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WO2017208210A1 - Anticorps anti-mcam et methodes d'utilisation associées - Google Patents

Anticorps anti-mcam et methodes d'utilisation associées Download PDF

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Publication number
WO2017208210A1
WO2017208210A1 PCT/IB2017/053289 IB2017053289W WO2017208210A1 WO 2017208210 A1 WO2017208210 A1 WO 2017208210A1 IB 2017053289 W IB2017053289 W IB 2017053289W WO 2017208210 A1 WO2017208210 A1 WO 2017208210A1
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Prior art keywords
antibody
mcam
seq
dose
amino acid
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English (en)
Inventor
Daniel Keith NESS
Kenneth Flanagan
Gene G. Kinney
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Prothena Biosciences Ltd
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Prothena Biosciences Ltd
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Publication of WO2017208210A1 publication Critical patent/WO2017208210A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • TH17 cells T helper 17 cells
  • EAE experimental autoimmune encephalomyelitis
  • TH17 cells have been reported to secrete a number of select cytokines including IL-17 and IL-22.
  • TH17 cells have been reported to undergo specific recruitment and infiltration of tissue.
  • MCAM has been reported to be expressed on TH17 cells and to bind laminin a4 as a ligand.
  • the invention provides methods of treating or effecting prophylaxis of a patient having or at risk of a neuroinflammatory or autoimmune disease, comprising administering to the patient a first regime of an MCAM antagonist which inhibits binding of MCAM to a laminin a4 chain, and a second regime of a second MCAM antagonist which inhibits binding of MCAM to a laminin a4 chain, wherein the first regime is effective to reduce T-cell MCAM levels in the patient's blood to less than 50% of pretreatment levels and the second regime is effective to maintain T-cell MCAM levels at less than 50% of pretreatment levels.
  • the T- cell MCAM levels are measured by fluorescence activated cell sorting (FACS).
  • the number of MCAM+ T-cells is measured. In some methods, the amount of MCAM expression on the surface of the T-cells is measured. In some methods, the T-cells are CD3+ cells.
  • each of the first and second MCAM antagonists is selected from the group consisting of an antibody, a small molecule, a peptide or a polypeptide. In some methods, the first and second MCAM antagonists are small molecules. In some methods, the first and second MCAM antagonists are different. In some methods, the first and second MCAM antagonists are antibodies. In some methods, the first and second MCAM antagonists are different. In some methods, the first and second MCAM antagonists are the same. In some methods, the first MCAM antagonist is an antibody and the second MCAM antagonist is a small molecule. In some methods, the first MCAM antagonist is a small molecule and the second MCAM antagonist is an antibody.
  • the first regime comprises administering a first dose of the antibody of about 10 to about 30 mg/kg, followed by a second regime of the antibody comprising administering a second dose that is greater than zero and less than the first dose, wherein the antibody comprises three Kabat heavy chain CDRs (SEQ ID NOS: 78-80, respectively) and three Kabat light chain CDRs (SEQ ID NOS: 73-75, respectively).
  • the antibody is administered at a dose of about 10 to about 30 mg/kg.
  • the antibody is administered at a dose of about 10 mg/kg.
  • the antibody is administered at a dose of about 15 mg/kg.
  • the antibody is administered at a dose of about 30 mg/kg.
  • the invention further provides a method of treating or effecting prophylaxis of a patient having or at risk of a neuroinflammatory or autoimmune disease, comprising
  • the first regime comprises administering a first dose of the first antibody of about 10 to about 30 mg/kg and the second regime comprises administering a second dose of the second antibody, wherein the second dose is greater than zero and less than the first dose.
  • the second dose is about 1 mg/kg to about 5 mg/kg. In some methods, the second dose is about 1 mg/kg. In some methods, the second dose is about 3 mg/kg. In some methods, the second dose is about 5 mg/kg.
  • At least one of the first and second antibodies comprises three Kabat heavy chain CDRs having the sequences of SEQ ID NOS: 78- 80, respectively and three Kabat light chain CDRs having the sequences of SEQ ID NOS: 73-75, respectively.
  • both antibodies comprise three Kabat heavy chain CDRs having the amino acid sequence of SEQ ID NOS: 78-80, respectively, and three Kabat light chain CDRs having the amino acid sequences of SEQ ID NOS: 73-75, respectively.
  • the first dose is administered once or on multiple occasions at first intervals of about 20 days to about 6 weeks; during the second regime, the second dose is administered on multiple occasions at second intervals of about 10 days to about 5 weeks; and the second dose is about 0.3 to less than about 10 mg/kg.
  • at least one of the first and second antibodies is at least 90% identical to SEQ ID NO: 161, and a mature light chain variable region at least 90% identical to SEQ ID NO: 123.
  • at least one of the first and second antibodies is a human IgGl kappa antibody comprising a mature heavy chain variable region of SEQ ID NO: 161 and a mature light chain variable region of SEQ ID NO: 123.
  • At least one of the first and second antibodies is administered as a component of a formulation comprising (a) the antibody at a concentration within a range from about 10 mM to about 50 mM; (b) one or more sugars and polyols ("sugar/polyol") selected from: (i) sucrose present at a concentration within the range from about 200 mM to about 260 mM; and (ii) trehalose present at a concentration within the range from about 200 mM to about 260 mM ; (c)histidine at 10-30 mM; and (d) polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight; wherein the pharmaceutical formulation is characterized by a pH within the range from about 5.5 to about 7.
  • both the first and second antibodies are human IgGl kappa antibodies comprising a mature heavy chain variable region of SEQ ID NO: 161 and a mature light chain variable region of SEQ ID NO: 123.
  • the formulation comprises the antibody at 40 mg/ml, histidine at 20 mM, sucrose at 220 mM, polysorbate at 0.2 g/L and pH 6.
  • the antibody is administered intravenously.
  • the first dose is administered once.
  • the second dose is about 1 mg/kg to about 6 mg/kg and the second intervals are about every 2 weeks to about monthly.
  • the second dose is administered at least four times.
  • the second dose is administered at least ten times.
  • the second dose is administered at a frequency and for a treatment period effective to maintain T- cell MCAM levels at less than 50% of pretreatment levels.
  • the second dose is administered for the rest of the patient's life.
  • the disease can be multiple sclerosis, psoriasis or psoriatic arthritis, Behcet's disease, Giant Cell Arteritis, Polymyalgia Rheumatica, or Takayasu's Arteritis.
  • the invention further provides methods of treating or effecting prophylaxis of a patient having or at risk of a neuroinflammatory or autoimmune disease, comprising
  • the antibody comprises three Kabat heavy chain CDRs having the amino acid sequences of SEQ ID NOS: 78-80, respectively) and three Kabat light chain CDRs having the amino acid sequences of SEQ ID NOS: 73-75, respectively).
  • the antibody comprises a mature heavy chain variable region of SEQ ID NO: 161 and a mature light chain variable region of SEQ ID NO: 123.
  • the antibody is administered as a component of a formulation comprising (a) the antibody at a concentration within a range from about 10 mM to about 50 mM;(b) one or more sugars and polyols
  • sucrose/polyol selected from:(i) sucrose present at a concentration within the range from about 200 mM to about 260 mM; and (ii) trehalose present at a concentration within the range from about 200 mM to about 260 mM ; (c) histidine at 10-30 mM; and (d)polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight; wherein the pharmaceutical formulation is characterized by a pH within the range from about 5.5 to about 7.
  • the formulation comprises the antibody at 40 mg/ml, histidine at 20 mM, sucrose at 220 mM, polysorbate at 0.2 g/L and pH 6.
  • Some methods comprise subcutaneously administering a 3 mg/kg dose of the antibody at intervals of about every 5 days to about every 2 weeks. In some methods, the intervals are about once per week. Some methods comprise subcutaneously administering a dose of the antibody in the range of about 150 mg to about 350 mg at intervals of about every 5 days to about every 2 weeks. In some methods, the intervals are about once per week. In some methods, the dose is in the range of about 200 mg to about 250 mg. In some methods, the dose is about 210 mg.
  • the invention further provides methods treating or effecting prophylaxis of a patient having or at risk of a neuroinflammatory or autoimmune disease, comprising administering to the patient a regime of an antibody which inhibits binding of MCAM to a laminin a4 chain, wherein the regime includes a dose higher than that required achieve a serum concentration of the antibody sufficient to saturate MCAM.
  • FIG. 1 depicts the identification of critical clones.
  • FIGS. 2A-C depicting the location and exposure of the 1141 and P145 residues of human MCAM.
  • FIGS. 3 A & B show the alignment of sequences of the variable heavy chains for the following: rat 2120.4.19 anti-MCAM antibody (2120.4.19.6_VH_topo_pro; SEQ ID NO: 114); 2120 VH1 humanized anti-MCAM antibody (h2120VHl ; SEQ ID NO: 115); 2120 VH2 humanized anti-MCAM antibody (h2120VH2; SEQ ID NO: 116); 2120 VH3 humanized anti-MCAM antibody (h2120VH3; SEQ ID NO: 117); 2120 VH4 humanized anti-MCAM antibody (h2120VH4; SEQ ID NO: 118); 2120 VH5 humanized anti-MCAM antibody
  • FIG. 3B shows the alignment of sequences of the variable light chains for the following: rat 2120.4.19.6 anti-MCAM antibody (2120.4.19.6_VL_topo_pro; SEQ ID NO: 120); 2120 VL1 humanized anti-MCAM antibody (h2120VLl SEQ ID NO: 121); 2120 VL2 humanized anti-MCAM antibody (h2120VL2 SEQ ID NO: 122); 2120 VL3 humanized anti- MCAM antibody (h2120VL3 SEQ ID NO: 123); and light chain human variable X84343 IGKV2-26*01 sequence used as the framework donor (X84343 VL SEQ ID NO: 124).
  • HVRs hypervariable regions grafted from the rat 2120.4.19.6 antibody to the variable light chain variable X84343 IGKV2-26*01 framework are boxed.
  • the bolded amino acid residues in the humanized antibody sequences differ from the corresponding residues in the rat antibody sequence.
  • the position of canonical and interface amino acid residues that may affect CDR contact or CDR structure are indicated by an asterisk.
  • FIG. 4A shows the alignment of sequences of the mature heavy chain variable regions for the following: rat 2120.4.19 anti-MCAM antibody (2120.4.19.6_VH_topo_pro; SEQ ID NO: 114); 2120 VH1.Q1E humanized anti-MCAM antibody (h2120VHl .QlE; SEQ ID NO: 157); 2120 VH2.Q1E humanized anti-MCAM antibody (h2120VH2.QlE; SEQ ID NO: 158); 2120 VH3.Q1E humanized anti-MCAM antibody (h2120VH3.QlE; SEQ ID NO: 159); 2120
  • VH4.Q1E humanized anti-MCAM antibody (h2120VH4.QlE; SEQ ID NO: 160); 2120
  • VH5.Q1E humanized anti-MCAM antibody (h2120VH5.QlE; SEQ ID NO: 161); and heavy chain human variable AF062133 IGHV2-26*01 sequence used as the framework donor
  • variable light chains for the following: rat 2120.4.19.6 anti-MCAM antibody (2120.4.19.6_VL_topo_pro; SEQ ID NO: 120); 2120 VL1 humanized anti-MCAM antibody (h2120VLl; SEQ ID NO: 121); 2120 VL2 humanized anti-MCAM antibody (h2120VL2; SEQ ID NO: 122); 2120 VL3 humanized anti- MCAM antibody (h2120VL3; SEQ ID NO: 123); and light chain human variable X84343 IGKV2-26*01 sequence used as the framework donor (X84343 VL SEQ ID NO: 124). Kabat numbering is used and hypervariable regions (HVRs) grafted from the rat 2120.4.19.6 antibody to the variable light chain variable X84343 IGKV2-26*01 framework are boxed.
  • HVRs hypervariable regions
  • FIGS. 5A-C show MCAM expression is dose- and time-dependently down regulated by PRX003 treatment in hMCAM transgenic mice as shown in a single mouse after single injections.
  • FIG. 5A shows MCAM expression at predose.
  • FIGS. 5B & C show MCAM expression levels at six hours and 96 hours.
  • FIGS. 6A & B show expression and subsequent reduction of MCAM on NK cells after a single PRX-003 dose (FIG. 6A) and time-dependently down regulated by PRX003 treatment in hMCAM transgenic mice as shown in multiple mice after repeated injections (FIG 6B).
  • FIGS. 7A & B show PRX003 selectively down regulates hMCAM and does not affect the viability of MCAM + cells.
  • FIG. 7B shows anti-MCAM clone 15 selectively down regulates mMCAM and does not affect the viability of MCAM + cells.
  • FIG. 8 shows dynamic changes in soluble MCAM induced by PRX003 are tightly coupled with cellular downregulation of MCAM.
  • FIGS. 9A-C show anti-MCAM lowers inflammation and clinical score in a mouse model of EAE, known to involve TH17 cells: treatment period (FIG. 9A); MCAM + cells (FIG. 9B); and, CD3 + /MCAM + T cells (FIG 9C).
  • FIGS. 10A-B show down regulation of MCAM on CD3+ lymphocytes in a dose- dependent manner in healthy adults by PRX003.
  • FIG. 10A depicts a line graph showing the percent pretreatment MCAM + expressing CD3+ lymphocytes (y-axis) detected 0-90 days post infusion (x-axis).
  • FIG. 10B depicts a line graph showing percent pretreatment MCAJVT " expressing CD3+ lymphocytes (x-axis) detected 0-2.5 days post infusion (y-axis).
  • FIG. 11 shows PRX003 treatment elevating the levels of soluble MCAM (sMCAM) in healthy adults.
  • the mean concentration of sMCAM detected (y-axis) is plotted against days post drug infusion (x-axis).
  • FIG. 12 shows in vivo stability studies of PRX003 in healthy adults.
  • the mean concentration of PRX003 detected in human serum (y-axis) is plotted against post infusion time points (x-axis).
  • FIG. 13 shows pharmacokinetic dynamics for PRX003 in healthy adults.
  • the bar graph shows drug clearance in (mL/h/kg) with increasing concentrations of PRX003.
  • FIGS. 14A-B show PRX003 inducing demargination of TH17 lymphocytes from a marginal pool in healthy adults.
  • FIG 14A shows a bar graph indicating a dose-dependent increase in MCAM expression on CD3+ lymphocytes in the blood with increasing PRX003 concentrations.
  • FIG. 14B shows a bar graph depicting a similar increase in MCAM expression measured by area under the curve (AUC) with increasing PRX003 concentrations.
  • Fig. 15 shows three mechanisms of action of MCAM antagonist PRX003.
  • PRX003 can block MCAM laminin a4 interaction thereby inhibiting extravasation into the vascular, induce demargination of TH17 cells already in vascular cell walls or tissue, and reduce expression of MCAM.
  • SEQ ID NO: 1 is the nucleic acid sequence encoding the mature light chain variable region of antibody clone 17.
  • SEQ ID NO: 2 is the amino acid sequence of the mature light chain variable region of antibody clone 17.
  • SEQ ID NO: 3 is the amino acid sequence of CDRL1 of the antibody clone 17.
  • SEQ ID NO: 4 is the amino acid sequence of CDRL2 of the antibody clone 17.
  • SEQ ID NO: 5 is the amino acid sequence of CDRL3 of the antibody clone 17.
  • SEQ ID NO: 6 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody clone 17.
  • SEQ ID NO: 7 is the amino acid sequence of the mature heavy chain variable region of antibody clone 17.
  • SEQ ID NO: 8 is the amino acid sequence of CDRH1 of the antibody clone 17.
  • SEQ ID NO: 9 is the amino acid sequence of CDRH2 of the antibody clone 17.
  • SEQ ID NO: 11 is the amino acid sequence of human MCAM Accession No. CAA48332.
  • SEQ ID NO: 12 is the nucleic acid sequence encoding the mature light chain variable region of antibody clone 15.
  • SEQ ID NO: 13 is the amino acid sequence of the mature light chain variable region of antibody clone 15.
  • SEQ ID NO: 14 is the amino acid sequence of CDRL1 of the antibody clone 15.
  • SEQ ID NO: 15 is the amino acid sequence of CDRL2 of the antibody clone 15.
  • SEQ ID NO: 16 is the amino acid sequence of CDRL3 of the antibody clone 15.
  • SEQ ID NO: 17 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody clone 15.
  • SEQ ID NO: 18 is the amino acid sequence of the mature heavy chain variable region of antibody clone 15.
  • SEQ ID NO: 19 is the amino acid sequence of CDRH1 of the antibody clone 15.
  • SEQ ID NO: 20 is the amino acid sequence of CDRH2 of the antibody clone 15.
  • SEQ ID NO: 21 is the amino acid sequence of CDRH3 of the antibody clone 15.
  • SEQ ID NO: 22 is the amino acid sequence of human MCAM domain 1 (residues 19- 129).
  • SEQ ID NO: 23 is the amino acid sequence of human MCAM domain 2 (residues 139-242).
  • SEQ ID NO: 24 is the amino acid sequence of human MCAM domain 3 (residues 244-321).
  • SEQ ID NO: 25 is the amino acid sequence of human MCAM domain 4 (residues 355-424).
  • SEQ ID NO: 26 is the amino acid sequence of human MCAM domain 5 (residues 430-510).
  • SEQ ID NO: 27 is the amino acid sequence of an a4-chain isoform of human laminin 411 (Accession No. NP001098676).
  • SEQ ID NO: 28 is the amino acid sequence of an a4-chain isoform of human laminin 411 (Accession No. CAA48332).
  • SEQ ID NO: 29 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1174.1.3.
  • SEQ ID NO: 30 is the amino acid sequence of the mature light chain variable region of antibody 1174.1.3.
  • SEQ ID NO: 31 is the amino acid sequence of CDRL1 of antibody 1174.1.3.
  • SEQ ID NO: 32 is the amino acid sequence of CDRL2 of antibody 1174.1.3.
  • SEQ ID NO: 33 is the amino acid sequence of CDRL3 of antibody 1174.1.3.
  • SEQ ID NO: 34 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1174.1.3.
  • SEQ ID NO: 35 is the amino acid sequence of the mature heavy chain variable region of antibody 1174.1.3.
  • SEQ ID NO: 36 is the amino acid sequence of CDRH1 of antibody 1174.1.3.
  • SEQ ID NO: 37 is the amino acid sequence of CDRH2 of antibody 1174.1.3.
  • SEQ ID NO: 38 is the amino acid sequence of CDRH3 of antibody 1174.1.3.
  • SEQ ID NO: 39 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1414.1.2.
  • SEQ ID NO: 40 is the amino acid sequence of the mature light chain variable region of antibody 1414.1.2.
  • SEQ ID NO: 41 is the amino acid sequence of CDRL1 of antibody 1414.1.2.
  • SEQ ID NO: 42 is the amino acid sequence of CDRL2 of antibody 1414.1.2.
  • SEQ ID NO: 43 is the amino acid sequence of CDRL3 of antibody 1414.1.2.
  • SEQ ID NO: 44 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1414.1.2.
  • SEQ ID NO: 45 is the amino acid sequence of the mature heavy chain variable region of antibody 1414.1.2.
  • SEQ ID NO: 46 is the amino acid sequence of CDRH1 of antibody 1414.1.2.
  • SEQ ID NO: 47 is the amino acid sequence of CDRH2 of antibody 1414.1.2.
  • SEQ ID NO: 48 is the amino acid sequence of CDRH3 of antibody 1414.1.2.
  • SEQ ID NO: 49 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1415.1.1.
  • SEQ ID NO: 50 is the amino acid sequence of the mature light chain variable region of antibody 1415.1.1.
  • SEQ ID NO: 51 is the amino acid sequence of CDRL1 of antibody 1415.1.1.
  • SEQ ID NO: 52 is the amino acid sequence of CDRL2 of antibody 1415.1.1.
  • SEQ ID NO: 53 is the amino acid sequence of CDRL3 of antibody 1415.1.1.
  • SEQ ID NO: 54 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1415.1.1.
  • SEQ ID NO: 55 is the amino acid sequence of the mature heavy chain variable region of antibody 1415.1.1.
  • SEQ ID NO: 56 is the amino acid sequence of CDRH1 of antibody 1415.1.1.
  • SEQ ID NO: 57 is the amino acid sequence of CDRH2 of antibody 1415.1.1.
  • SEQ ID NO: 58 is the amino acid sequence of CDRH3 of antibody 1415.1.1.
  • SEQ ID NO: 59 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1749.1.3.
  • SEQ ID NO: 60 is the amino acid sequence of the mature light chain variable region of antibody 1749.1.3.
  • SEQ ID NO: 61 is the amino acid sequence of CDRL1 of antibody 1749.1.3.
  • SEQ ID NO: 62 is the amino acid sequence of CDRL2 of antibody 1749.1.3.
  • SEQ ID NO: 63 is the amino acid sequence of CDRL3 of antibody 1749.1.3.
  • SEQ ID NO: 64 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1749.1.3.
  • SEQ ID NO: 65 is the amino acid sequence of the mature heavy chain variable region of antibody 1749.1.3.
  • SEQ ID NO: 66 is the amino acid sequence of CDRH1 of antibody 1749.1.3.
  • SEQ ID NO: 67 is the amino acid sequence of CDRH2 of antibody 1749.1.3.
  • SEQ ID NO: 68 is the amino acid sequence of CDRH3 of antibody 1749.1.3.
  • SEQ ID NO: 69 is the nucleic acid sequence encoding a mature light chain variable region of antibody 2120.4.19.
  • SEQ ID NO: 70 is the amino acid sequence of the mature light chain variable region of antibody 2120.4.19 set forth in SEQ ID NO: 69.
  • SEQ ID NO: 71 is the amino acid sequence of a mature light chain variable region of antibody 2120.4.19.
  • SEQ ID NO: 72 is the amino acid sequence of a mature light chain variable region of antibody 2120.4.19.
  • SEQ ID NO: 73 is the amino acid sequence of CDRL1 of antibody 2120.4.19.
  • SEQ ID NO: 74 is the amino acid sequence of CDRL2 of antibody 2120.4.19.
  • SEQ ID NO: 75 is the amino acid sequence of CDRL3 of antibody 2120.4.19.
  • SEQ ID NO: 76 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 2120.4.19.
  • SEQ ID NO: 77 is the amino acid sequence of the mature heavy chain variable region of antibody 2120.4.19.
  • SEQ ID NO: 78 is the amino acid sequence of CDRH1 of antibody 2120.4.19.
  • SEQ ID NO: 79 is the amino acid sequence of CDRH2 of antibody 2120.4.19.
  • SEQ ID NO: 80 is the amino acid sequence of CDRH3 of antibody 2120.4.19.
  • SEQ ID NO: 81 is a nucleic acid sequence encoding a mature light chain variable region of antibody 2107.4.10.
  • SEQ ID NO: 82 is the amino acid sequence of the mature light chain variable region of antibody 2107.4.10 set forth in SEQ ID NO: 81.
  • SEQ ID NO: 83 is a nucleic acid sequence encoding a mature light chain variable region of antibody 2107.4.10.
  • SEQ ID NO: 84 is the amino acid sequence of the mature light chain variable region of antibody 2107.4.10 set forth in SEQ ID NO: 83.
  • SEQ ID NO: 85 is the amino acid sequence of CDRL1 of antibody 2107.4.10.
  • SEQ ID NO: 86 is the amino acid sequence of CDRL2 of antibody 2107.4.10.
  • SEQ ID NO: 87 is the amino acid sequence of CDRL3 of antibody 2107.4.10.
  • SEQ ID NO: 88 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 2107.4.10.
  • SEQ ID NO: 89 is the amino acid sequence of the mature heavy chain variable region of antibody 2107.4.10.
  • SEQ ID NO: 90 is the amino acid sequence of CDRH1 of antibody 2107.4.10.
  • SEQ ID NO: 91 is the amino acid sequence of CDRH2 of antibody 2107.4.10.
  • SEQ ID NO: 92 is the amino acid sequence of CDRH3 of antibody 2107.4.10.
  • SEQ ID NO: 93 is the amino acid sequence of the mature heavy chain variable region of antibody 1749.1.3.
  • SEQ ID NO: 94 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 1749 version 1 (VH1).
  • SEQ ID NO: 95 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 1749 version 2 (VH2).
  • SEQ ID NO: 96 is the amino acid sequence of the heavy chain variable framework donor U96282 VH.
  • SEQ ID NO: 97 is the amino acid sequence of the mature light chain variable region of antibody 1749.1.3.
  • SEQ ID NO: 98 is the amino acid sequence of the mature light chain variable region of humanized antibody 1749 version 1 (VL1).
  • SEQ ID NO: 99 is the amino acid sequence of the mature light chain variable region of humanized antibody 1749 version 2 (VL2).
  • SEQ ID NO: 100 is the amino acid sequence of the light chain variable framework donor X02990 VL.
  • SEQ ID NO: 101 is the amino acid sequence of the mature heavy chain variable region of antibody 2107.4.10.18.
  • SEQ ID NO: 102 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 1 (VH1).
  • SEQ ID NO: 103 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 2 (VH2).
  • SEQ ID NO: 104 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 3 (VH3).
  • SEQ ID NO: 105 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 4A (VH4A).
  • SEQ ID NO: 106 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 5A (VH5A).
  • SEQ ID NO: 107 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 6 (VH6).
  • SEQ ID NO: 108 is the amino acid sequence of the heavy chain variable framework donor AF062133 VH.
  • SEQ ID NO: 109 is the amino acid sequence of the mature light chain variable region of antibody 2107.4.10.18.
  • SEQ ID NO: 110 is the amino acid sequence of the mature light chain variable region of humanized antibody 2107 version 1 (VL1).
  • SEQ ID NO: 111 is the amino acid sequence of the mature light chain variable region of humanized antibody 2107 version 2 (VL2).
  • SEQ ID NO: 112 is the amino acid sequence of the mature light chain variable region of humanized antibody 2107 version 3 (VL3).
  • SEQ ID NO: 113 is the amino acid sequence of the light chain variable framework donor U86803.
  • SEQ ID NO: 114 is the amino acid sequence of the mature heavy chain variable region of antibody 2120.4.19.6.
  • SEQ ID NO: 115 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 1 (VH1).
  • SEQ ID NO: 116 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 2 (VH2).
  • SEQ ID NO: 117 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 3 (VH3).
  • SEQ ID NO: 118 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 4 (VH4).
  • SEQ ID NO: 119 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 5 (VH5).
  • SEQ ID NO: 120 is the amino acid sequence of the mature light chain variable region of antibody 2120.4.19.6.
  • SEQ ID NO: 121 is the amino acid sequence of the mature light chain variable region of humanized antibody 2120 version 1 (VL1).
  • SEQ ID NO: 122 is the amino acid sequence of the mature light chain variable region of humanized antibody 2120 version 2 (VL2).
  • SEQ ID NO: 123 is the amino acid sequence of the mature light chain variable region of humanized antibody 2120 version 3 (VL3).
  • SEQ ID NO: 124 is the amino acid sequence of the light chain variable framework donor X84343_VL.
  • SEQ ID NO: 125 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 126 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 127 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 128 is the amino acid sequence of a humanized heavy chain/light chain framework region.
  • SEQ ID NO: 129 is the amino acid sequence of a humanized light chain framework [0153]
  • SEQ ID NO: 130 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 131 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 132 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 133 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 134 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 135 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 136 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 137 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 138 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 139 is the amino acid sequence of CDRHl of humanized antibody 2120 version 3 (VH3).
  • SEQ ID NO: 140 is the amino acid sequence of CDRHl of humanized antibody 2120 version 4 (VH4).
  • SEQ ID NO: 141 is the amino acid sequence of CDRHl of humanized antibody 2120 version 5 (VH5).
  • SEQ ID NO: 142 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 143 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 144 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 145 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 146 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 147 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 148 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 149 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 150 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 151 is the amino acid sequence of CDRHl of humanized antibody 2107 version 1 (VHl).
  • SEQ ID NO: 152 is the amino acid sequence of CDRHl of humanized antibody 2107 version 4 (VH4).
  • SEQ ID NO: 153 is the amino acid sequence of CDRH3 of humanized antibody 2120 1-5 (VH1-VH5).
  • SEQ ID NO: 154 is the amino acid sequence of a humanized light chain framework region.
  • SEQ ID NO: 155 is the amino acid sequence of a humanized heavy chain framework region.
  • SEQ ID NO: 156 is the amino acid sequence of the mature heavy chain variable region of antibody 2120.4.19.Q1E, wherein position 1 (Kabat numbering) is occupied by E.
  • SEQ ID NO: 157 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 1 QIE (VHI.QIE), wherein position 1 (Kabat numbering) is occupied by E.
  • SEQ ID NO: 158 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 2 QIE (VH2.Q1E), wherein position 1 (Kabat numbering) is occupied by E.
  • SEQ ID NO: 159 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 3 QIE (VH3.Q1E), wherein position 1 (Kabat numbering) is occupied by E.
  • SEQ ID NO: 160 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 4 QIE (VH4.Q1E), wherein position 1 (Kabat numbering) is occupied by E.
  • SEQ ID NO: 161 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 5 QIE (VH5.Q1E), wherein position 1 (Kabat numbering) is occupied by E.
  • SEQ ID NO: 162 is the nucleic acid sequence encoding an exemplary signal peptide that can be fused to a mature heavy chain or mature light chain variable region.
  • SEQ ID NO: 163 is the amino acid sequence of the exemplary signal peptide encoded by the nucleic acid sequence of SEQ ID NO: 162.
  • SEQ ID NO: 164 is the nucleic acid sequence encoding an exemplary signal peptide that can be fused to a mature heavy chain or mature light chain variable region.
  • SEQ ID NO: 165 is the amino acid sequence of the exemplary signal peptide encoded by the nucleic acid sequence of SEQ ID NO: 164.
  • SEQ ID NO: 166 is the nucleic acid sequence encoding an exemplary signal peptide that can be fused to a mature heavy chain or mature light chain variable region.
  • SEQ ID NO: 167 is the amino acid sequence of the exemplary signal peptide encoded by the nucleic acid sequence of SEQ ID NO: 166.
  • SEQ ID NO: 168 is the amino acid sequence of a humanized 2120 light chain constant region, with Arginine at the N-terminus.
  • SEQ ID NO: 169 is the amino acid sequence of a humanized 2120 light chain constant region, without Arginine at the N-terminus.
  • SEQ ID NO: 170 is the amino acid sequence of a humanized 2120 heavy chain constant region.
  • SEQ ID NO: 171 is the amino acid sequence of a BIP version heavy chain Glm3 allotype constant region.
  • SEQ ID NO: 172 is the amino acid sequence of a BIP version heavy chain Glm3 allotype constant region.
  • SEQ ID NO: 173 is the amino acid sequence of a mature light chain region of humanized antibody 2120 version 3 (VL3 + light chain constant region).
  • SEQ ID NO: 174 is the amino acid sequence of a mature heavy chain region of humanized antibody 2120 version 5 (VH5 + BIP version heavy chain Glm3 allotype constant region).
  • SEQ ID NO: 175 is the amino acid sequence of a mature heavy chain region of humanized antibody 2120 version 5 (VH5 + BIP version heavy chain Glm3 allotype constant region).
  • SEQ ID NO: 176 is the amino acid sequence of a mature heavy chain region of humanized antibody 2120 version 5 QIE (VH5.Q1E + BIP version heavy chain Glm3 allotype constant region).
  • SEQ ID NO: 177 is the amino acid sequence of a mature heavy chain region of humanized antibody 2120 version 5 QIE (VH5.Q1E + BIP version heavy chain Glm3 allotype constant region).
  • SEQ ID NO: 178 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 4B (VH4B).
  • SEQ ID NO: 179 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 5B (VH5B).
  • Monoclonal antibodies are typically provided in isolated form. This means that an antibody is typically at least 50% w/w pure of proteins and other macromolecules arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of pharmaceutical acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95 or 99% w/w pure of proteins and other macromolecules from production or purification.
  • Specific binding of a monoclonal antibody to its target antigen means an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M "1 . Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that a monoclonal antibody binds one and only one target.
  • the basic antibody structural unit is a tetramer of subunits.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide.
  • the variable region without the signal peptide is sometimes referred to as a mature variable region.
  • a light chain mature variable region means a light chain variable region without the light chain signal peptide.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 or more amino acids.
  • each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 From N- terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • Kabat Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901- 917 (1987); Chothia et al., Nature 342:878-883 (1989).
  • Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number (e.g., H83 means position 83 by Kabat numbering in the mature heavy chain variable region; likewise position L36 means position 36 by Kabat numbering in the mature light chain variable region).
  • Kabat numbering is used throughout in referring to positions in the variable region of an antibody unless explicitly stated otherwise.
  • antibody includes intact antibodies and antigen binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, diabodies, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.
  • antibody also includes a bispecific antibody, and/or a chimeric antibody, and/or a humanized antibody.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al, J.
  • the two different heavy/light chain pairs may include a humanized heavy chain/light chain pair and a heavy chain/light chain pair specific for a different epitope.
  • one heavy chain light chain pair is a humanized antibody as further disclosed below and the heavy light chain pair is from an antibody that binds to a receptor expressed on the blood brain barrier, such as an insulin receptor, an insulin-like growth factor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, or a transferrin receptor (Fnden et al, PNAS 88:4771-4775, 1991; Fnden et al, Science 259:373-377, 1993).
  • a bispecific antibody can be transferred cross the blood brain barrier by receptor-mediated transcytosis. Brain uptake of the bispecific antibody can be further enhanced by engineering the bi-specific antibody to reduce its affinity to the blood brain barrier receptor.
  • Exemplary bispecific antibodies can also be (1) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al, Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so called “dock and lock” molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different
  • Examples of platforms useful for preparing bispecific antibodies include but are not limited to BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star) , Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab).
  • epitope refers to a site on an antigen to which an antibody binds.
  • An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2- dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
  • An "antagonist" antibody or other binding agent is one which inhibits a biological activity of the antigen it binds. Such antibodies may substantially or completely inhibit the biological activity of the antigen.
  • biological activity and “biologically active” with regard to MCAM refer to its ability to specifically bind its ligand (a laminin a4 chain, e.g., the a4 chain of laminin 411) and/or to facilitate the infiltration of MCAM-expressing cells, e.g., TH17 cells, into the ligand (a laminin a4 chain, e.g., the a4 chain of laminin 411) and/or to facilitate the infiltration of MCAM-expressing cells, e.g., TH17 cells, into the
  • inhibitor means an agent decreases the biological activity of at least one target, for example MCAM. Such an inhibitor inhibits the activity of at least one target by at least about at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95% or at least 100%.
  • a "subject” includes a human or other mammalian subject that receives either prophylactic or therapeutic treatment.
  • amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non- conservative substitutions constitute exchanging a member of one of these classes for a member of another.
  • Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
  • a subject antibody region e.g., the entire mature variable region of a heavy or light chain
  • compositions or methods "comprising" one or more recited elements may include other elements not specifically recited.
  • a composition that comprises antibody may contain the antibody alone or in combination with other ingredients.
  • Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
  • the term “about” encompasses values within a standard margin of error of measurement (SEM) of a stated value. If used to qualify a range, both ends of the range are so qualified. In the context of dosages, the term about can indicate errors in measurement or rounding to a whole number in caclulating a dosage.
  • 2120.4.19 refers to a rodent derived monoclonal antibody clone having a mature variable heavy chain corresponding to SEQ ID NO: 114 and a mature variable light chain corresponding to SEQ ID NO: 120.
  • Humanized 2120 or “hu2120” refers to humanized variants of the 2120.4.19 clone. Statistical significance means p ⁇ 0.05.
  • the present application discloses among other things dosage regimes, and monitoring regimes for patients treated with antibodies inhibiting MCAM interaction with laminin a4. Methods of treatment with inhibitors of MCAM expression are also provided.
  • Natural human wild-type MCAM (melanoma cell adhesion molecule, also known as CD 146 and MUC18) is a protein of 646 amino acids having the following amino acid sequence:
  • EKYIDLRH (SEQ ID NO: 11). [0224] (GenBank database under Accession Number AAA20922.1 (CAA48332)). MCAM is a cell surface glycoprotein belonging to the immunoglobulin superfamily involved in cell adhesion, and in cohesion of the endothelial monolayer at intercellular junctions in vascular tissue. It also promotes tumor progression of many cancers, such as solid tumors, including melanoma and prostate cancer. It is known to interact in a homotypic/homophilic manner and may also bind to other ligands.
  • the human MCAM includes five immunoglobulin domains (1 : amino acid residues 24-129; 2: amino acid residues 139-242; 3: amino acid residues 244-330; 4: amino acid residues 335-424; and 5: amino acid residues 430-510), shown as SEQ ID NOS: 22- 26. Approximately residues 24-559 constitute the extracellular domain.
  • MCAM Unless otherwise apparent from the context, reference to MCAM or its fragments includes the natural human wildtype amino acid sequences indicated above, and human allelic variants thereof.
  • Laminin a4 refers to one of the polypeptide chains found in laminin molecules, which are expressed in the basal lamina (of the basement membrane), a protein network foundation for most cells and organs. Laminins are known to bind to cell membranes through plasma membrane molecules and contribute to cell attachment.
  • the laminin a4 chain typically forms a complex with a laminin ⁇ -chain, and a laminin ⁇ -chain.
  • the laminin a4 chain is found in numerous laminin molecules including laminin 411 (laminin 8 or ⁇ 4 ⁇ 1 ⁇ 1); laminin 421 (laminin 9 or ⁇ 4 ⁇ 2 ⁇ 1), and laminin 423 (laminin 14 or ⁇ 4 ⁇ 2 ⁇ 3).
  • laminin 411 refers to a trimeric polypeptide complex made up of three polypeptide subunits or chains: a4-chain, a ⁇ -chain, and a ⁇ -chain.
  • the present methods employ antibodies to MCAM that fully or partially inhibits its binding to laminin a4. Although practice of the invention is not dependent on understanding of mechanism, it is believed such inhibition inhibits MCAM-expressing cells e.g.., a TH17 cells from infiltrating or migrating into a subject's tissue. Examples of such antibodies are described in WO/2012/170071, WO 2014/039975, WO2015/136470, WO2015/136469 and US
  • a humanized antibody is a genetically engineered antibody in which the CDRs from a non-human "donor” antibody (i.e., 2120.4.19) are grafted into human "acceptor” antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539, Carter, US 6,407,213, Adair, US 5,859,205 6,881,557, Foote, US 6,881,557).
  • the acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence.
  • the human acceptor antibody sequences can optionally be selected from among the many known human antibody sequences to provide a high degree of sequence identity (e.g., 65-85% identity) between a human acceptor sequence variable region frameworks and corresponding variable region frameworks of a donor antibody chain.
  • a humanized antibody is an antibody having some or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences.
  • a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences.
  • a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences.
  • a humanized antibody comprises a humanized heavy chain and a humanized light chain.
  • a CDR in a humanized antibody is substantially from a corresponding CDR in a non- human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs, except CDRHl can have up to two substitutions and CHDRH2 can have substitutions at positions H60-65.
  • variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85%, 90%, 95% or 100% of corresponding residues defined by Kabat are identical.
  • humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5 CDRs) from a mouse antibody (e.g., Pascalis et al, J. Immunol. 169:3076, 2002; Vajdos et al, Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol.
  • CDR residues not contacting antigen and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from regions of Kabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/or empirically, or as described in Gonzales et al, Mol. Immunol. 41 : 863, 2004.
  • the amino acid occupying the position can be an amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence.
  • the number of such substitutions of acceptor for donor amino acids in the CDRs to include reflects a balance of competing considerations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.
  • the 2120.4.19 rat antibody against MCAM was disclosed in WO2014/039975 and is defined herein by SEQ ID NOs: 69-80. Residues 145, 167, 175, 206, 207, 216 and 225 of human MCAM contribute most to the epitope of the 2120.4.19 antibody (WO2015/136469 and WO2015/136470). Chimeric, veneered, and humanized forms of the 2120.4.19 antibody were also disclosed in the '975 application. The disclosed humanized forms are defined herein as SEQ ID NOs: 115-119, 121-123, 139-141, and 153.
  • any permutation of a humanized heavy chain and humanized light chain represented by these SEQ ID NOS. can be used in some aspects of the present invention, such as pharmaceutical compositions and formulations.
  • Additional humanized forms of the 2120.4.19 antibody in which glutamine is substituted to glutamic acid at position 1 (Kabat numbering) of the heavy chain variable region i.e. Q1E
  • the Q1E substitution in the heavy chain variable region is a conservative substitution not expected to produce a substantial effect on the binding characteristics of the antibody, but which can improve antibody stability.
  • Such antibodies include antibodies comprising a heavy chain variable region comprising Kabat CDR1 of SEQ ID NO: 78: GFSLTSNGVS; Kabat CDR2 of SEQ ID NO: 79: AISSGGTTYYNSAFKS; and Kabat CDR3 of SEQ ID NO: 80: RYGYGW YFDF .
  • Some antibodies comprise a light chain variable region comprising Kabat CDR1 of SEQ ID NO: 73: KASQNIYNSLA; Kabat CDR2 of SEQ ID NO: 74: NANSLQT; and Kabat CDR3 of SEQ ID NO: 75: QQFYSGYT.
  • Some such antibodies comprise an N32S substitution or an N32Q substitution in Kabat CDR1 of SEQ ID NO: 78, and some comprise a G33A substitution in Kabat CDR1 of SEQ ID NO: 78. These substitutions have been found to offer improved characteristics including an increase in antibody affinity and potency.
  • Other provided anti-MCAM antibodies are antibodies in which the mature heavy chain variable region has at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 161, and the mature light chain variable region has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 123.
  • Some such antibodies include three heavy chain and three light chain CDRs entirely or substantially identical to the CDR regions of the donor 2120.4.19 antibody. If not identical, CDRs preferably have substitutions at a type and position defined herein, such as in the previous paragraph.
  • the CDR regions can be defined by any conventional definition (e.g., Chothia) but are preferably as defined by Kabat.
  • any of the above described antibodies can be humanized antibodies.
  • Some humanized antibodies comprise a mature heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO: 161 (which are the same as the CDRs of SEQ ID NO: 156) except that position 32 (Kabat numbering) can be N, S, or Q, and position 33 (Kabat numbering) can be G or A, and a mature light chain variable region comprising the three Kabat CDRs of SEQ ID NO: 123 (which are the same as the CDRs of SEQ ID NO: 120), preferably wherein the mature heavy chain variable region is at least 90% identical to SEQ ID NO: 161, and preferably wherein the mature light chain variable region is at least 90% identical to SEQ ID NO: 123.
  • Any such antibody can have either Q or E (i.e., Q1E substitution) at position HI by Kabat numbering.
  • the antibodies provided herein having a Q1E substitution in the mature heavy chain variable region include antibodies comprising a mature heavy chain variable region having the amino acid sequence of SEQ ID NO: 156 (i.e., 2120.4.19.Q1E), SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO: 161.
  • Some such antibodies comprise a mature light chain variable region having the amino acid sequence designated SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123.
  • the mature heavy chain and light chain variable regions can be combined in any possible permutation.
  • An exemplary combination is an antibody that comprises the mature heavy chain variable region having the amino acid sequence of SEQ ID NO: 161, and the mature light chain variable region having the amino acid sequence designated SEQ ID NO: 123.
  • Forms of these antibodies without the Q1E substitution such as have been described in PCT/US2013/058773, can also be used in some aspects of the invention, such as pharmaceutical compositions and formulations.
  • antibodies in which the heavy chain mature variable region has at least 90%, 95%, 96%, 97%, 98%, 99% or 100 % sequence identity to the amino acid sequence of any of SEQ ID NO: 156 (i.e., 2120.4.19.Q1E), SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO: 161 and the light chain has at least 90%,95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123.
  • Such antibodies are preferably humanized. Any such antibody can have either Q or E (i.e., Q1E substitution) at position HI by Kabat numbering.
  • Variants of disclosed SEQ ID NOs typically differ from the mature heavy chain and light chain variable region sequences by a small number (e.g., typically no more than 1, 2, 3, 5 or 10 in either the light chain or heavy chain mature variable region framework, or both) of replacements, deletions or insertions. Any changes are preferably conservative substitutions.
  • chimeric and veneered forms of the 2120.4.19 antibody are also provided.
  • a chimeric antibody is an a nti body in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence.
  • a veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T- cell epitopes, for example exposed residues with residues from the corresponding positions of a human antibody sequence (Padlan, Mol. Immunol. 28:489, 1991).
  • the result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions.
  • Human antibodies against MCAM are provided by a variety of techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, above, or otherwise, to have the same epitope specificity as a particular non-human antibody, such as 2120.4.19. Human antibodies can also be screened for a particular epitope specificity by using only a fragment of MCAM as the target antigen, and/or by screening antibodies against a collection of deletion mutants of MCAM.
  • Methods for producing human antibodies include the trioma method of Oestberg et al, Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al, US Patent 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al., W093/12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991) and phage display methods (see, .e.g.
  • the inhibitory activity of the MCAM antibodies or other antagonists described herein can be assayed by various methods including competitive binding assays with antibodies that bind the same or a substantially similar epitope (e.g., m2120) and blocking of MCAM binding with its ligand, the laminin a4 chain of laminin 411.
  • MCAM-expressing cells are (a) incubating with a recombinant polypeptide comprising a laminin a4 chain, e.g., an a4 chain of laminin 411, in the presence or absence of a candidate antibody; (b) monitoring the level of binding of the laminin a4 to the cells, e.g.
  • An alternate screening protocol involves the use of a population of cells expressing a laminin a4 chain, which can be incubated with MCAM, in the presence and absence of a candidate antibody, and binding of MCAM to the cell population monitored. If the binding of MCAM to the cell population in the presence of the candidate antibody is lower than in its absence, the candidate antibody is an MCAM antagonist.
  • FACS fl uorescence-activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • the MCAM antagonists identified based on their ability to inhibit the binding of MCAM to its ligand, e.g., a laminin a4 chain, are candidates for the treatment of inflammatory conditions characterized by infiltration of MCAM-expressing cells.
  • the inhibitory activity of an MCAM antibody can also be assessed in vivo.
  • An example of a methodology for assessing the inhibitory activity of an MCAM antibody is with an experimental autoimmune encephalomyelitis (EAE) model.
  • EAE is a disease that is generated in laboratory animals to produce symptoms similar to those of multiple sclerosis (MS) in humans. See, e.g., Bauer et al., Proc. Natl Acad. Sci. USA 106: 1920-1925 (2009).
  • EAE is generally produced by injecting animals with different proteins from the central nervous system of other animals, for example, extracts of myelin basic protein and whole spinal cord or brain tissue, or with T cells that specifically react to myelin.
  • EAE is commonly used to follow the course of relapsing or progressive forms of MS.
  • EAE has been served as a suitable animal model to both develop therapeutic agents for MS and study the specific disease processes of MS. See, e.g., Gold et al., Brain 129: 1953-1971 (2006); see also Steinman et al, Ann. Neurol. 60: 12-21 (2006).
  • mice are immunized with PLP 139-151 peptide to induce EAE. After disease onset, mice are treated intraperitoneally with either a candidate anti-MCAM antibody or isotype control, and every day thereafter. Mice are monitored daily and scored for in a blinded manner, and body weights were obtained every 2-3 days. A delay in relapse and significant reduction in symptom severity in mice treated with a candidate MCAM antibody is indicative of a successful candidate antibody.
  • the heavy and light chain variable regions of chimeric, veneered or humanized antibodies can be linked to at least a portion of a human constant region.
  • the choice of constant region depends, in part, whether antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis and/or complement dependent cytotoxicity are desired.
  • human isotopes IgGl and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not.
  • Human IgGl and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4.
  • Light chain constant regions can be lambda or kappa.
  • One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al, US Patent No. 5,624,821 ; Tso et al, US Patent No. 5,834,597; and Lazar et al, Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al, J. Biol. Chem.
  • substitutions include a Gin at position 250 and/or a Leu at position 428 (EU numbering is used in this paragraph for the constant region) for increasing the half-life of an antibody. Substitution at any or all of positions 234, 235, 236 and/or 237 reduce affinity for Fey receptors, particularly FcyRI receptor (see, e.g., US
  • alanine substitution at positions 234, 235, and 237 of human IgGl can be used for reducing effector functions.
  • Some antibodies have alanine substitution at positions 234, 235 and 237 of human IgGl for reducing effector functions.
  • positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g., US 5,624,821).
  • a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgGl is used.
  • positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine.
  • positions 234 and 235 are substituted with alanine, such as in SEQ ID NO: 172.
  • the isotype is human IgG2 or IgG4.
  • An exemplary human light chain kappa constant region has the amino acid sequence of SEQ ID NO: 168.
  • N-terminal arginine of SEQ ID NO: 168 can be omitted, in which case light chain kappa constant region has the amino acid sequence of SEQ ID NO: 169.
  • An exemplary human IgGl heavy chain constant region has the amino acid sequence of SEQ ID NO: 170 (with or without the C-terminal lysine).
  • Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which heavy and light chain mature variable domains are linked through a spacer.
  • Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes.
  • another heavy chain constant region is of IgGl Glm3 allotype and has the amino acid sequence of SEQ ID NO: 171.
  • Another heavy chain constant region has the amino acid sequence of SEQ ID NO: 171 except that it lacks the C-terminal lysine.
  • Another heavy chain constant region has the amino acid sequence of SEQ ID NO: 172.
  • Yet another heavy chain constant region has the amino acid sequence of SEQ ID NO: 172 except that it lacks the C- terminal lysine.
  • the invention further provides nucleic acids encoding any of the above constant regions.
  • nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to the constant region.
  • Antibodies can be produced by recombinant expression.
  • Nucleic acids encoding the antibodies can be codon-optimized for expression in the desired cell-type (e.g., CHO or Sp2/0).
  • Recombinant nucleic acid constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally-associated or heterologous promoter regions.
  • the expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the crossreacting antibodies.
  • the vector or vectors encoding the antibody chains can also contain a selectable gene, such as dihydrofolate reductase, to allow amplification of copy number of the nucleic acids encoding the antibody chains.
  • E. coli is a prokaryotic host particularly useful for expressing antibodies, particularly antibody fragments.
  • Microbes, such as yeast are also useful for expression. Saccharomyces is an example of a yeast host, with suitable vectors having expression control sequences, an origin of replication, termination sequences and the like as desired.
  • Typical promoters include 3- phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilizations.
  • Mammalian cells can be used for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH
  • suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0. It can be advantageous to use nonhuman cells.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al, Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Suitable expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).
  • cell pools can be screened for growth productivity and product quality in serum-free media. Top-producing cell pools can then be subjected to FACS-based single-cell cloning to generate monoclonal lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be advantageous. Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore. A selected clone can then be banked in multiple vials and stored frozen for subsequent use.
  • antibodies can be purified according to standard procedures of the art, including protein A capture, column chromatography (e.g., hydrophobic interaction or ion exchange), low-pH for viral inactivation and the like (see generally, Scopes, Protein Purification (Springer- Verlag, NY, 1982)).
  • the antagonist is an alternative binding agent that binds to MCAM or laminin a4.
  • alternative binding agents may include, for example, any of the engineered protein scaffolds known in the art.
  • Such scaffolds include, for example, anticalins, which are based upon the lipocalin scaffold, a protein structure characterized by a rigid beta- barrel that supports four hypervariable loops which form the ligand binding site. Novel binding specificities are engineered by targeted random mutagenesis in the loop regions, in combination with functional display and guided selection (Skerra (2008) FEBS J. 275: 2677-2683).
  • Suitable scaffolds may include, for example, adnectins, or monobodies, based on the tenth extracellular domain of human fibronectin III (Koide and Koide (2007) Methods Mol. Biol. 352: 95-109); affibodies, based on the Z domain of staphylococcal protein A (Nygren et al. (2008) FEBS J. 275: 2668-2676)); DARPins, based on ankyrin repeat proteins (Stumpp et al. (2008) Drug. Discov. Today 13: 695-701); fynomers, based on the SH3 domain of the human Fyn protein kinase (Grabulovski et al. (2007) J. Biol. Chem.
  • the binding agent is an anticalin, an adnectin, an affibody, a DARPin, a fynomer, an affitin, an affilin, an avimer, a cysteine-rich knottin peptide, or an engineered Kunitz-type inhibitor.
  • Libraries of peptides or other small molecules can also be screened for suitability.
  • Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and
  • oligocarbamates Large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which is incorporated by reference for all purposes). Peptide libraries can also be generated by phage display methods. See, e.g., DevLin, WO 91/18980. V. INHIBITORS OF MCAM EXPRESSION
  • the Examples show that treatment with a naked (i.e., unconjugated) anti-MCAM antibody reduces expression of MCAM on the surface of CD3 T cells.
  • Inhibitors of MCAM expression can achieve the same effect through a different mechanism.
  • Agents that can be designed to inhibit expression of a gene include zinc finger proteins, talons, siRNA, anti-sense RNA and Cas9 enzyme directed by a guide RNA. Such agents can bind to DNA interfering with transcription or bind to and/or cleave mRNA interfering with expression.
  • the antibodies or other antagonists of the invention can be used for treating or effecting prophylaxis of subjects having (e.g., meeting art- recognized criteria, such as those of the DSM-IV-TR or DSM-V) or at elevated risk relative to the general population of an autoimmune disease, neuroinflammatory disease and cancer among others. Elevated risk can be assessed from presence of one or more genetic or biochemical markers associated with the disease, or one or more symptoms consistent with the disease but insufficient to allow a definite diagnosis.
  • the above mentioned categories or disease are not necessarily mutually exclusive of one another; for example, multiple sclerosis can be classified as neuroinflammatory or autoimmune.
  • Some specific exemplary diseases treatable by the present methods include multiple sclerosis, Parkinson's disease, allergic contact dermatitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, inflammatory bowel disease, Crohn's disease, ankylosing spondylitis, and cancer, particularly, solid tumors, such as melanoma.
  • antibodies or other antagonists function at least in part by inhibiting the interaction of MCAM expressed on T cells (e.g., TH17 cells) and laminin a4 chain, e.g., an a4 chain of laminin 411 expressed on the surface of an endothelial cell.
  • Antibody-drug conjugates can have additional mechanisms of action including the cytotoxic or cytostatic effect of the linked agent, typically after uptake within the targeted cell. Antibody-drug conjugates may also induce tumor- associated macrophage toxicity.
  • Neuroinflammatory conditions are characterized by CNS inflammation and/or cell/tissue damage.
  • the indicia can include increased glial activation, increased proinflammatory cytokine/chemokine levels (e.g., TNFa, INFy, IL- ⁇ ), increased blood-brain- barrier permeability, and/or increased immune cell (e.g., leukocyte) recruitment/invasion to the CNS.
  • the neuro inflammation is often chronic associated with chronic activation of cells of the immune system (i.e., autoimmune-associated neuro inflammation) but can alternatively or additional have acute episodes.
  • RR-MS Relapsing- remitting MS
  • RR-MS Relapsing- remitting MS
  • RR-MS represents about 85% of all newly diagnosed subjects.
  • the definition of relapse requires the new symptom or sign to be present for at least 24 hours, to not be associated with a fever or intercurrent illness (such as the "flu" or a urinary tract infection), because an elevated body temperature can unmask silent or old lesions.
  • PP-MS Primary progressive
  • SP-MS secondary progressive form
  • PR-MS progressive relapsing form
  • Diagnosis of MS is usually based on a medical history, a neurologic exam and various tests, including magnetic resonance imaging (MRI), evoked potentials (EP) and spinal fluid analysis.
  • MRI magnetic resonance imaging
  • EP evoked potentials
  • spinal fluid analysis A definitive diagnosis of MS requires evidence of damage in at least two separate areas of the central nervous system (CNS), which includes the brain, spinal cord and optic nerves and evidence that the damage occurred at least one month apart and exclusion of all other possible diagnoses.
  • CNS central nervous system
  • the present methods can also be used prophylactically to treat individually having at least one sign or symptom of MS placing them at increased risk of progression to MS compared with the general population of healthy individuals.
  • the methods can be used to treat individuals who have had one attack (also called a relapse or an exacerbation) of MS-like symptoms— referred to as a clinically-isolated syndrome (CIS), who may or may not go on to develop MS.
  • CIS clinically-isolated syndrome
  • Individuals at risk of developing MS can also be identified by presence of an antibody to the protein KIR4.1 in their serum, among other methods.
  • Neuroinflammatory disease also includes Parkinson's disease.
  • Symptoms of Parkinson's disease include tremor (e.g., trembling in hands, arms, legs, jaw, and face); rigidity or stiffness of the limbs and trunk; bradykinesia or slowness of movement; postural instability or impaired balance and coordination; depression and other emotional changes; difficulty in swallowing, chewing, and speaking; urinary problems or constipation; skin problems; sleep disruptions. Parkinson's disease can be diagnosed from such symptoms, and/or brain scans and/or other tests to rule out other diseases.
  • Cancers can be hematopoietic malignancies or solid tumors, i.e., masses of cells that result from excessive cell growth or proliferation, either benign or malignant, including pre-cancerous legions. Cancers can be benign, malignant, or metastatic. Metastatic cancer refers to a cancer that has spread from the place where it first started to another place in the body. Tumors formed by metastatic cancer cells are called a metastatic tumor or a metastasis, which is a term also used to refer to the process by which cancer cells spread to other parts of the body. In general, metastatic cancer has the same name and same type of cancer cells as the original, or primary, cancer.
  • cancers include solid tumors, such as melanoma, carcinoma, blastoma, and sarcoma. Cancers also include hematologic malignancies, such as leukemia or lymphoid malignancies, such as lymphoma. More particular examples of such cancers include squamous cell cancer, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • hematologic malignancies such as leukemia or lymphoid malignancies, such as lymphoma. More particular examples of such cancers include
  • Autoimmune diseases include systemic autoimmune diseases, organ- or tissue-specific autoimmune diseases, and diseases that exhibit autoimmune-type expressions. In these diseases, the body develops a cellular and/or humoral immune response against one of its own antigens, leading to destruction of that antigen and potentially crippling and/or fatal
  • TH17 cells a lineage T helper cells characterized by production of interleukin (IL)-17 and IL-22, have been reported to enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis in humans and experimental autoimmune encephalomyelitis (EAE) in mice. See, e.g., Cua et al., Nature 421 : 744-748 (2003); Ivonov et al., Cell 126: 1121-1133 (2006).
  • TH17 cells may initiate or propagate an inflammatory response by their specific recruitment to and infiltration of tissue.
  • autoimmune diseases include Graves' disease, Hashimoto's thyroiditis, autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus (type 1 diabetes), insulin-resistant diabetes mellitus (type 2 diabetes), immune-mediated infertility, autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, autoimmune thrombocytopenic purpura, pernicious anemia, myasthenia gravis, Guillain-Barre syndrome, stiff man syndrome, acute rheumatic fever, sympathetic ophthalmia, Goodpasture's syndrome, autoimmune uveitis, temporal arteritis, Bechet's disease, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, primary biliary cirrhosis, autoimmune he
  • GCA Giant cell arteritis onset occurs at age 50 onward with a mean age of diagnosis of 72. About 190,000 subjects present with the disease. This disease frequently manifests as an analgesic resistant headache (60-90%) and visual loss (12-40%). Symptoms show a dramatic initial response to glucocorticoids. GCA is diagnosed from clinical presentation, patient characteristics, blood test markers of inflammation and most characteristically, presence of giant multinucleate cells in vessel wall biopsies most usually proximate to the internal elastic membrane.
  • GCA is caused by infiltration of TH17 and TH1 cells into a blood vessel wall. Histo- pathological lesions are observed in all layers of the artery leading to segmental and focal panarteritis with a polymorphic cell infiltrate that includes T cells, macrophages and
  • TH17 cells are the precursors of giant cells (Samson Clin Exp Rheumatol. 2013 Jan-Feb;31(l Suppl 75):S65-73. Epub 2013 Apr 19.). Therefore antibodies of the disclosure can inhibit GCA by inhibiting migration of TH17 or THl cells into blood vessel walls or conversion of TH17 cells to giant cells, among other mechanism.
  • An animal model for GCA is available for testing antibodies. The animal model is formed by transferring human temporal arterial specimens subcutaneously into SCK) mice (Deng et al., Circ. Res. 104, 488-495 (2009)).
  • a cellular model is also available for analyzing the effect of potential drugs on migration and proliferation of smooth muscle cells in the vasculature. This models uses smooth muscle cells on a MatrigelTM matrix.
  • PMR Polymyalgia rheumatica
  • GCA GCA
  • subjects have pain or stiffness, usually in the neck, shoulders, upper arms and hips, but which may occur all over the body.
  • the pain can be very sudden, or can occur gradually over a period. It may be caused by an inflammatory condition of blood vessels such as temporal arteritis.
  • Elevated ESR and C-reactive protein are characteristic of PMR as is a rapid response to low dose corticosteroids, which are the conventional treatment for PMR.
  • PMR can occur concurrently or separately than GCA.
  • GCA giant cell arteritis
  • Most inflammation is at the level of the synovium and bursae, with MRI studies revealing periarticular inflammation as well as bursitis in the bursae associated with both the shoulder and hip girdles.
  • Systemic macrophage and T-cell activation are characteristic of both GCA and PMR. Patients often have an elevated IL-6 level which is likely responsible for the systemic
  • a decrease in the level of circulating IL-6 correlates with remission of clinical symptoms.
  • age of onset is 50 year or later with a mean of 72 years.
  • Takayasu's arteritis is a rare type of vasculitis related to GCA. It is a form of large vessel granulomatous vasculitis with massive intimal fibrosis and vascular narrowing, affecting often young or middle-aged women of Asian descent. It mainly affects the aorta (the main blood vessel leaving the heart) and its branches, as well as the pulmonary arteries. Females are about 8-9 times more likely to be affected than males. Those with the disease often notice symptoms between 15 and 30 years of age Takayasu's arteritis can also lead to arm or chest pain and high blood pressure and eventually to heart failure or stroke. The goal of treatment is to relieve inflammation in the arteries and prevent potential complications. Signs and symptoms of Takayasu's arteritis include: arm or leg weakness or pain with use (claudication),
  • MRA magnetic resonance angiography
  • CTA computed tomography angiography
  • DSA arterial angiography
  • Treatments include corticosteroids, methotrexate, azathioprine, adalimumab, etenercept, [0280]
  • the present methods can also be used to inhibit growth or metastasis of cancer.
  • Cancers can be hematopoietic malignancies or solid tumors, i.e., masses of cells that result from excessive cell growth or proliferation, either benign or malignant, including pre-cancerous legions. Cancers can be benign, malignant, or metastatic. Metastatic cancer refers to a cancer that has spread from the place where it first started to another place in the body. Tumors formed by metastatic cancer cells are called a metastatic tumor or a metastasis, which is a term also used to refer to the process by which cancer cells spread to other parts of the body. In general, metastatic cancer has the same name and same type of cancer cells as the original, or primary, cancer.
  • cancers include solid tumors, such as melanoma, carcinoma, blastoma, and sarcoma. Cancers also include hematologic malignancies, such as leukemia or lymphoid malignancies, such as lymphoma. More particular examples of such cancers include squamous cell cancer, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • hematologic malignancies such as leukemia or lymphoid malignancies, such as lymphoma. More particular examples of such cancers include
  • Antibodies or inhibitors of expression are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disease being treated (i.e., a positive treatment response).
  • an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disease being treated (i.e., a positive treatment response).
  • a therapeutically effective regime If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime.
  • therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient.
  • therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.
  • the dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.
  • Exemplary dosages for an antibody are 0.3-50 mg /kg body weight. Exemplary dosage ranges include 0.3-1 mg/kg, 1-3 mg/kg, 3-10 mg/kg, 10-30 mg/kg and 30-50 mg/kg. Exemplary dosage regimes for an antibody includes 0.3 1, 3, 10, 30 or 50 mg/kg. Dosages of antibody are usually administered on multiple occasions separated by an interval.
  • the interval can be, for example, 2-5 weeks, e.g., every 2 weeks, every four weeks, or every month.
  • Dosages of antibodies can be administered on at least three occasions, at least ten occasions, at least until a positive treatment response is detected, at least until steady state levels of antibody in the plasma are reached (i.e., the same peaks and troughs between dosages within experimental errors), for at least one year, for at least 10 years or for the remaining life of the patient.
  • the same dose of antibody is administered at the same intervals, although in other patients the dose or frequency can be increased or decreased depending on treatment response and side effects observed.
  • Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular.
  • Preferred route of administration are intravenous infusion or subcutaneous administration.
  • administration can be, for example, by infusion over a period such as 30-90 min.
  • Some exemplary regimes include 3-10 mg/kg every two weeks and 20-30 mg/kg of antibody every four weeks by intravenous infusion.
  • other routes of administration can be performed, optionally with adjustment of the dose or frequency to achieve the same area under the curve as an intravenous infusion.
  • the data provided in the examples provide evidence that administration of PRX003 or other MCAM-laminin a4 antagonist not only inhibits extravasation of TH17 lymphocytes from the vascular into tissue, where they exert inflammatory effects, but can also effectively reverse this process with TH17 lymphocytes in the vascular wall or tissue returning to the peripheral circulation, as shown in Fig. 15.
  • the process in which TH17 lymphocytes return to the peripheral circulation is referred to as demargination. Because infiltration of TH17 lymphocytes into the vascular tissue causes or promotes neuroinflammatory or autoimmune disease, demargination is an additional mechanism by which antagonists of MCAM laminin a4 binding can effect treatment or prophylaxis of such diseases.
  • the present data show that demargination is greater at higher dosages of an antagonist above the amount of antagonist required to saturate MCAM binding sites.
  • treatment of neuroinflammatory and autoimmune diseases can be effected by performing a loading regime followed by a maintenance regime.
  • the loading regime serves to effect demargination and then lower levels of T-cell MCAM below pretreatment levels.
  • the maintenance regime serves to maintain MCAM below treatment levels.
  • the loading regime can be used to maximize demargination and then reduce the level of T-cell MCAM to less than 50%, 60%, 70%, 80% or 90% of the pretreatment level and the maintenance regime can be used to maintain the level of T-cell MCAM at below 50%, 60%, 70%, 80% or 90% of the pretreatment level respectively.
  • T-cell levels of MCAM are preferably measured on CD3+ cells. The amount of MCAM on the surface of such cells can be measured by
  • the amount of MCAM on the surface of cells can be measured from the number of cells detectably expressing any MCAM or the total amount of MCAM expressed.
  • the loading and maintenance regimes can involve administering different dosages of the same antagonist, in which case the loading regime can use a higher dose than the
  • the loading and maintenance regimes can be used the same antagonist but with different frequencies of administration, the frequency being lower for the maintenance regime.
  • different antagonists can be used for the loading and maintenance regimes.
  • the loading regime can be with an antibody, such as PRX003, and the maintenance regime with a small molecule (up to 600 kDa), a peptide (up to 20 amino acids) or polypeptide (regardless of length).
  • the loading regime can be a first antibody and the maintenance regime a second antibody, or the loading regime can be a first small molecule and the maintenance regime a second small molecule
  • the loading regime can involve single or multiple administrations of an antagonist, but usually involves administration on no more than 4 doses. One administration is often enough.
  • the maintenance regime usually involves multiple administration, for example, at least four times, at least ten times or the rest of a patient's life.
  • the maintenance regime can be administered at recurring intervals, for example, weekly, fortnightly, monthly or quarterly, or can be administered as needed to maintain T cell MCAM levels below a desired threshold level relative to preteatment as discussed above.
  • the loading and maintenance regimes can be by the same or different routes of administration.
  • the loading regime can be intravenous and the maintenance regime subcutaneous or vice versa.
  • An exemplary method administers about 10-50 or 10-30 mg/kg PRX003 or other antibody antagonist of MCAM binding to laminin a4 as a loading regime and a lower but nonzero dose of the same or different antibody antagonist of MCAM binding to laminin a4 binding as a maintenance regime.
  • the dose in the maintenance regime can be about 1 mg/kg to about 5 mg/kg, including dosages of about 1 /mg/kg. 3 mg/kg and 5 mg/kg.
  • the dose in the loading regime can be about 10 to about 30 mg/kg including about 10, 15, 20, 25 or 30 mg/kg and ranges of 10-15, 15-20, 20-25 or 25-30 mg/kg.
  • the loading regime involves administering an antibody antagonist once or multiple time at intervals of about 20 days to about 6 weeks, and the maintenance regime involves administering an antibody antagonist on multiple occasions at intervals of about 10 days to about 5 weeks, at a dose of about 0.3 to 10 mg/kg.
  • the maintenance regime is about 1 mg/kg to about 6 mg/kg or about 1-3, 3-6, 2-5, or 3-4 mg/kg at intervals of about every 2 weeks to about monthly.
  • the maintenance regime is administered at least 4 or at least ten times, or for the life of the patient.
  • the maintenance regime can also be administered at intervals and duration as needed to maintain a T- cell MCAM level below a threshold.
  • the maintenance regime can be a regime in which an MCAM laminin a4 antagonist is administered at or below the minimum concentration necessary to saturate its target antigen (i.e., MCAM or laminin a4) within 24 hr of administering an initial dose.
  • the saturating concentration can be determined empirically by administering different doses of an antibody, extracting T-cells (or CD3 lymphocyte subset) without dissociating antibody, and testing the ability of the lymphocytes or a CD3+ fraction thereof to bind labelled antibody.
  • the minimum dose of antibody administered to the test subject that reduces binding of labeled antibody to background is a saturating dose.
  • the saturating serum concentration of antibody is about 1-3 ug/ml serum.
  • PRX003 at 3 mg/k every four weeks provides a subsaturating dose.
  • PRX003 at 10 mg/kg every 2 weeks or 25 mg/kg every four weeks provides a saturating dose.
  • An exemplary regimes involves subcutaneously administering PRX003 at a 3 mg/kg dose at intervals of about every 5 days to about every 2 weeks. Optionally the intervals are about once per week.
  • Another exemplary regimes involves subcutaneously administering a dose of the antibody in the range of about 150 mg to about 350 mg at intervals of about every 5 days to about every 2 weeks.
  • the intervals are about once per week.
  • the dose is in the range of about 200 mg to about 250 mg, such as about 210 mg.
  • a loading regime can be a regime which an MCAM laminin a4 antagonist is administered at a dose that achieves an above saturating level at is mean serum concentration, for example at a dose of about 10-30 mg/kg.
  • a saturating serum concentration is achieved by administering a 3 mg/kg dose of PRX003 or other antibody antagonist of MCAM- laminin a4 at intervals of about every 5 days to about every 2 weeks.
  • the intervals are about once per week.
  • All of the above regimes can be used with any of the antibodies or other antagonist described in the application, and particularly for PRX003. Such antibodies can be incorporated within the various formulations described.
  • the above regimes can be used for treatment of any of the disorders disclosed herein, such as multiple sclerosis, psoriasis or psoriatic arthritis, Behcet's disease, giant cell arteritis, polymyalgia rheumatic or Takayau's arteritis.
  • Treatment with antibodies or other inhibitors disclosed herein can be combined with other treatments effective against the disorder being treated.
  • Combination treatments can be formulated for administered separately.
  • Some examples of treatments useful for combination therapies include methotrexate, steroids, NSAIDs, or any combination thereof.
  • Additional therapeutic agents for treatment of multiple sclerosis include one or more of the following: teriflunomide, interferon beta- la, interferon beta- lb, glatiramer acetate, fingolimod, and mitoxantrone, or a corticosteroid, such as prednisone, methylprednisolone, or dexamethasone.
  • Additional therapeutic agents for cancer include alkylating agents such as carmustine, chlorambucil, cisplatin, carboplatin, oxaliplatin, procarbazine, and cyclophosphamide;
  • antimetabolites such as fluorouracil, floxuridine, fludarabine, gemcitabine, methotrexate and hydroxyurea
  • natural products including plant alkaloids and antibiotics such as bleomycin, doxorubicin, daunorubicin, idarubicin, etoposide, mitomycin, mitoxantrone, vinblastine, vincristine, and Taxol (paclitaxel) or related compounds such as Taxotere®
  • the topoisomerase 1 inhibitor irinotecan temozolomide and Gliadel®, carmustine
  • inhibitors of tyrosine kinases such as Gleevec®, Sutent® (sunitinib malate), Nexavar® (sorafenib) and Tarceva® (erlotinib) or Iressa® (gefitinib); inhibitors of angiogenesis; and monoclonal antibodies, including Herceptin® against the HER2 antigen; Avast
  • Additional agents for treating Parkinson's disease include including levodopa, benzaseride, carbidopa, dopamine agonists, non-ergot dopamine agonists, catechol-O-methyl (“COMT”) inhibitors such as, for example, entacopone or tolcopone, monoamine oxidase (“MAO”) inhibitors, such as, for example, rasagaline, amantadine, or anticholinergic agents
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
  • compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries.
  • the formulation depends on the route of administration chosen.
  • antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection).
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection).
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Formulations comprise an antibody or other antagonist described herein, a buffer, one or more sugars and/or polyols and a surfactant, and have a pH within the range from about 5.5 to about 7.
  • the formulations can be prepared for storage in liquid form or in lyophilized form. When stored in lyophilized form, the formulations can be reconstituted with a liquid (e.g., sterile water) to the concentrations and properties described herein.
  • a lyophilized composition When a lyophilized composition is said to be reconstitutable by adding water to generate a formulation of specified component concentrations and pH, it is meant that the lyophilized formulation can be so reconstituted simply by addition of water (i.e., without supplying additional amounts of components or adding acid or base to change the pH).
  • concentrations and properties of a prelyophilized liquid formulation can also be in accordance with those described below if the lyophilized formulation is reconstituted to the same volume as the formulation prelyophilization. If the volume is different, then concentrations of formulations should be adjusted proportionally. For example, if the reconstituted volume is half the prelyophilization volume, then the concentrations of components in the prelyophilization formulation should be half the
  • the antibody is resuspended in a formulation as described below, temporarily frozen for storage prelyophilization, lyophilized, and reconstituted with water to the same concentrations as prelyophilization.
  • a formulation should preferably stabilize the antibody throughout freezing, lyophilization, storage, and reconstitution as well as being suitable for parenteral administration.
  • a purified antibody is resuspended at about 40 mg/mL in a formulation and stored frozen at -40°C in bags. Bags are thawed at room temperature for 3 hours and the contents are pooled.
  • the formulation is sterile filtered through a 0.2 micron sterile filer. Vials are filled with 5.4 mL of the formulation and lyophilized.
  • Lyophilized vials are stored at 2-8°C. Lyophilized vials are reconstituted by adding sterile water (e.g., approximately 5.0 to 5.4 mL sterile water, depending on the formulation). Five mL of the reconstituted product is then added into the port of an IV bag containing 20-100 mL of normal saline, lactated Ringers solution, or 5% dextrose solution or the like for intravenous infusion into a patient.
  • sterile water e.g., approximately 5.0 to 5.4 mL sterile water, depending on the formulation. Five mL of the reconstituted product is then added into the port of an IV bag containing 20-100 mL of normal saline, lactated Ringers solution, or 5% dextrose solution or the like for intravenous infusion into a patient.
  • Some formulations include a bulking agent, which may or may not be the same as the sugar/polyol component.
  • the formulations are sterile, for example, as accomplished by sterile filtration using a 0.2 ⁇ or a 0.22 ⁇ filter.
  • the formulations are also generally stable by low to undetectable levels of fragmentation and/or aggregation as further defined below on freezing and thawing.
  • Still other formulations are stable following reconstitution of a lyophilized cake for at least three months at about 40°C. In some formulations, less than about 5% of the antibody is present as an aggregate in the formulation.
  • the antibody is present at a concentration within the range from about 5 mg/mL to about 100 mg/mL. In some formulations, the antibody is present at a concentration within the range from about 5 mg/mL to about 50 mg/mL. In some formulations, the antibody is present at a concentration within the range from about 25 mg/mL to about 50 mg/mL. For example, the antibody may be present at a concentration of about 35-45 mg/mL or about 40 mg/mL. The antibody may be present in a sterile liquid dosage form of about 50 mg/vial to about 500 mg/vial, or greater. The antibody may be present in a lyophilized dosage form of about 40 mg/vial to about 500 mg/vial. For example, the antibody may be present in a sterile liquid or lyophilized dosage form of about 250-350 mg/vial or about 200 mg/vial.
  • the formulation can comprise any of the antibodies described herein.
  • the formulated antibody is an antibody comprising: (i) a mature heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO: 161 except that position 32 (Kabat numbering) can be N, S, or Q, and position 33 (Kabat numbering) can be G or A, wherein the mature heavy chain variable region is at least 90% identical to SEQ ID NO: 161, and (ii) a mature light chain variable region comprising the three Kabat CDRs of SEQ ID NO: 123, and being at least 90% identical to SEQ ID NO: 123.
  • position 1 (Kabat numbering) of the mature heavy chain variable region can be occupied by E.
  • the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO: 161 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123.
  • the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 161 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 123.
  • the formulated antibody is an isolated anti-MCAM antibody described herein.
  • the isolated anti-MCAM antibody binds to human MCAM (SEQ ID NO: 11) at an epitope including amino acid residue 141.
  • Buffers are used in the disclosed formulations to achieve a suitable pH for the antibody, such as, for example, histidine, succinate, and citrate buffers.
  • Some formulations have a pH within the range from about 5.5 to about 7, for example, a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.
  • Some formulations have a pH of between about 5.5 to about 6.5.
  • Some formulations have a pH of about 6.0 and other formulations have a pH of about 6.5.
  • histidine buffer is present at a concentration within the range from about 10 mM to about 30 mM, for example, at a concentration of about 15-25 mM or about 20 mM.
  • Suitable sugars and/or polyols for the formulations include trehalose and sucrose, or a combination thereof.
  • Sugars/polyols serve as bulking agents, lyoprotecting agent, and/or tonicity adjusting agents.
  • some formulations include trehalose present at a concentration within the range from about 200 mM to about 260 mM, or sucrose present at a concentration within the range from about 200 mM to about 260 mM.
  • Some formulations include trehalose present at a concentration of about 220 mM.
  • Other formulations include sucrose present at a concentration of about 220 mM.
  • Some such formulations are characterized by an osmolality in the range of about 250-400, 300-400, or 300-350 mOsm/kg, such as, for example, 287 or 295 mOsm/kg.
  • Formulations can contain a surfactant to reduce antibody aggregation and absorption to surfaces.
  • Suitable surfactants include polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight.
  • Polysorbate 20 protects against marked increases in aggregation or turbidity that would otherwise occur in formulations of antibodies.
  • the polysorbate 20 may be present at a concentration within the range from about 0.01% to about 0.05%.
  • the concentration can be 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%.
  • polysorbate 20 is present at a concentration within the range of about from about 0.05 g/L, 0.1 g/L, 0.15 g/L, 0.2 g/L, 0.25 g/L, 0.3 g/L, 0.35 g/L, 0.4 g/L, 0.45 g/L, or 0.5 g/L.
  • Some formulations include polysorbate 20 at a concentration of 0.2 g/L.
  • An exemplary formulation (liquid, prelyophilization or reconstituted after lyophilization) is characterized by a pH within the range from about 5.5 to about 7 and includes: (a) an antibody described herein, at a concentration within the range from about 10 mg/mL to about 50 mg/mL; (b) a histidine buffer present at a concentration within the range from about 10 mM to about 30 mM; (c) one or more sugars and polyols ("sugar/polyol”) selected from trehalose present at a concentration within the range from about 200 mM to about 260 mM, and sucrose present at a concentration within the range from about 200 mM to about 260 mM; and (d) polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight.
  • an antibody described herein at a concentration within the range from about 10 mg/mL to about 50 mg/mL
  • a histidine buffer present at a concentration within the range from about 10
  • the formulation can include: (a) any antibody described herein; (b) a histidine buffer at a concentration of about 20 mM; (c) sucrose at a concentration of about 220 mM; (d) polysorbate 20 at a concentration of about 0.02%; and a pH of about 6.0.
  • the formulation can include: (a) any antibody described herein; (b) a histidine buffer at a concentration of about 20 mM; (c) trehalose at a concentration of about 220 mM; (d) polysorbate 20 at a concentration of about 0.02%; and a pH of about 6.5.
  • Some lyophilized formulations include: (a) an antibody described herein; (b) histidine buffer; (c) trehalose or sucrose; and (d) polysorbate 20.
  • the lyophilized formulation can include about 200 mg of the antibody.
  • Some lyophilized formulations are capable of being reconstituted with sterile water.
  • Some lyophilized formulations include 100-300 or 150-250 mg antibody, 10 to 20 or 14 to 16 mg of histidine, 300 to 450 or 350 to 400 mg sucrose, and 0.5 to 1.5 mg or 0.75 to 1.25 mg polysorbate 20.
  • lyophilized formulations include 100 to 300 or 150 to 250 mg antibody, 10 to 20 or 14 to 16 mg of histidine, 360 to 500 or 400 to 450 mg trehalose dehydrate, and 0.5 to 1.5 mg or 0.75 to 1.25 mg polysorbate 20.
  • An exemplary lyophilized formulation includes 200 mg of an antibody, 15.5 mg of histidine, 376 mg sucrose, and 1 mg polysorbate 20.
  • Another exemplary lyophilized formulation includes 200 mg of an antibody, 15.5 mg of histidine, 416 mg trehalose dihydrate, and 1 mg polysorbate 20.
  • Some such formulations can be reconstituted to a volume of about 5 mL.
  • Other lyophilized formulations include the same components in the same proportions as any disclosed in this paragraph but in different amounts (e.g., 400 mg antibody, 31 mg histidine, 752 mg sucrose, and 2 mg polysorbate 20).
  • Lyophilized formulations can be reconstituted to an antibody concentration of about 30-50 or 35-45 mg/mL, for example to about 40 mg/mL; (b) a histidine buffer present at a concentration of about 10-30 or 15-25 mM, for example about 20 mM; (c) sucrose or trehalose present at a concentration of about 160-330 or 200-260 mM, for example about 220 mM; (d) polysorbate 20 present at a concentration of about 0.1-0.3 or 0.15 to 0.25 g/L, for example about 0.2 g/L; and (e) a pH of about 5.5-6.5, for example about 6.0 (if sucrose is present) or 6.5 (if trehalose is present).
  • Liquid or reconstituted lyophilized formulations are preferably substantially isotonic, implying an osmolality of about 250-350 mOsm/kg water. Some formulations have an osmolality of 270-300 mOsm/kg. Some formulations have an osmolality of about 287 or about 295 mOsm/kg. Liquid or reconstituted lyophilized formulations can also be hypertonic > 350 mOsm/kg water or hypotonic ( ⁇ 250 mOsm/kg water).
  • any of the formulations described can be made without pharmaceutical excipients, carriers or the like, other than those described as being components herein.
  • Such a formulation can be described as consisting of the recited components, or consisting essentially of the recited components if insignificant amounts of other components not affecting the properties of the formulation are present.
  • Formulations are preferably made under good manufacturing practices (GMP) approved or approvable by the FDA for preparation of drugs for administration to humans.
  • antibody formulations having stability at 38°C-42°C (e.g., as assessed by high performance size exclusion chromatography (HPSEC)) for at least about 30 days, for at least about 3 months, or longer. Such formulations may also have stability at 20°C- 24°C for at least about 1 year, and/or stability at 2°C-4°C for at least about 3 years. Stability of lyophilized formulations is assessed for storage in the lyophilized state. A formulation is considered stable if, after incubation at one or more of these specified combinations of time and temperature, it meets the below definition for low to undetectable fragmentation and/or low to undetectable aggregation.
  • HPSEC high performance size exclusion chromatography
  • the disclosed formulations exhibit low to undetectable levels of antibody aggregation and/or fragmentation, or a low or undetectable increase in fragmentation and/or aggregation above an initial level (e.g., less than about 5% aggregation).
  • a formulation having low to undetectable levels of fragmentation contains at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, of the total protein, for example, in a single peak as determined by hydrophobic interaction chromatography, or in two peaks (one corresponding to each of the antibody heavy chains and antibody light chains) by reduced Capillary Gel Electrophoresis (rCGE), representing the non-degraded antibody, and containing no other single peaks having more than 5%, more than 4%, more than 3%, more than 2%, more than 1%, or more than 0.5% of the total protein each.
  • rCGE reduced Capillary Gel Electrophoresis
  • a formulation having low to undetectable levels of aggregation contains no more than about 15%, no more than about 10%, no more that about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or no more than about 0.5% aggregation by weight protein, as measured by high performance size exclusion chromatography (HPSEC). For example, in some formulations, less than about 5% of the antibody is present as an aggregate.
  • Stable formulations also show little or no loss of biological activity(ies) having, for example, binding affinity measurable by ELISAs and/or additional functional assay, that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of an initial measurable value.
  • Patients receiving treatment with an anti-MCAM antibody can be monitored using the level of MCAM expressed by CD3+ T cells or level of MCAM extracellular domain in blood as markers of a positive treatment response.
  • a decreased level of MCAM expressed on CD3+ T cells and/or an increased level of MCAM in blood indicates a positive treatment response.
  • MCAM on CD3+ cells can be detected by an immuno assay, employing an antibody that specifically binds to MCAM.
  • the antibody should have a different epitope specificity than the anti-MCAM antibody or other antagonist used for treatment.
  • the assay employs two antibodies, one to recognize CD3+ T cells, the other to detect MCAM expression. Dual color FACS provides a suitable format for such detection.
  • Soluble MCAM can be detected in blood including whole blood or any fraction thereof, such as plasma or serum, also by immuno assay, such as ELISA. Preferably, such assays are performed before treatment and on one or more occasions after commencing treatment to determine whether levels of MCAM change responsive to treatment.
  • the level of change of MCAM if any responsive to treatment can be used as an indicator of whether to continue or change treatment.
  • patients experiencing a positive treatment response i.e., decreased expression of MCAM on CD3+ cells or increased expression in blood
  • patients not experiencing a positive treatment response may have treatment changed by increasing the dose or increasing the frequency of administration, or particularly if such measures still do not result in a positive response to treatment, may have treatment discontinued.
  • a positive response to treatment or otherwise maybe but one of several factors determined in setting future treatment, but in general a higher percentage of patients having a positive treatment response continue treatment with an anti-MCAM antibody than patients not having a positive treatment response.
  • MCAM-Fc was generated by fusing the extracellular domain of murine MCAM to human IgG and produced in CHO cells using standard techniques. Lou/M rats were immunized with 100 ⁇ g of MCAM-Fc protein in CFA (1 : 1 volume). Rats were boosted two times at two week intervals with MCAM- Fc protein in incomplete Freund's adjuvant (IF A) (1 : 1 volume). Hybridomas were generated from immunized rats using standard protocols and clones were selected by Clonepix. CHO cells were transfected with the full length murine MCAM gene and selected for stable expression using neomycin and standard techniques.
  • Parental CHO cells (MCAM negative) were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) using standard techniques and mixed at a 1 : 1 ratio with unlabeled MCAM transfected CHO cells. Hybridoma supernatants were incubated with this mixture of cells for 30 minutes and binding of potential MCAM specific antibodies was detected with a fluorescently labeled anti-rat secondary antibody (Jackson Immuno) by flow cytometry.
  • CFSE carboxyfluorescein succinimidyl ester
  • hMCAM- Fc was generated by fusing the extracellular domain of human MCAM to human IgG and produced in CHO cells using standard techniques. Lou/M rats were immunized with 250 ⁇ g of hMCAM-Fc protein in CFA (1 : 1 volume). Rats were boosted two times at two week intervals with hMCAM-Fc protein in incomplete Freund's adjuvant (IF A) (1 : 1 volume). Hybridomas were generated from immunized rats using standard protocols and clones were selected by Clonepix. CHO cells were transfected with the full length human MCAM gene and selected for stable expression using neomycin and standard techniques.
  • Parental CHO cells (MCAM negative) were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) using standard techniques and mixed at a 1 : 1 ratio with unlabeled human MCAM transfected CHO cells. Hybridoma supernatants were incubated with this mixture of cells for 30 minutes and binding of potential human MCAM specific antibodies was detected with a fluorescently labeled anti-rat secondary antibody (Jackson Immuno) by flow cytometry.
  • CFSE carboxyfluorescein succinimidyl ester
  • hMCAM-Fc was generated by fusing the extracellular domain of human MCAM to human IgG and produced in CHO cells using standard techniques.
  • Balb/c mice were immunized with 50 ⁇ g of hMCAM-Fc protein in CFA (1 : 1 volume). Mice were boosted two times at two week intervals with hMCAM-Fc protein in incomplete Freund's adjuvant (IF A) (1 : 1 volume).
  • IF A incomplete Freund's adjuvant
  • Hybridomas were generated from immunized mice using standard protocols and clones were selected by Clonepix. CHO cells were transfected with the full length human MCAM gene and selected for stable expression using neomycin and standard techniques. Parental CHO cells (MCAM negative) were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) using standard techniques and mixed at a 1 : 1 ratio with unlabeled human MCAM transfected CHO cells. Hybridoma supernatants were incubated with this mixture of cells for 30 minutes and binding of potential human MCAM specific antibodies was detected with a fluorescently labeled anti-mouse secondary antibody (Jackson Immuno) by flow cytometry.
  • CFSE carboxyfluorescein succinimidyl ester
  • RNA was isolated from hybridoma cells using RNAquous-4PCR kit (Ambion), and was used for cDNA synthesis.
  • First and second strand cDNA was synthesized using methods modified from Marathon cDNA amplification (Clontech) with the cDNA adaptor ligated to the 5 '-end of the obtained dscDNA.
  • the reverse specific primer was designed based on the specific antibody isotype constant region sequence for both heavy and light chains, and was used along with the adaptor primer in the PCR amplification of both VL and VH fragments using Pfu Ultra DNA polymerase (Stratagene).
  • the amplified PCR product was cloned into pCR-Blunt-TOPO (Invitrogen), and the nucleotide sequence was determined. The sequences of the identified clones were compared for percent identity within the VL and VH sequences.
  • ELISA was performed using a commercial kit (R&D Systems).
  • Mouse and rat monoclonal antibodies directed against human MCAM protein were generated as described in Materials and Methods above. The specific binding between the monoclonal antibody and human MCAM was confirmed by assessing the monoclonal antibody's ability to bind to cells transfected with human MCAM. For this, untransfected cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and mixed with unlabeled human MCAM transfected cells. Untransfected cells could, therefore, be differentiated.
  • CFSE carboxyfluorescein succinimidyl ester
  • the anti-human MCAM monoclonal antibodies were used to test their ability to block the binding of human MCAM to its ligand.
  • Human MCAM-Fc protein (5 ⁇ g/mL) was pre-incubated with isotype control antibody, or 10 ⁇ g/mL of the test monoclonal antibody for 30 minutes in PBS. The mixture was added to healthy spinal cord tissue sections and subsequently characterized by fluorescence microscopy as described in Materials and Methods above.
  • CHOKl parental CHO cells
  • CHO cells transfected with a human MCAM gene were preincubated with CHO culture media (DMEM), recombinant laminin 411 (10 ⁇ g/ml), or recombinant laminin 511 (i.e., laminin 10 ( ⁇ 5 ⁇ 1 ⁇ 1)) (10 ⁇ g/ml) at 37°C for 45 minutes.
  • DMEM CHO culture media
  • laminin 411 10 ⁇ 5 ⁇ 1 ⁇ 1
  • laminin 511 i.e., laminin 10 ( ⁇ 5 ⁇ 1 ⁇ 1)
  • the 87 independent mouse fusion clones and 26 independent rat fusion clones described in Example 1 above as being capable of (i) binding to human MCAM, and (ii) blocking the interaction between human MCAM and the a-4 chain of laminin were further characterized as follows.
  • IC50 quantitation for the ability of the monoclonal antibody to block the binding of human MCAM to the a-4 chain of laminin was determined as follows. CHO cells expressing human MCAM were incubated with an anti-human MCAM antibody (at various concentrations) for 30 minutes at 4 degrees Celsius. Unbound antibody was then washed away, and the cells were incubated with recombinant human laminin 411 at 20 ug/ml for 45 minutes at 37 degrees Celsius.
  • Unbound laminin was then washed away, and the laminin bound to the surface of the cells was detected with fluorescently labeled anti-laminin antibodies. After washing, the amount of laminin bound to the surface was detected by flow cytometry, and IC50s were calculated based on the mean fluorescent intensity.
  • Amino acid and nucleic acid sequences of the heavy and light chains of these antibodies, and their hypervariable regions, are provided in SEQ ID NOs: 29-92. More specifically, in the above assay, IC50s for the monoclonal antibody clones 1174.1.3, 1414.1.2, 1415.1.1, 1749.1.3, 2120.4.19, and 2107.4.10 were determined to be 0.469 ug/ml, 0.431 ug/ml, 0.307 ug/ml, 0.545 ug/ml, 0.888 ug/ml, and 0.290 ug/ml, respectively.
  • Figure 3 depicts the alignment of various 2120 heavy and light chain sequences. Residue numbering is according to Kabat numbering. Different mutations to the framework (FR) amino acid residues involved in CDR formation and antigen binding were identified depending upon the version of antibody.
  • Exemplary mutations of the 2120 antibodies are depicted in Fig. 3 A (boxed residues in CDR-H1 (S30T), between CDR-H1 and CDR-H2 (I37V and L48I), and between CDR-H2 and CDR-H3 (K71R) affect CDR contact; and S30T, 137V, L48I, and K71R mutations combined with an additional mutation after CDR-H2 (T68S) affect CDR contact); and Fig.
  • N-deamidation motifs mutations to the asparagines or glycine were introduced into the standard version.
  • the various humanized V regions were synthesized with a heterologous signal sequence and cloned into expression vectors containing human CK (VL) or human IgGl (VH).
  • VL human CK
  • VH human IgGl
  • the heavy and light chain plasmids were co-transfected into 293F cells with the FreeStyleTM MAX transfection regent (Invitrogen) according to the manufacturer's protocol.
  • the expressed antibody was purified with protein A PhyTip columns (Phynexus) and quantified via OD280.
  • ELISA plates were coated with recombinant hMCAM-His, and blocked with casein buffer to prevent non-specific binding.
  • Biotinylated rodent or chimeric antibody was added at a subsaturating concentration, in the presence or absence of 3x increasing concentrations of unlabeled competitor (humanized antibody, rodent, or chimeric). After washing to remove unbound antibody, streptavidin HRP was added to allow detection of the biotinylated antibody.
  • the ELISA was developed with TMB substrate and the OD450 was measured. The IC50 of the unlabeled competitor was determined using the GraphPad Prism5 software.
  • Table 5 summarizes the design of humanized sequences.
  • the heavy and light chain plasmids were co-transfected into 293F cells with the FreeStyleTM MAX transfection regent (Invitrogen) according to the manufacturer's protocol.
  • the expressed antibody was purified with protein A PhyTip columns (Phynexus) and quantified via OD280.
  • the apparent affinities of the humanized antibodies were compared to the parental rodent or chimeric antibody in a competitive ELISA according to the following protocol:
  • ELISA plates were coated with recombinant hMCAM-His, and blocked with casein buffer to prevent non-specific binding.
  • Biotinylated rodent or chimeric antibody was added at a subsaturating concentration, in the presence or absence of 3x increasing concentrations of unlabeled competitor (humanized antibody, rodent, or chimeric). After washing to remove unbound antibody, streptavidin HRP was added to allow detection of the biotinylated antibody.
  • the ELISA was developed with TMB substrate and the OD450 was measured. The IC50 of the unlabeled competitor was determined using the GraphPad Prism5 software.
  • affinities were measured using the ForteBio Octet Red.
  • Anti-human Fc sensors were used to capture the humanized antibodies, and several concentrations of hMC AMHis analyte were used to determine the affinity using a 1 : 1 fitting model.
  • the potencies of the antibodies were measured in the laminin/FACS assay according to the following protocol: recombinant laminin 411 (Biolaminate) was added to hMC AM expressing CHO cells in the presence or absence of varying concentrations of the humanized, rodent, or chimeric antibodies. Following incubation for 30-45 minutes, the cells were washed and anti-laminin conjugated to AF650 (NovusBio) was added to detect the bound laminin. The cells were run on a flow cytometer to measure the laminin binding signal.
  • Table 6 provides the constructs used for transfection.
  • Table 8 shows the relative affinities of the humanized antibodies compared to the rodent parent as measured by ForteBio and competitive ELISA, as well as the expression levels for the first round of transfections.
  • Table 9 shows the measured affinity by ForteBio, competitive ELISA, and functional blocking data (laminin/FACS assay) compared to the rodent parent, as well as the expression levels, from the second round of transfections.
  • variants of the rat and humanized versions of the 2120.4.19 antibody mature heavy chain variable regions were constructed. Variants of 2120.4.19, h2120VHl, h2120VH2, h2120VH3, h2120VH4, and h2120VH5 were constructed having a glutamine to glutamic acid substitution at position HI (Kabat numbering) (Fig. 4A). These variants are referred to as 2120.4.19. Q 1 E, h2120VH1. Q 1 E, h2120 VH2. Q 1 E, h2120VH3. Q 1 E,
  • h2120VH4.QlE, and h2120VH5.QlE are shown in SEQ ID NOS: 156-161.
  • the humanized versions identified by SEQ ID NOs: 157-161 are depicted in the alignment in Fig. 4A.
  • Various rat and humanized antibodies can be constructed using the modified variable heavy chains, including: h2120VHl .QlE+h2120VLl ; h2120VHl.QlE +h2120VL2; h2120VHl.QlE
  • Example 5 MCAM expression is dose- and time-dependently downregulated by PRX003 treatment in hMCAM transgenic mice
  • Example 7 Dynamic changes in soluble MCAM induced by PRX003 are tightly coupled with cellular downregulation of MCAM
  • EAE known to involve TH17 cells.
  • MCAM is expressed primarily on circulating NK cells in the hMCAM transgenic mouse and is expressed at a low level on T cells and neutrophils, a pattern that differs from that seen in humans (because of an unknown mechanism).
  • the off -target tissue cross-reactivity profile e.g., PRX003 binding to vascular, adipocyte, and smooth muscle cells
  • PRX003 binding to vascular, adipocyte, and smooth muscle cells
  • PRX003 humanized anti-MCAM antibody with mature heavy chain variable region of SEQ ID NO: 161 and mature light chain variable region of SEQ ID NO: 123 and human IgGl kappa isotype
  • Method A total of 9 chimpanzees (4 males and 5 females) were dosed on Day 1 via slow IV infusion of vehicle or PRX003.
  • Results Prescreening for CD3+MCAM+ T Cells. Twenty chimpanzees were prescreened to select 9 animals for enrollment based on an appropriate population of circulating CD3+MCAM+ T cells, along with other factors including health status and housing social order. Among the 9 animals selected, the number of CD3+MCAM+ T cells ranged from 7 to 15% of the total CD3+CD3+ T cell population. An additional prestudy collection from the enrolled animals at Day -7 confirmed the prescreening values.
  • Serum PRX003 Levels Blood was collected by venipuncture predose and at 5 minutes, 8, 24, 48, 96, 168, 240, 336, and weekly thereafter up to 1680 hours (10 weeks) post dose on Day 1 for pharmacokinetics evaluation. None of the serum samples from the vehicle control group contained quantifiable serum concentrations of PRX003. All animals in the 1.5, 5, 15 and 50 mg/kg dose groups were exposed to PRX003 following a single IV infusion. Serum PRX003 concentrations in all treated animals were below the limit of quantitation (BLQ) by the 1344-hour (8-week) time point.
  • Serum ADA levels Serum anti-PRX003 antibody (ADA) assessments are summarized in Table 12.
  • Serum samples from the 1512-hour time point were initially screened for ADA levels because there was no measurable PRX003 in serum from any animal at this time point to interfere with the assay.
  • a low titer (1 : 100) of ADA was confirmed in one treated animal (5 mg/kg male) at this time point.
  • predose 504, 1008 hours
  • no ADA titers were detected and there was no apparent meaningful effect on exposures or PK profile.
  • ADA anti-drug antibody
  • F female
  • M male
  • MCAM Occupancy and Expression on Circulating T Cells Initial evaluation of MCAM occupancy revealed saturable binding by PRX003 (i.e. no labeled PRX003 signal by FACS) on circulating T cells during the first 24 hours in all dose groups, beginning at 5 minutes (0.083 hours) post end-of-infusion (EOI), which corresponded with serum PRX003 levels (>30 ⁇ g/mL) that were well above experimental concentrations (1-3 ⁇ g/mL) required to saturate MCAM in chimpanzee whole blood, in vitro.
  • PRX003 i.e. no labeled PRX003 signal by FACS
  • Serum Soluble MCAM levels Serum Soluble MCAM levels. Serum soluble MCAM increased in a dose-dependent fashion, with respect to both magnitude and duration, following a single IV infusion of PRX003. Levels of soluble MCAM began to rise in all dose groups by 8 hours, and continued to increase up to -150% over baseline levels by 48 hours at the low dose and up to -400% by 840 hours (5 weeks) at the high dose. Within 2-3 weeks after peak levels were observed, soluble MCAM had returned to baseline levels, which generally corresponded with serum PRX003 at or near subsaturating concentrations for binding MCAM+ T cells. The vehicle-treated control animal shows normal variability of soluble MCAM levels.
  • MCAM Expression in Skin Skin punch biopsies (5 mm diameter, area above pectoralis muscle) were taken from all animals at 672 hours following a single IV infusion of vehicle or PRX003 to assess MCAM expression in various cell types, including endothelium, smooth muscle and adipose. Despite dose-dependent reductions in levels of MCAM expression on circulating T cells at this time point, no treatment differences in MCAM levels were detected on non-circulating skin cells, even at the highest dose level.
  • the PK/PD model for MCAM expression that was developed in the chimpanzee was used to estimate human doses that 1) maintain a partial MCAM expression inhibition with an every other week (q2wk) dose interval; 2) maintain full MCAM expression inhibition with a q2wk dose interval, and 3) maintain full MCAM expression inhibition with an every fourth week (q4wk) dose interval.
  • a clinically pragmatic dose regimen can be within this range.
  • the model demonstrates that 3 mg/kg-q2wk, 10 mg/kg-q2wk, and 25 mg/kg-q4wk achieve these respective PD targets. These predictions form the basis for a clinical trial dose range that extends up to 30 mg/kg. Nonclinical safety supports this planned dose range.
  • NOAEL no-observed-adverse-effect-level
  • Body mass index (B-MI) between 18-32 kg/m2 with a minimum weight of 45 kg
  • Vascular diseases including, but not limited to, blood clotting disorders, atherosclerosis, aneurysms, and renal artery disease Hypotension (systolic blood pressure [BP] ⁇ 85 millimeters of mercury [mmHg]) at Baseline (Day 1 /Visit 2) predose or a known history or documentation of hypotension on more than one occasion within 3 months prior to Baseline (Day 1 /Visit 2) predose or a known history or documentation of hypotension on more than one occasion within 3 months prior to Baseline (Day
  • Clinically significant systemic infection e.g., chronic or acute infection, urinary tract infection, upper respiratory infection
  • recurrent or chronic infection e.g., viral infections [including hepatitis B or C, human immunodeficiency virus (HIV)], bacterial infections, systemic fungal infections, or syphilis
  • DSM-IV-TR Diagnostic and Statistical Manual of Mental Disorders IV Text Revision
  • Subjects with adequately controlled depression for at least 6 months are not excluded; however, suicidal ideation or attempt at any time within the past year is exclusionary.
  • any of the following laboratory abnormalities at Screening (Visit 1): o Total bilirubin (unless attributed to Gilbert's syndrome) >1.5 times the upper limit of normal (x ULN), alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >2 ⁇ ULN o Serum creatinine >133 ⁇ /L (1.5 mg/dL) o Hemoglobin ⁇ 11.5 g/dL for males or ⁇ 10.0 g/dL for females, absolute neutrophil count of ⁇ 1500 ⁇ L (with the exception of a documented history of a chronic benign neutropenia), or platelet count of ⁇ 120,000 ⁇ L
  • the PRX003 drug product is supplied as a lyophilized powder for solution for infusion. It is a white to yellowish powder, presented in a Type I glass vial with a nominal fill volume of 5 mL.
  • PRX003 drug product is to be reconstituted with water for injection (WFI) to a concentration of 40 mg/mL in a buffered, isotonic, preservative- free solution.
  • WFI water for injection
  • the formulation is composed of 20 mM Histidine buffer, 220 mM Sucrose and 0.2 g/L Polysorbate 20 at pH 6.0.
  • the container closure system consists of a Type I 20/25 mL (20R) glass vial, elastomeric stopper suitable for lyophilization, and aluminum seal with white colored plastic button.
  • composition The qualitative and quantitative composition of a unit vial of the PRX003 drug product (200 mg/vial) is described in Table 13.
  • Overfill An overfill of at least 0.2 mL is applied and the vials are filled to a target fill volume of 5.4 mL ⁇ 0.2 mL to permit an extractable dose of 200 mg per vial, upon reconstitution of the lyophilized cake with 5 mL WFI. In this case the concentration is approximately 40 mg/mL.
  • Example 15 Dose-dependent down regulation of MCAM in healthy adults by PRX003
  • PRX003 was administered by intravenous infusion.
  • Asian+ White 0 (0) 0 (0) 0 (0) 1 (16.7) 0 (0) 0 (0) 1 (2.5)
  • FIG. 10A shows the percentage of MCAM cell surface expression relative to its pretreatment level for various dosages of PRX003.
  • Fig. 10A shows an initially higher level of MCAM after administering antibody that reduces to about 20% of pretreatment level and then returns to pretreatment level.
  • FIG. 10B shows the percentage of MCAM expression relative to pretreatment levels for various dosages of PRX003 0-2.5 day(s) post infusion. There is an initial dose-dependent increase in MCAM expression that declines to pretreatment level within 2.5 days, with higher doses taking longer to return to the pretreatment level.
  • FIG. 11 shows the levels of sMCAM ng/mL detected in serum after 0-90 days post infusion with PRX003 at 0 mg/kg (placebo), 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg. The level initially increases in treatment groups followed by return to pretreatment levels.
  • FIG. 12 shows the mean concentration of PRX003 in ⁇ g/mL of serum observed after 0-28 days post infusion with PRX003 at various treatment concentrations.
  • the clearance rates (CL) of PRX003 were determined for healthy adults as shown in FIG. 13.
  • the half-life for 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg was determined to be 3.8, 17, 44, 81, and 189 hr, respectively; see, Table 15 below.
  • Consistent with rapid distribution of PRX003 to its intended target (MCAM) high drug clearance was observed at low doses of PRX003 and high doses of PRX003 shows clearance rates similar to the reported clearance rate for IgG; see, FIG.13.
  • FIG. 14A depicts the dose-dependent increase in peak MCAM expression with increasing concentrations of PRX003.
  • FIG. 14B this effect is depicted by the area under the curve (AUC), where MCAM expression increases with increasing concentrations of PRX003.
  • Example 16 PRX003 safety and tolerability in healthy adults.
  • Table 16 shows the most common treatment-emergent adverse effects (TEAEs) their prevalence (%) and severity (>Grade 3) among the patient cohort.
  • the most common TEAE reported was headaches with no severe effects (10%), while balance disorder, seasonal allergy, and viral URT infections, were all reported at the same level, namely (6.7%) with no severe effects.

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Abstract

L'invention concerne des anticorps anti-MCAM qui inhibent la capacité du MCAM humain à se lier à une chaîne alpha-4 de laminine, des schémas posologiques, des schémas de surveillance et des schémas de traitement avec un agent inhibant l'expression de MCAM.
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