mouse monoclonal antibodies MAB1 and MAB2, several Alanine mutants were generated (Fig. 4A.). The data illustrated in Fig. 4B. demonstrated that binding of MAB 1 and MAB 2 to IL-2 was abolished in protein A-4 and therefore that these mAbs bind a region of IL-2 that contains amino acids 8 and 9 (KK).
  • Example 2 Creation of IL-2 Modulator Antibodies MAB3.
  • MAB4. MAB5, MAB6, MAB7 and MAB8
  • This example describes the generation of human antibodies MAB3, MAB4, MAB5, MAB 6, MAB 7, and MAB 8 by engineering the murine monoclonal IL2 neutralizing antibody MAB1 to have greater sequence homology to a human germline antibody.
  • MAB3, MAB4, MAB5, MAB6, MAB7, and MAB8 retain the epitope specificity, affinity, and cynomolgus macaque IL2 cross-reactivity of the parental murine antibody, MAB1.
  • MAB3, MAB3, MAB4, MAB5, MAB6, MAB7, and MAB8 have much higher homology to the human germline sequence than the original murine antibody and should therefore be better tolerated by the human immune system.
  • Mouse monoclonal MAB 1 was HumaneeredTM (engineered to contain sequences that have high homology to human germline sequences) to bring its protein sequence closer to a human germline sequence and decrease its immunogenicity. HumaneeringTM technology is available through KaloBios of South San Francisco (on the worldwide web at
  • Antibody HumaneeringTM generates human antibodies with V-region sequences that have high homology to a human germline sequence while still retaining the specificity and affinity of the parent or reference antibody (U.S. Patent Publ. 2005/0255552 and 2006/0134098).
  • the process first identifies the minimum antigen binding specificity determinants (BSDs) in the heavy and light chain variable regions of a reference Fab (typically sequences within the heavy chain CDR3 and the light chain CDR3). As these heavy and light chain BSDs are maintained in all libraries constructed during the
  • full chain libraries in which an entire light or heavy chain variable region is replaced with a library of human sequences
  • cassette libraries in which a portion of the heavy or light chain variable region of the mouse Fab is replaced with a library of human sequences
  • a bacterial secretion system is used to express members of the library as antibody Fab fragments, and the library is screened for Fabs that bind antigen using a colony lift binding assay (CLBA). Positive clones are further characterized to identify those with the highest affinity.
  • Identified human cassettes supporting binding in the context of residual murine sequences are the combined in a final library screen to generate completely human V-regions.
  • the resulting HumaneeredTM Fabs have V-segment sequences derived from human libraries, retain the short BSD sequences identified within the CDR3 regions, and have human germline Framework 4 regions. These Fabs are converted to full IgGs by cloning the variable regions of the heavy and light chains into IgG expression vectors. Fully
  • HumaneeredTM antibodies generated in this process retain the binding specificity of the parent, murine antibody, typically having equivalent or higher affinity for the antigen that the parent antibody recognizes, and have V-regions with a high degree of sequence identity to human germline antibody genes at the protein level.
  • variable region DNA from murine monoclonal MAB 1 was amplified by Circular cDNA RACE method. Briefly, cDNA was synthesized from the RNA obtained from the hybridoma cell line using mouse immunoglobulin constant region gene specific primers, mIgG2b-pO (Po-5 ' TTTACCCGGAGACCGGGAGATGGTCTTCTTCAGG-3 ' , SEQ ID NO:83) and CK-pO (Po-5 ' ACACTC ATTCCTGTTGAAGCTCTTGAC AATGG-3 ' , SEQ ID NO:84). The resulting single strained cDNA was circularized using Circuligase II.
  • Heavy chain variable region was amplified from the circularized cDNA with mIgG2b CH3-f (5 ' -CTGAAAAATTACTACCTGAAGAAGACC-3 ' , SEQ ID NO:85) and mIgG2b CHl-r (5 ' GTGTGCACACTGCTGGACAGG-3 ' , SEQ ID NO:86).
  • Light chain variable region was amplified from the same cDNA with CK-f (5'-CACTCACAAGACATCAACTTCACC-3 ⁇ SEQ ID NO:87) and CK-r (5 ' -GATACAGTTGGTGCAGCATCAGCC-3 ' , SEQ ID NO:88). Variable heavy and light chain products were inserted into a pcDNA3.1 vector and sequence verified.
  • the MAB-1 VH and VK genes were synthesized to optimize expression in E. coli. Subsequently the optimized MAB-l-VH was cloned into KB 1292-His (modified version of KB 1292 that encodes a C-terminal flexible linker and 6-His tag of amino acid sequence AAGASHHHHHH (SEQ ID NO:89) on CHI) at Ncol (5') and Kpnl (3'); The optimized MAB-l-VK was cloned into KB1296b at Ncol (5') and Hindlll (3').
  • Fab fragments were expressed by secretion from E. coli using KaloBios expression vectors. Cells were grown in 2 x YT medium to an OD500 of -0.6. Expression was induced by adding IPTG to 100 ⁇ and shaking for 4 hours at 33°C. Assembled Fab was obtained from periplasmic fractions by osmotic lysis and purification by affinity chromatography using Ni-NTA columns HisTrap HP columns; GE Healthcare catalog #17-5247-01) according to standard methods. Fabs were eluted in buffer containing 500 mM imidazole and thoroughly dialyzed against PBS pH7.4 without calcium and magnesium.
  • Proleukin (Chiron) was used for all ELISA assays. Typically, proleukin diluted in PBS pH 7.4 was bound to a 96-well microtiter plate at 100 ng/well by overnight incubation at 4°C. After being rinsed three times with PBST, the plate was blocked with a solution of 1% BSA in PBS for one hour at 37°C, and then rinsed once with PBST. Fab-containing cell medium or diluted, purified Fab (50 ⁇ ) was then added to each well. After a one-hour incubation at 37°C, or overnight incubation at 4 °C, the plate was rinsed three times with PBST.
  • Anti-human-kappa chain HRP conjugate (Sigma #A7164) diluted 1 :5000 in PBST (50 ⁇ ) was added to each well, and the plate was incubated for 45 min at room temperature. The plate was washed three times with PBST, then 100 ⁇ L ⁇ of SureBlue TMB substrate (KPL #52-00-03) was added to each well and the plate was incubated for about 10 min at room temperature. The plate was read at 650 nm in a spectrophotometer. Affinity titration ELISA
  • an affinity titration ELISA was developed. This assay combines two consecutive ELISA steps: the first one, using goat anti-human Fab (Jackson ImmunoResearch Lab #109- 005-097) capture and goat anti-human Kappa (Sigma #A7164) detection, measures Fab concentrations in cell culture medium to normalize the amount of Fab used in the second antigen titration ELISA; the second ELISA, a normal antigen specific ELISA, generates an antigen binding dilution curve with the same amount of starting Fab. By comparing the dilution curves of different clones the high affinity clones are identified.
  • VH cassette libraries Two VH cassette libraries were constructed by overlap PCR: front- end cassette library (MAB 1 VHFE-01 ) and middle cassette library (MABlVHMD-01).
  • MABlVHFE-01 has human VHl germline sequences in FRl, FR2 and CDRl combined with reference CDR2-FR3 and BSD. Parental amino acid residues were introduced at the human IL2 contacting positions as predicted by the structure model in the CDRl ;
  • Each Vh cassette library was cloned into vector KB1292-His at Ncol (5') and Kpnl (3').
  • Vk full-chain library (MAB lVK-FcL-01).
  • the Vk full-chain library was cloned into vector KB1296-B (modified version of KaloBios vector KB1296 which has a silent Hindlll site added in FR4) at Ncol (5') and Hindlll (3').
  • Vh or Vk plasmid libraries were then combined with the complementary chain from the optimized reference Fab (MAB lopVK or MABl opVH (e.g., each Vh cassette library was combined with the optimized reference Vk, and the Vk full-chain library was combined with the optimized reference VH) by digestion with BssHll and CM and subsequent ligation to create libraries of dicistronic vectors expressing full Fabs.
  • optimized reference Fab MAB lopVK or MABl opVH
  • Colony lift assay identified a number of MABIVH-MD clones that supported human IL2 binding. Two of these clones had similar affinity toward human IL2 judged by ForteBio Octet analysis. No MAB1VH front-end and MAB 1VK full-chain clones binding to human IL2 were identified from colony lift assay. Thus, two mutagenic libraries were constructed. The first library was MABlVH-gFcL-01, in which the amino acid residues in VH-CDR1 were identical to that of the parental murine sequence and positions, H41, H43, and H44 in the FR2 were mutated to either the parental murine residue or human VH1-46 residue.
  • This mutagenic segment was combined to human VH1-46 FR1, FR3 and a CDR2 sequence with parental murine residues at the human IL2 contacting position predicted by the theoretic structure model.
  • the second mutagenic library was MABlVK-gFcL-02, in which residues at positions, L27c, L27d, and L30 in VK CDR1 and positions L36 and L37 in FR2 were mutated to the parental murine residues; residues at positions, L46, L48, L50, L53, and L54 were mutated to either the parental murine residue or the selected human germline residue.
  • This mutagenic segment was embedded within the human VKII Al frame work sequence.
  • MAB lopVH to generate Fab producing units. Since these two libraries had limited diversity, antigen specific ELISA was employed to identify human IL2 reactive clones and the clones were ranked by antigen affinity titration ELISA.
  • VH full-chain clones were selected from MABlVH-gFcL-01 for kinetic measurement by ForteBio Octet. They all had substantial affinity reduction. Further mutagenesis was carried out. The VH-CDR2 was restored to that of the parent, and the beneficial residues in the FR3 region identified from the selected MABIVH-MD clones were used. Three VH full-chain clones that had similar KD to that of the MAB lopVH were identified. One clone MAB lVH-1, which has one "R" residue replacement at position H82a, and has 88% homology to VH1-46, was kept for further development.
  • VK full-chain clones were selected from MAB 1 VK-gFcL-02 for kinetic measurement by ForteBio Octet.
  • MABlVK-dl5 which has minor reduced Kon, but overall similar KD to that of MAB lopVK and 91% homology to VKII-A1, was kept for further development.
  • An affinity maturation library was generated using the "round the horn site directed mutagenesis" protocol with 5 separate pairs of primers, each containing a "NNK” degenerated trinucleotide coden at the intended mutation site.
  • This VK affinity maturation library was paired with MABlVH-1.
  • Members in this library all have high % of homology to human germline VH1-46 and VKII Al. Colony lift assay was performed to screen the library for high affinity proleukin binders.
  • the deamidation site "NG" in the CDR2 MAB 1 VH-1 was removed by replacing the glysine with an alanine.
  • the "NG” to “NA” conversions was accomplished using the "round- the-horn site directed mutagenesis” protocol.
  • the forward primer has the degenerate "NNK” trinucleotide coden starting at the glycine site.
  • the reverse primer primes the complementary strain starting at the asparagine site.
  • the PCR reactions were performed with lOOng of MAB lVH-1 template, 0.2 ⁇ of each primer, 200 ⁇ dNTPs, and 2.5U of pfuUltrall DNA polymerase (Strategene) in a 50 ⁇ 1 reaction volume.
  • the PCR conditions were 94°C for 3 min for 1 cycle; 94°C for 15 seconds, 52°C for 20 seconds, and 65°C for 5minutes for 30 cycles; and finally, 1 cycle at 72°C for 5 minutes.
  • Dpnl (2U) was added to the PCR reaction and incubated at 37°C for 30 minutes to remove the template.
  • the amplified MAB lVH-1 heavy chain variants were separated by a 1% SYBR gel and purified using a Qiagen Gel
  • the gel purified PCR product was treated with T4 DNA polynucleotide kinase, ligated and transformed into DH5a chemically competent cells (Invitrogen) under ampicillin selection. A total of 86 clones were tested for binding to proleukin. Affinity titration ELISA showed that among 32 proleukin positive clones alanine and serine substitution of the original glycine resulted minimum reduction in affinity.
  • the clone with the alanine replacement MAB1VH-NA was used to generate the first Humaneered (human engineered) MAB 1, MAB3. Serine substitution forms a weak deamidation, "NS", site.
  • VH from MAB 1 VH-NA and VK from MAB 1 VK6 were cloned into a single dicistronic Fab expression plasmid to form MAB3-Fab.
  • a look through single position saturation mutagenesis approach targeting the individual residues in MAB3 VH CDR2 and CDR3 and MAB 3 VK CDR3 at positions, L90, L91, L92, and L93 was taken to further improve MAB3's affinity to human IL2.
  • the diversity of this affinity maturation library is 560. Two thousand individual clones were evaluated. Sample supernatants from one ml micro-expression were subjected to proleukin and human Fab ELISA. The ratio of A650- proleukin/A650-Fab was used to rank the top affinity binders. The top twelve clones, which had better A650-proleukin/A650-Fab ratio than that of the MAB3 Fab were selected.
  • VH and VK sequences of MAB l, MAB3 and the twelve affinity maturation variants were cloned into the mammalian expression vectors, pRS5a-hIgGl-s and pRS5a- hKappa-s by the SLIC protocol.
  • MAB l VH and VK were amplified with the following primers:
  • MAB l VK F - GCTTCCGGACACCACCGGTGATGTTGTGCTGACCCAGACT (SEQ ID NO:92), MAB 1 VK R - GCTGGGAGCGGCCACCGTACGTTTTATTTCCAACTTTGTC (SEQ ID NO:93).
  • VH and VK sequences of MAB3 and the affinity maturation variants were amplified with the following primers:
  • the 5 '-twenty nucleotides of these amplification primers overlap with the mammalian expression vectors at the cloning junctions.
  • the amplified VH and VK DNA fragments were separated by a 1% SYBR gel and purified using a Qiagen Gel Purification Kit.
  • the human IgGl heavy chain expression vector pRS5a-hIgGl-s and the human kappa chain expression vector pRS5a-hKappa-s were linearized with EcoRI and Kpnl and purified by QIAgen PCR DNA purification kit.
  • the fully HumaneeredTM MAB3 and the twelve affinity maturation variants were produced by co-transfection of the heavy and light chain mammalian expression plasmids into 293 Freestyle cells using 293fectin transfection reagent (Invitrogen #51-0031) according to the manufacturer' s protocol.
  • Antibody was purified from 293 Freestyle cells supernatants using a 5-mL HiTrap Protein A HP column (GE Healthcare #17-0403-03). Antibody was eluted using IgG Elution Buffer (Pierce #21004), and buffer exchanged into PBS by dialysis. Protein A affinity chromatography was performed on an AKTA-FPLC liquid chromatography system (GE Healthcare).
  • MAB 3 IgG and its' affinity maturation variants have very low K cff below the detection limit of the ForteBio octet instrument.
  • a competition ELISA was performed. Proleukin at 2 ⁇ g/ml PBS was immobilized onto a maxisorb plate. The plate was rinsed three times with PBST and blocked with 1% BSA.
  • MAB 3 was biotinylated following the protocol from Pierce Sulfo-NHS-LC- Biotin kit.
  • Biotinylated MAB3 at 1.25 nM final concentration was premixed with MAB3 or one of the twelve affinity maturation variants at various concentrations range from 200 nM to 0.1 nM in a separate dilution plate. The mixture was added to the plate. The captured biotinylated MAB3 was detected by Streptavidin-HRP conjugates (GE Healthcare,
  • RPN1231) diluted 1:2000 in PBST.
  • Competition by the non-labeled MAB 3 and its affinity maturation variants resulted in decreased capture of the biotinylated MAB3 and reduced signal.
  • Mo7e cells were starved for 15 hours in serum free media.
  • Human IL-2 and MAB1 or 8 were mixed with human IL-2 and added to the starved Mo7e cells. After 20 mins incubation in a 37°C tissue culture incubator the cells were fixed by addition of a 1 : 1 volume of pre-warmed BD Phosflow Fix Buffer I (BD # 557870) to the cell suspension. The cells were incubated at 37°C for 10 minutes. After washing the cells in Staining Media +5% FBS they were permeabilized by slowly adding 100 ⁇ cold Phosflow Perm Buffer III, (BD #558050). The cells were incubated for 30 minutes on ice and then washed twice with Staining Media +5% FBS. Alexa Fluor 647 Mouse Anti-STAT5 (BD #612599) was added at 20 ⁇ / 1X10 6 cells. After incubating the cells at room temperature for 30 minutes in the dark the samples were analyzed by FACS.
  • the heavy and light chain variable region sequences from murine monoclonal MAB1 were amplified by Circular cDNA RACE method and cloned into a pcDNA3.1 vector and sequence verified.
  • the theoretic MAB 1 VH and VK protein sequences translated from the respective DNA sequences were largely (90% or greater) verified at the protein level using a ThermoElectron LTQ-Orbitrap Mass Spectrometer.
  • the heavy and light chain variable regions of MAB1 were then cloned into KaloBios vectors in order to create the reference Fab: MABlrFab.
  • the Fab, MABlrFab has intact murine V-regions from MAB 1 fused with human CHI constant regions.
  • an optimized Fab, MABlopFab was constructed.
  • Several framework amino acid residues in MAB lrFab were changed to human germline residues in MABlopFab.
  • MAB lopFab in FR1 and FR3 are those specified by the PCR primers used to amplify the human V-segment repertoire and thus are present in all members of the HumaneeredTM V- region libraries.
  • the optimized reference Fab is constructed to assess whether or not any of the changes to human germline alter the properties of Fab binding.
  • the affinity constants (Ka (1/Ms), Kd (1/s), and KD (M) of MAB lrFab, MABlopFab was assayed using the ForteBio Octed QK system and Streptavidin High Binding Biosensors coated with biotinylated hGITR-hFc. Compared with MAB lrFab, MABlopFab, had only a minor loss in K D (Table 1.) indicating that the amino acid changes in MABlrFab are tolerated.
  • MAB 3 is an improved Fab over MABlVk6.
  • the deamidation site "NG" in the VHCDR2 was replaced by "NA”.
  • Affinity improved variants with single amino acid substitutions in the VHCDR2, but not in VH CDR3 or VK CDR3, were identified by human IL2 and Fab ELISA. These variants were converted to full IgG. Additional variants combining two or three amino acids change were generated. Competition ELISA was performed to compare the binding affinity of these variants in IgG format. Four affinity improved variants MAB4, MAB5, MAB6, and MAB7 were selected.
  • the parental mouse antibody MAB 1 binds to human and cynomolgus monkey but not rodent IL-2.
  • Fig. 1B-C and Fig. 2B-C. show that, like MAB 1, the HumaneeredTM antibodies MAB3, 4, 5, 6, 7 and 8 were able to bind in a similar manner to both human and cynomolgus monkey IL-2.
  • the crystal structure shown in Fig. 5A. shows that the epitope recognized by the MAB lrFab resides at the extreme N-terminus of IL-2 and encompasses all of the unstructured region (residues Alal , Pro2, Thr3, Ser4, Ser5, Ser6, Thr7, Lys8, Lys9 of mature peptide) prior to helix A and several residues on the same face of helix A (residues Glnl l , Leul2, Glul5, Hisl6, Leul9).
  • the residues comprising the IL-2 epitope and MAB lrFab paratope are summarized below in Table 4.
  • IL-2 has been shown to induce STAT5 phosophorylation in Mo7e cells.
  • By incubating the IL-2 with either MAB1 or MAB8 we can modulate the levels of STAT5 phosphorylation.
  • the presence of MAB1 or MAB8 causes a loss of STAT5 phosphorylation (Fig 7A.), however the antibodies causes only a slight repression of STAT5 phosphorylation on cells expressing IL-2RocPy (Fig 7B).
  • CDRL1 of MAB 1 4 SQSLFDSDGKTY
  • CDRL2, MAB1 5 LVS
  • CDRL3, MAB1 6 GTHFPF
  • CDRL1 of MAB 1 9 KSSQSLFDSDGKTYLN
  • CDRL2 CDRL2, MAB1 10: LVSKLDS
  • CDRL3, MAB1 11 WQGTHFPFT
  • CDRL1 of MAB2 4 SQSLFDSDGKTY
  • CDRL2, MAB2 5 LVS
  • CDRL3, MAB2 6 GTHFPF
  • CDRL1 of MAB2 9 KSSQSLFDSDGKTYLN
  • CDRL2 CDRL2, MAB2 10: LVSKLDS
  • CDRL3, MAB2 11 WQGTHFPFT
  • CDRL1 of MAB3 24 SQSLRDSDGKTY
  • CDRL2 CDRL2
  • MAB3 5 LVS
  • CDRL3, MAB3 6 GTHFPF
  • CDRL1 of MAB3 26 RSSQSLRDSDGKTYLN
  • CDRL2 CDRL2, MAB3 10: LVSKLDS
  • CDRL3, MAB3 11 WQGTHFPFT
  • CDRL1 of MAB4 24 SQSLRDSDGKTY
  • CDRL2 CDRL2
  • MAB4 5 LVS
  • CDRL3, MAB4 6 GTHFPF
  • CDRL1 of MAB4 26 RSSQSLRDSDGKTYLN
  • CDRL2 CDRL2, MAB4 10: LVSKLDS
  • CDRL3, MAB4 11 WQGTHFPFT
  • CDRL1 of MAB5 24 SQSLRDSDGKTY
  • CDRL2 CDRL2
  • MAB5 5 LVS
  • CDRL3, MAB5 6 GTHFPF
  • CDRL1 of MAB5 26 RSSQSLRDSDGKTYLN
  • CDRL2 CDRL2, MAB5 10: LVSKLDS
  • CDRL3, MAB5 11 WQGTHFPFT
  • CDRL1 of MAB6 24 SQSLRDSDGKTY
  • CDRL2 CDRL2
  • MAB6 5 LVS
  • CDRL3, MAB6 6 GTHFPF
  • CDRL1 of MAB6 26 RSSQSLRDSDGKTYLN
  • CDRL2 CDRL2, MAB6 10: LVSKLDS
  • CDRL3, MAB6 11 WQGTHFPFT
  • CDRL1 of MAB7 24 SQSLRDSDGKTY
  • CDRL2 CDRL2
  • MAB7 5 LVS
  • CDRL3, MAB7 6 GTHFPF
  • CDRL1 of MAB7 26 RSSQSLRDSDGKTYLN
  • CDRL2 CDRL2, MAB7 10: LVSKLDS
  • CDRL3, MAB7 11 WQGTHFPFT
  • CDRL1 of MAB8 59 SQSLRDSSGKTY
  • CDRL2 CDRL2
  • MAB8 5 LVS
  • CDRL3, MAB8 6 GTHFPF
  • CDRL1 of MAB8 60 RSSQSLRDSSGKTYLN
  • CDRL2 CDRL2, MAB8 10: LVSKLDS
  • CDRL3, MAB8 11 WQGTHFPFT
  • VL, MAB8 62 VL, MAB8 62:

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Abstract

La présente invention concerne des anticorps conçus par l'homme qui se lient spécifiquement à l'IL-2 et développent des cellules Treg lorsqu'ils sont complexés à l'IL-2, ainsi que des procédés et des compositions d'utilisation de ces anticorps.
PCT/US2013/055157 2012-08-15 2013-08-15 Anticorps d'interleukine 2 et complexes d'anticorps WO2014028748A1 (fr)

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Cited By (8)

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WO2015109212A1 (fr) * 2014-01-17 2015-07-23 Pfizer Inc. Anticorps anti-il-2 et compositions et utilisations de ceux-ci
WO2015140172A3 (fr) * 2014-03-17 2015-12-10 Bundesrepublik Deutschland Letztvertreten Durch Das Robert Koch-Institut Vertreten Durch Seinen Präsidenten Médicament destiné à être utilisé dans un procédé d'induction ou d'extension d'une réponse immunitaire cytotoxique cellulaire
WO2017070561A1 (fr) * 2015-10-23 2017-04-27 Pfizer Inc. Anticorps anti-il-2, compositions les contenant et leurs utilisations
WO2019131964A1 (fr) 2017-12-27 2019-07-04 協和発酵キリン株式会社 Variant d'il-2
WO2021164722A1 (fr) * 2020-02-21 2021-08-26 江苏恒瑞医药股份有限公司 Anticorps anti-il-2 et fragment de liaison à l'antigène de celui-ci et utilisation médicale de ceux-ci
WO2021245130A1 (fr) * 2020-06-03 2021-12-09 Ascendis Pharma Oncology Division A/S Séquences d'il-2 et leurs utilisations
JP2022028645A (ja) * 2018-12-21 2022-02-16 エフ・ホフマン－ラ・ロシュ・アクチェンゲゼルシャフト Ｃｄ３に結合する抗体
WO2023025249A1 (fr) * 2021-08-25 2023-03-02 江苏恒瑞医药股份有限公司 Composition pharmaceutique contenant une protéine de fusion

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ONUR BOYMAN ET AL: "The role of interleukin-2 during homeostasis and activation of the immune system", NATURE REVIEWS IMMUNOLOGY, vol. 12, no. 3, 1 March 2012 (2012-03-01), pages 180 - 190, XP055080466, ISSN: 1474-1733, DOI: 10.1038/nri3156 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015109212A1 (fr) * 2014-01-17 2015-07-23 Pfizer Inc. Anticorps anti-il-2 et compositions et utilisations de ceux-ci
US10688165B2 (en) 2014-03-17 2020-06-23 Bundesrepublik Deutschland letztvertreten durch das Robert-Koch-Institut vertreten durch seinen Präsidenten Medicament for use in a method of inducing or extending a cellular cytotoxic immune response
WO2015140172A3 (fr) * 2014-03-17 2015-12-10 Bundesrepublik Deutschland Letztvertreten Durch Das Robert Koch-Institut Vertreten Durch Seinen Präsidenten Médicament destiné à être utilisé dans un procédé d'induction ou d'extension d'une réponse immunitaire cytotoxique cellulaire
US10849963B2 (en) 2014-03-17 2020-12-01 Bundesrepublik Deutschland letztvertreten durch das Robl Koch-Institut vertreten durch seinen Präsidenten Medicament for use in a method of inducing or extending a cellular cytotoxic immune response
US10738113B2 (en) 2015-10-23 2020-08-11 The Regents Of The University Of California Anti-IL-2 antibodies and compositions and uses thereof
JP7030689B2 (ja) 2015-10-23 2022-03-07 ファイザー インコーポレイティッド 抗ｉｌ－２抗体ならびにその組成物及び使用
US11459385B2 (en) 2015-10-23 2022-10-04 The Regents Of The University Of California Anti-IL-2 antibodies and compositions and uses thereof
CN109071648A (zh) * 2015-10-23 2018-12-21 辉瑞有限公司 抗il-2抗体及其组合物和用途
US10138298B2 (en) 2015-10-23 2018-11-27 The Regents Of The University Of California Anti-IL-2 antibodies and compositions and uses thereof
CN109071648B (zh) * 2015-10-23 2022-07-19 辉瑞有限公司 抗il-2抗体及其组合物和用途
WO2017070561A1 (fr) * 2015-10-23 2017-04-27 Pfizer Inc. Anticorps anti-il-2, compositions les contenant et leurs utilisations
JP2018537962A (ja) * 2015-10-23 2018-12-27 ファイザー インコーポレイティッド 抗ｉｌ−２抗体ならびにその組成物及び使用
KR20200103681A (ko) 2017-12-27 2020-09-02 쿄와 기린 가부시키가이샤 Il-2 개변체
WO2019131964A1 (fr) 2017-12-27 2019-07-04 協和発酵キリン株式会社 Variant d'il-2
JP2022028645A (ja) * 2018-12-21 2022-02-16 エフ・ホフマン－ラ・ロシュ・アクチェンゲゼルシャフト Ｃｄ３に結合する抗体
WO2021164722A1 (fr) * 2020-02-21 2021-08-26 江苏恒瑞医药股份有限公司 Anticorps anti-il-2 et fragment de liaison à l'antigène de celui-ci et utilisation médicale de ceux-ci
WO2021245130A1 (fr) * 2020-06-03 2021-12-09 Ascendis Pharma Oncology Division A/S Séquences d'il-2 et leurs utilisations
US11879001B2 (en) 2020-06-03 2024-01-23 Ascendis Pharma Oncology Division A/S Conjugate comprising an IL-2 moiety
WO2023025249A1 (fr) * 2021-08-25 2023-03-02 江苏恒瑞医药股份有限公司 Composition pharmaceutique contenant une protéine de fusion

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