ANTI-MCAM ANTIBODIES AND ASSOCIATED METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC 119(e) of US Provisional Application No. 62/345,732 filed June 3, 2016, which is incorporated by reference in its entirety for all purposes

REFERENCE TO A SEQUENCE LISTING

[0002] The Sequence Listing written in file 497104SEQLIST.txt is 154 kilobytes, was created on June 2, 2017, and is hereby incorporated by reference.

BACKGROUND

[0001] A subset of CD3+ T cells, termed TH17 cells (T helper 17 cells), has been implicated in the pathogenesis of a number of autoimmune diseases and neuroinflammatory conditions involving CNS infiltration of T cells, such as multiple sclerosis and the animal model, experimental autoimmune encephalomyelitis (EAE). 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.

SUMMARY OF THE CLAIMED INVENTION

[0002] 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. In some methods, the T- cell MCAM levels are measured by fluorescence activated cell sorting (FACS). In some methods, 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. In some methods, 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.

[0003] In some methods, 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). In some methods, the antibody is administered at a dose of about 10 to about 30 mg/kg. In some methods, the antibody is administered at a dose of about 10 mg/kg. In some methods, the antibody is administered at a dose of about 15 mg/kg. In some methods, the antibody is administered at a dose of about 30 mg/kg.

[0004] 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

administering to the patient a first regime of a first antibody which inhibits binding of MCAM to a laminin a4 chain, and a second regime of a second antibody which inhibits binding of MCAM to a laminin a4 chain, wherein 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. In some methods, 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. In some methods, 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. In some methods, 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. In some methods, during the first regime, 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. In some methods, 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. In some methods, 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. In some methods, 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. In some methods, 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. In some methods, 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. In some methods, the antibody is administered intravenously. In some methods, the first dose is administered once. In some methods, 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. In some methods, the second dose is administered at least four times. In some methods, the second dose is administered at least ten times. In some methods, 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. In some methods, the second dose is administered for the rest of the patient's life.

[0005] In any of the methods, the disease can be multiple sclerosis, psoriasis or psoriatic arthritis, Behcet's disease, Giant Cell Arteritis, Polymyalgia Rheumatica, or Takayasu's Arteritis.

[0006] The invention further 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 regime of an antibody which inhibits binding of MCAM to a laminin a4 chain, wherein the regime is effective to maintain a serum concentration of the antibody sufficient to saturate MCAM. In some methods, the serum concentration is at least 1 ug/ml of the antibody. In some methods, 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). In some methods, 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. In some methods, 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

("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. In some methods, 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.

[0007] 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.

[0008] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 depicts the identification of critical clones. The mean 2120.4.19 binding value plotted as a function of its mean surface expression value (gray diamonds). Thresholds of <30% monoclonal antibody reactivity and >50% mouse sera binding were applied to identify clones (black diamonds) that were negative for antibody binding but positive for surface expression

[0010] FIGS. 2A-C. FIG. 2A a homology model of human MCAM, represented by a ribbon diagram. FIG. 2B depicts a partial alignment of human BCAM, human MCAM, and mouse MCAM sequences indicating residues of interest at position 141 (1141) and position 145 (P145) of human MCAM. FIG. 2C depicts a ribbon diagram depicting the location and exposure of the 1141 and P145 residues of human MCAM.

[0011] FIGS. 3 A & B. FIG. 3 A shows 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

(h2120VH5; SEQ ID NO: 119); and heavy chain human variable AF062133 IGHV2-26*01 sequence used as the framework donor (AF062133 VFI; SEQ ID NO: 108). Kabat numbering is used and hypervariable regions (HVRs) grafted from the rat 2120.4.19.6 antibody to the variable heavy chain variable AF062133 IGHV2-26*01 framework are boxed. The S30T, I37V, L48I and K71R mutations combined with (i) mutations of the boxed N/D residues in CDR-H1, e.g., N32S (VH3); N32Q (VH4); or G33A (VH5)), provides an N deamidation mutant. 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. Residues where mutations were focused due to the presence of N-deamination sites or N-glycosylation sites are shown in the bracketed box.

[0012] 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). 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. 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.

[0013] 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

(AF062133 VFI; SEQ ID NO: 108). Kabat numbering is used and hypervariable regions (HVRs) grafted from the rat 2120.4.19.6 antibody to the variable heavy chain variable AF062133 IGHV2-26*01 framework are boxed. The position Q1E substitution is outlined by a box. [0014] FIG. 4B 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). 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.

[0015] 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.

[0016] 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).

[0017] FIGS. 7A & B. FIG. 7A shows 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.

[0018] FIG. 8 shows dynamic changes in soluble MCAM induced by PRX003 are tightly coupled with cellular downregulation of MCAM.

[0019] 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).

[0020] 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).

[0021] 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).

[0022] 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).

[0023] 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.

[0024] 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.

[0025] 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.

BRIEF DESCRIPTION OF THE SEQUENCES

[0026] SEQ ID NO: 1 is the nucleic acid sequence encoding the mature light chain variable region of antibody clone 17.

[0027] SEQ ID NO: 2 is the amino acid sequence of the mature light chain variable region of antibody clone 17.

[0028] SEQ ID NO: 3 is the amino acid sequence of CDRL1 of the antibody clone 17. [0029] SEQ ID NO: 4 is the amino acid sequence of CDRL2 of the antibody clone 17.

[0030] SEQ ID NO: 5 is the amino acid sequence of CDRL3 of the antibody clone 17.

[0031] SEQ ID NO: 6 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody clone 17.

[0032] SEQ ID NO: 7 is the amino acid sequence of the mature heavy chain variable region of antibody clone 17.

[0033] SEQ ID NO: 8 is the amino acid sequence of CDRH1 of the antibody clone 17.

[0034] 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.

[0035] SEQ ID NO: 12 is the nucleic acid sequence encoding the mature light chain variable region of antibody clone 15.

[0036] SEQ ID NO: 13 is the amino acid sequence of the mature light chain variable region of antibody clone 15.

[0037] SEQ ID NO: 14 is the amino acid sequence of CDRL1 of the antibody clone 15.

[0038] SEQ ID NO: 15 is the amino acid sequence of CDRL2 of the antibody clone 15.

[0039] SEQ ID NO: 16 is the amino acid sequence of CDRL3 of the antibody clone 15.

[0040] SEQ ID NO: 17 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody clone 15.

[0041] SEQ ID NO: 18 is the amino acid sequence of the mature heavy chain variable region of antibody clone 15.

[0042] SEQ ID NO: 19 is the amino acid sequence of CDRH1 of the antibody clone 15.

[0043] SEQ ID NO: 20 is the amino acid sequence of CDRH2 of the antibody clone 15. [0044] SEQ ID NO : 21 is the amino acid sequence of CDRH3 of the antibody clone 15.

[0045] SEQ ID NO: 22 is the amino acid sequence of human MCAM domain 1 (residues 19- 129).

[0046] SEQ ID NO: 23 is the amino acid sequence of human MCAM domain 2 (residues 139-242).

[0047] SEQ ID NO: 24 is the amino acid sequence of human MCAM domain 3 (residues 244-321).

[0048] SEQ ID NO: 25 is the amino acid sequence of human MCAM domain 4 (residues 355-424).

[0049] SEQ ID NO: 26 is the amino acid sequence of human MCAM domain 5 (residues 430-510).

[0050] SEQ ID NO: 27 is the amino acid sequence of an a4-chain isoform of human laminin 411 (Accession No. NP001098676).

[0051] SEQ ID NO: 28 is the amino acid sequence of an a4-chain isoform of human laminin 411 (Accession No. CAA48332).

[0052] SEQ ID NO: 29 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1174.1.3.

[0053] SEQ ID NO: 30 is the amino acid sequence of the mature light chain variable region of antibody 1174.1.3.

[0054] SEQ ID NO: 31 is the amino acid sequence of CDRL1 of antibody 1174.1.3.

[0055] SEQ ID NO: 32 is the amino acid sequence of CDRL2 of antibody 1174.1.3.

[0056] SEQ ID NO: 33 is the amino acid sequence of CDRL3 of antibody 1174.1.3. [0057] SEQ ID NO: 34 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1174.1.3.

[0058] SEQ ID NO: 35 is the amino acid sequence of the mature heavy chain variable region of antibody 1174.1.3.

[0059] SEQ ID NO: 36 is the amino acid sequence of CDRH1 of antibody 1174.1.3.

[0060] SEQ ID NO: 37 is the amino acid sequence of CDRH2 of antibody 1174.1.3.

[0061] SEQ ID NO: 38 is the amino acid sequence of CDRH3 of antibody 1174.1.3.

[0062] SEQ ID NO: 39 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1414.1.2.

[0063] SEQ ID NO: 40 is the amino acid sequence of the mature light chain variable region of antibody 1414.1.2.

[0064] SEQ ID NO: 41 is the amino acid sequence of CDRL1 of antibody 1414.1.2.

[0065] SEQ ID NO: 42 is the amino acid sequence of CDRL2 of antibody 1414.1.2.

[0066] SEQ ID NO: 43 is the amino acid sequence of CDRL3 of antibody 1414.1.2.

[0067] SEQ ID NO: 44 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1414.1.2.

[0068] SEQ ID NO: 45 is the amino acid sequence of the mature heavy chain variable region of antibody 1414.1.2.

[0069] SEQ ID NO: 46 is the amino acid sequence of CDRH1 of antibody 1414.1.2.

[0070] SEQ ID NO: 47 is the amino acid sequence of CDRH2 of antibody 1414.1.2.

[0071] SEQ ID NO: 48 is the amino acid sequence of CDRH3 of antibody 1414.1.2. [0072] SEQ ID NO: 49 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1415.1.1.

[0073] SEQ ID NO: 50 is the amino acid sequence of the mature light chain variable region of antibody 1415.1.1.

[0074] SEQ ID NO: 51 is the amino acid sequence of CDRL1 of antibody 1415.1.1.

[0075] SEQ ID NO: 52 is the amino acid sequence of CDRL2 of antibody 1415.1.1.

[0076] SEQ ID NO: 53 is the amino acid sequence of CDRL3 of antibody 1415.1.1.

[0077] SEQ ID NO: 54 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1415.1.1.

[0078] SEQ ID NO: 55 is the amino acid sequence of the mature heavy chain variable region of antibody 1415.1.1.

[0079] SEQ ID NO: 56 is the amino acid sequence of CDRH1 of antibody 1415.1.1.

[0080] SEQ ID NO: 57 is the amino acid sequence of CDRH2 of antibody 1415.1.1.

[0081] SEQ ID NO: 58 is the amino acid sequence of CDRH3 of antibody 1415.1.1.

[0082] SEQ ID NO: 59 is the nucleic acid sequence encoding the mature light chain variable region of antibody 1749.1.3.

[0083] SEQ ID NO: 60 is the amino acid sequence of the mature light chain variable region of antibody 1749.1.3.

[0084] SEQ ID NO: 61 is the amino acid sequence of CDRL1 of antibody 1749.1.3.

[0085] SEQ ID NO: 62 is the amino acid sequence of CDRL2 of antibody 1749.1.3.

[0086] SEQ ID NO: 63 is the amino acid sequence of CDRL3 of antibody 1749.1.3. [0087] SEQ ID NO: 64 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 1749.1.3.

[0088] SEQ ID NO: 65 is the amino acid sequence of the mature heavy chain variable region of antibody 1749.1.3.

[0089] SEQ ID NO: 66 is the amino acid sequence of CDRH1 of antibody 1749.1.3.

[0090] SEQ ID NO: 67 is the amino acid sequence of CDRH2 of antibody 1749.1.3.

[0091] SEQ ID NO: 68 is the amino acid sequence of CDRH3 of antibody 1749.1.3.

[0092] SEQ ID NO: 69 is the nucleic acid sequence encoding a mature light chain variable region of antibody 2120.4.19.

[0093] 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.

[0094] SEQ ID NO: 71 is the amino acid sequence of a mature light chain variable region of antibody 2120.4.19.

[0095] SEQ ID NO: 72 is the amino acid sequence of a mature light chain variable region of antibody 2120.4.19.

[0096] SEQ ID NO: 73 is the amino acid sequence of CDRL1 of antibody 2120.4.19.

[0097] SEQ ID NO: 74 is the amino acid sequence of CDRL2 of antibody 2120.4.19.

[0098] SEQ ID NO: 75 is the amino acid sequence of CDRL3 of antibody 2120.4.19.

[0099] SEQ ID NO: 76 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 2120.4.19.

[0100] SEQ ID NO: 77 is the amino acid sequence of the mature heavy chain variable region of antibody 2120.4.19.

[0101] SEQ ID NO: 78 is the amino acid sequence of CDRH1 of antibody 2120.4.19. [0102] SEQ ID NO: 79 is the amino acid sequence of CDRH2 of antibody 2120.4.19.

[0103] SEQ ID NO: 80 is the amino acid sequence of CDRH3 of antibody 2120.4.19.

[0104] SEQ ID NO: 81 is a nucleic acid sequence encoding a mature light chain variable region of antibody 2107.4.10.

[0105] 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.

[0106] SEQ ID NO: 83 is a nucleic acid sequence encoding a mature light chain variable region of antibody 2107.4.10.

[0107] 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.

[0108] SEQ ID NO: 85 is the amino acid sequence of CDRL1 of antibody 2107.4.10.

[0109] SEQ ID NO: 86 is the amino acid sequence of CDRL2 of antibody 2107.4.10.

[0110] SEQ ID NO: 87 is the amino acid sequence of CDRL3 of antibody 2107.4.10.

[0111] SEQ ID NO: 88 is the nucleic acid sequence encoding the mature heavy chain variable region of antibody 2107.4.10.

[0112] SEQ ID NO: 89 is the amino acid sequence of the mature heavy chain variable region of antibody 2107.4.10.

[0113] SEQ ID NO: 90 is the amino acid sequence of CDRH1 of antibody 2107.4.10.

[0114] SEQ ID NO: 91 is the amino acid sequence of CDRH2 of antibody 2107.4.10.

[0115] SEQ ID NO: 92 is the amino acid sequence of CDRH3 of antibody 2107.4.10.

[0116] SEQ ID NO: 93 is the amino acid sequence of the mature heavy chain variable region of antibody 1749.1.3. [0117] SEQ ID NO: 94 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 1749 version 1 (VH1).

[0118] SEQ ID NO: 95 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 1749 version 2 (VH2).

[0119] SEQ ID NO: 96 is the amino acid sequence of the heavy chain variable framework donor U96282 VH.

[0120] SEQ ID NO: 97 is the amino acid sequence of the mature light chain variable region of antibody 1749.1.3.

[0121] SEQ ID NO: 98 is the amino acid sequence of the mature light chain variable region of humanized antibody 1749 version 1 (VL1).

[0122] SEQ ID NO: 99 is the amino acid sequence of the mature light chain variable region of humanized antibody 1749 version 2 (VL2).

[0123] SEQ ID NO: 100 is the amino acid sequence of the light chain variable framework donor X02990 VL.

[0124] SEQ ID NO: 101 is the amino acid sequence of the mature heavy chain variable region of antibody 2107.4.10.18.

[0125] SEQ ID NO: 102 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 1 (VH1).

[0126] SEQ ID NO: 103 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 2 (VH2).

[0127] SEQ ID NO: 104 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 3 (VH3).

[0128] SEQ ID NO: 105 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 4A (VH4A). [0129] SEQ ID NO: 106 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 5A (VH5A).

[0130] SEQ ID NO: 107 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 6 (VH6).

[0131] SEQ ID NO: 108 is the amino acid sequence of the heavy chain variable framework donor AF062133 VH.

[0132] SEQ ID NO: 109 is the amino acid sequence of the mature light chain variable region of antibody 2107.4.10.18.

[0133] SEQ ID NO: 110 is the amino acid sequence of the mature light chain variable region of humanized antibody 2107 version 1 (VL1).

[0134] SEQ ID NO: 111 is the amino acid sequence of the mature light chain variable region of humanized antibody 2107 version 2 (VL2).

[0135] SEQ ID NO: 112 is the amino acid sequence of the mature light chain variable region of humanized antibody 2107 version 3 (VL3).

[0136] SEQ ID NO: 113 is the amino acid sequence of the light chain variable framework donor U86803.

[0137] SEQ ID NO: 114 is the amino acid sequence of the mature heavy chain variable region of antibody 2120.4.19.6.

[0138] SEQ ID NO: 115 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 1 (VH1).

[0139] SEQ ID NO: 116 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 2 (VH2).

[0140] SEQ ID NO: 117 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 3 (VH3). [0141] SEQ ID NO: 118 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 4 (VH4).

[0142] SEQ ID NO: 119 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2120 version 5 (VH5).

[0143] SEQ ID NO: 120 is the amino acid sequence of the mature light chain variable region of antibody 2120.4.19.6.

[0144] SEQ ID NO: 121 is the amino acid sequence of the mature light chain variable region of humanized antibody 2120 version 1 (VL1).

[0145] SEQ ID NO: 122 is the amino acid sequence of the mature light chain variable region of humanized antibody 2120 version 2 (VL2).

[0146] SEQ ID NO: 123 is the amino acid sequence of the mature light chain variable region of humanized antibody 2120 version 3 (VL3).

[0147] SEQ ID NO: 124 is the amino acid sequence of the light chain variable framework donor X84343_VL.

[0148] SEQ ID NO: 125 is the amino acid sequence of a humanized heavy chain framework region.

[0149] SEQ ID NO: 126 is the amino acid sequence of a humanized heavy chain framework region.

[0150] SEQ ID NO: 127 is the amino acid sequence of a humanized heavy chain framework region.

[0151] 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.

[0154] SEQ ID NO: 131 is the amino acid sequence of a humanized light chain framework region.

[0155] SEQ ID NO: 132 is the amino acid sequence of a humanized light chain framework region.

[0156] SEQ ID NO: 133 is the amino acid sequence of a humanized heavy chain framework region.

[0157] SEQ ID NO: 134 is the amino acid sequence of a humanized heavy chain framework region.

[0158] SEQ ID NO: 135 is the amino acid sequence of a humanized heavy chain framework region.

[0159] SEQ ID NO: 136 is the amino acid sequence of a humanized heavy chain framework region.

[0160] SEQ ID NO: 137 is the amino acid sequence of a humanized heavy chain framework region.

[0161] SEQ ID NO: 138 is the amino acid sequence of a humanized heavy chain framework region.

[0162] SEQ ID NO: 139 is the amino acid sequence of CDRHl of humanized antibody 2120 version 3 (VH3).

[0163] SEQ ID NO: 140 is the amino acid sequence of CDRHl of humanized antibody 2120 version 4 (VH4).

[0164] SEQ ID NO: 141 is the amino acid sequence of CDRHl of humanized antibody 2120 version 5 (VH5). [0165] SEQ ID NO: 142 is the amino acid sequence of a humanized light chain framework region.

[0166] SEQ ID NO: 143 is the amino acid sequence of a humanized light chain framework region.

[0167] SEQ ID NO: 144 is the amino acid sequence of a humanized light chain framework region.

[0168] SEQ ID NO: 145 is the amino acid sequence of a humanized light chain framework region.

[0169] SEQ ID NO: 146 is the amino acid sequence of a humanized light chain framework region.

[0170] SEQ ID NO: 147 is the amino acid sequence of a humanized light chain framework region.

[0171] SEQ ID NO: 148 is the amino acid sequence of a humanized light chain framework region.

[0172] SEQ ID NO: 149 is the amino acid sequence of a humanized light chain framework region.

[0173] SEQ ID NO: 150 is the amino acid sequence of a humanized light chain framework region.

[0174] SEQ ID NO: 151 is the amino acid sequence of CDRHl of humanized antibody 2107 version 1 (VHl).

[0175] 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). [0177] SEQ ID NO: 154 is the amino acid sequence of a humanized light chain framework region.

[0178] SEQ ID NO: 155 is the amino acid sequence of a humanized heavy chain framework region.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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. [0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] SEQ ID NO: 168 is the amino acid sequence of a humanized 2120 light chain constant region, with Arginine at the N-terminus.

[0192] SEQ ID NO: 169 is the amino acid sequence of a humanized 2120 light chain constant region, without Arginine at the N-terminus.

[0193] SEQ ID NO: 170 is the amino acid sequence of a humanized 2120 heavy chain constant region.

[0194] SEQ ID NO: 171 is the amino acid sequence of a BIP version heavy chain Glm3 allotype constant region.

[0195] SEQ ID NO: 172 is the amino acid sequence of a BIP version heavy chain Glm3 allotype constant region.

[0196] 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).

[0197] 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). [0198] 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).

[0199] 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).

[0200] 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).

[0201] SEQ ID NO: 178 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 4B (VH4B).

[0202] SEQ ID NO: 179 is the amino acid sequence of the mature heavy chain variable region of humanized antibody 2107 version 5B (VH5B).

DEFINITIONS

[0203] 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.

[0204] 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. [0205] 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. Thus, for example, 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.

[0206] 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. Within light and heavy chains, 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. (See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7, incorporated by reference in its entirety for all purposes).

[0207] The mature variable regions of each light/heavy chain pair form the antibody binding site. Thus, 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. From N- terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of 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.

[0208] The term "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.

[0209] The term "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.

Immunol. 148: 1547-53 (1992)). In some bispecific antibodies, 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.

[0210] In some bispecific antibodies, 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). Such 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. Reduced affinity for the receptor resulted in a broader distributioin in the brain (see, e.g., Atwal. et al. Sci. Trans. Med. 3, 84ra43, 2011 ; Yu et al. Sci. Trans. Med. 3, 84ra44, 2011).

[0211] 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-Ig™) 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 Fab fragment; (5) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fc-region. 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).

[0212] The term "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).

[0213] 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.

[0214] The terms "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

CNS. "Inhibit" 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%. [0215] A "subject" includes a human or other mammalian subject that receives either prophylactic or therapeutic treatment.

[0216] For purposes of classifying amino acids substitutions as conservative or

nonconservative, 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.

[0217] 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.

[0218] Compositions or methods "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition that comprises antibody may contain the antibody alone or in combination with other ingredients.

[0219] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[0220] Unless otherwise apparent from the context, 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.

[0221] The terms "2120.4.19", "m2120", "mouse 2120" antibody refer 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.

DETAILED DESCRIPTION

I. GENERAL

[0222] 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.

II. TARGET MOLECULES

[0223] 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:

MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEVGSTAL

LKCGLS Q S QGNLSHVD WF S VHKEKRTLIFRVRQGQGQ SEPGE YEQ

RLSLQDRGATLALTQVTPQDERIFLCQGKRPRSQEYRIQLRVYKAP

EEPNIQVNPLGIPVNSKEPEEVATCVGRNGYPIPQVIWYKNGRPLK

EEKNRVHIQSSQTVESSGLYTLQSILKAQLVKEDKDAQFYCELNYR

LPSGNHMKESREVTVPVFYPTEKVWLEVEPVGMLKEGDRVEIRCL

ADGNPPPHFSISKQNPSTREAEEETTNDNGVLVLEPARKEHSGRYE

CQAWNLDTMISLLSEPQELLVNYVSDVRVSPAAPERQEGSSLTLTC

EAESSQDLEFQWLREETDQVLERGPVLQLHDLKREAGGGYRCVA

S WSIPGLNRTQLVKLAIFGPPWMAFKERKVWVKENMVLNLS CEA

SGHPRPΉSWNVNGTASEQDQDPQRVLSTLNVLVTPELLETGVECT

ASNDLGKNTSILFLELVNLTTLTPDSNTTTGLSTSTASPHTRANSTS

TERKLPEPESRGWIVAVrVCILVLAVLGAVLYFLYKKGKLPCRRS

GKQEITLPPSRKTELWEVKSDKLPEEMGLLQGS S GDKRAPGDQG

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.

[0225] 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.

[0226] 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 β-chain, and a laminin γ-chain. The laminin a4 chain is found in numerous laminin molecules including laminin 411 (laminin 8 or α4β1γ1); laminin 421 (laminin 9 or α4β2γ1), and laminin 423 (laminin 14 or α4β2γ3). There are two main isoforms of the human laminin a4-chain: GenBank Accession Nos. NP001098676 and NP001098677 (SEQ ID NOS:27 and 28, respectively). "Laminin 411" refers to a trimeric polypeptide complex made up of three polypeptide subunits or chains: a4-chain, a βΐ -chain, and a γΐ -chain.

III. ANTIBODIES

A. Antibody specificity

[0227] 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

14/656,596, each incorporated by reference in its entirety.

[0228] Some such antibodies are humanized. 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. Thus, 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. Similarly 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. Similarly 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. Other than nanobodies and dAbs, 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. The 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. [0229] Although 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.

36: 1079-1091, 1999; Tamura et al, Journal of Immunology, 164: 1432-1441, 2000).

[0230] In some antibodies only part of the CDRs, namely the subset of CDR residues required for binding, termed the SDRs, are needed to retain binding in a humanized antibody. 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. In such humanized antibodies at positions in which one or more donor CDR residues is absent or in which an entire donor CDR is omitted, 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.

[0231] 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. The disclosed forms including 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. [0232] 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) are disclosed in US 14/656,596 filed March 12, 2015 and herein. 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.

[0233] Unless otherwise apparent from the context, the following description includes the humanized antibodies disclosed in WO2014/039975 and the Q1E variants disclosed in

US14/656,596.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] Further provided are 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.

[0239] 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. [0240] Also provided are chimeric and veneered forms of the 2120.4.19 antibody. 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.

[0241] 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.

[0242] 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.

[0243] 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. Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332.

[0244] Chimeric, hu manized (including veneered) and human antibodies are typically produced by recombinant expression as described above. B. Methods of Screening Antibodies or other antagonists for Activity

[0245] 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.

[0246] For example, the activity of MCAM antibodies to inhibit the interaction between MCAM and the laminin a4 chain of laminin 411 can be screened as follows. 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. by fluorescence microscopy or flow cytometry; and (c) identifying said candidate antibody as an inhibitor the MCAM/laminin a4 interaction if the level of laminin a4 binding is lower in the presence than in the absence of the candidate antibody. 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.

[0247] Other methods of monitoring include fl uorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA).

[0248] 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.

[0249] 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).

[0250] The effects of MCAM blockade on disease progression can be examined in a therapeutic model of EAE in which TH17 polarization occurs in vivo. 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.

C. Selection of Constant Region

[0251] 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. For example, 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.

[0252] One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the 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. 279:6213, 2004). Exemplary 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

6,624,821). An 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. Optionally, 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). In some antibodies, 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. In some antibodies, a mutation at one or more of positions 318, 320, and 322 by EU numbering of human IgGl is used. In some antibodies, positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine, such as in SEQ ID NO: 172. In some antibodies, 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. The 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.

[0253] Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes. Thus, for example, 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. [0254] The invention further provides nucleic acids encoding any of the above constant regions. Optionally, such nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to the constant region.

D. Expression of Recombinant Antibodies

[0255] 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.

[0256] 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.

[0257] Mammalian cells can be used for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH

Publishers, NY, 1987). A number of 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).

[0258] Having introduced vector(s) encoding antibody heavy and light chains into cell culture, 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.

[0259] Once expressed, 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)).

[0260] Methodology for commercial production of antibodies including codon optimization, selection of promoters, tra nscription elements, and terminators, serum-free single cell cloning, cell banking, use of selection markers for amplification of copy number, CHO terminator, serum free single cell cloning, improvement of protein titers (see, e.g., US 5,786,464, US 5,888,809, US 6,063,598, US 6,114,148, US 7,569,339, WO2004/050884, WO2005/019442,

WO2008/012142, WO2008/012142, WO2008/107388, and WO2009/027471).

IV. OTHER ANTAGONISTS

[0261] In other embodiments, the antagonist is an alternative binding agent that binds to MCAM or laminin a4. These 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). Other 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. 282: 3196-3204); affitins, based on Sac7d from Sulfolobus acidolarius (Krehenbrink et al. (2008) J. Mol. Biol. 383: 1058-1068); affilins, based on human y-B-crystallin (Ebersbach et al. (2007) J. Mol. Biol. 372: 172-185); avimers, based on the A domains of membrane receptor proteins (Silverman et al. (2005) Biotechnol. 23: 1556-1561); cysteine-rich knottin peptides (Kolmar (2008) FEBS J. 275: 2684- 2690); and engineered Kunitz-type inhibitors (Nixon and Wood (2006) Curr. Opin. Drug.

Discov. Dev. 9: 261-268). For review, see Gebauer and Skerra (2009) Curr. Opin. Chem. Biol. 13: 245-255. In other embodiments, 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.

[0262] Libraries of peptides or other small molecules (kDa <600) 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

[0263] 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 (in this case human MCAM) 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.

VI. DISEASES AMENABLE TO TREATMENT

[0264] 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. Although practice of the methods is not dependent on understanding of mechanism, it is believed that in some methods 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. [0265] 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-Ιβ), 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.

[0266] Multiple sclerosis is a preferred disease for treatment in any of its at least four subtypes. Relapsing- remitting MS (RR-MS) is the most common form of MS and is characterized by clearly defined exacerbations/relapses (acute attacks) followed by partial or complete recovery. There is no disease progression between the relapse periods. Initially (at the time of diagnosis) 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.

[0267] Primary progressive (PP-MS) is continuous from the beginning without clear relapses. There can be plateaus (periods of stabilization). 10-15% of all MS subjects are in this group and it tends to occur in older aged individuals. The female to male ratio is equal in this group, unlike other forms where females predominant by about 2: 1. Also PP-MS tends to present with fewer cerebral MRI changes and more myelopathy/spinal cord related changes.

[0268] A secondary progressive form (SP-MS) starts as a RR-MS and later steady progression occurs with or without relapses. Approximately 50% of relapsing-remitting subjects progress to the secondary progressive form.

[0269] A progressive relapsing form (PR-MS), occurring in about 5% of individuals, is progressive from the onset with superimposed relapses (with or without recovery).

[0270] 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. 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. As well as therapeutically treating subjects having a diagnosis of MS by art- recognized criteria, 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. For example, 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. 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.

[0271] 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.

[0272] The present methods can 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. Examples of cancer 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.

[0273] 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

consequences. The cellular response if present can be B-cell or T-cell or both. 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.

[0274] Examples of 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 hepatitis, autoimmune oophoritis, fibromyalgia, polymyositis, dermatomyositis, ankylosing spondylitis, Takayashu arteritis, panniculitis, pemphigoid, vasculitis of unknown origin, anca negative vasculitis, anca positive vasculitis, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, scleroderma, systemic necrotizing vasculitis, Wegener's granulomatosis, CREST syndrome, antiphospholipid syndrome, Sjogren's syndrome, eosinophilic gastroenteritis, atypical topical dermatitis, cardiomyopathy, post-infectious syndromes, postinfectious endomyocarditis, celiac disease, multiple sclerosis, sarcoidosis, and psoriasis.

[0275] Giant cell arteritis (GCA) 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.

[0276] 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

multinucleated giant cells, a fragmented internal elastic lamina and intimal hyperplasia. 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 Matrigel™ matrix.

[0277] Polymyalgia rheumatica abbreviated as PMR, is a related condition to GCA, in which 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. [0278] PMR can occur concurrently or separately than GCA. Approximately 15% of patients with PMR develop giant cell arteritis (GCA), and 40-50% of patients with GCA have associated PMR. 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

inflammatory response in both GCA and PMR. A decrease in the level of circulating IL-6 correlates with remission of clinical symptoms. As with GCA, age of onset is 50 year or later with a mean of 72 years.

[0279] 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),

Lightheadedness or dizziness, fainting, headaches, memory problems, trouble thinking, shortness of breath, visual problems, high blood pressure, difference in blood pressure between arms, a difficult-to-find or absent pulse in the wrists; too few red blood cells (anemia), chest pain, abdominal pain. The condition is characterized by segmental and patchy granulomatous inflammation of the aorta and its major derivative branches. This inflammation leads to arterial stenosis, thrombosis, and aneurysms. There is also irregular fibrosis of the blood vessels due to chronic vasculitis, leading to sometimes massive intimal fibrosis (fibrosis of the inner section of the blood vessels). Prominent narrowing due to inflammation, granuloma, and fibrosis is often seen in arterial studies such as magnetic resonance angiography (MRA), computed tomography angiography (CTA), or arterial angiography (DSA). 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. Examples of cancer 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.

[0281] 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). 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. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, 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. [0282] 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. For some patients, 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.

[0283] 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. Intravenous

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. However, 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.

[0284] 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. [0285] 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. Thus, 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. For example, 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

fluorescence activated cell sorting. 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.

[0286] 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

maintenance regime. Alternatively or additionally, the loading and maintenance regimes can used the same antagonist but with different frequencies of administration, the frequency being lower for the maintenance regime. Alternatively, different antagonists can be used for the loading and maintenance regimes. For example, 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). Alternatively, 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. For example, the loading regime can be intravenous and the maintenance regime subcutaneous or vice versa.

[0287] 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. In an exemplary method, 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. Optionally, 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. Optionally, 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.

[0288] 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. For PRX003, 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. Optionally, the intervals are about once per week. Optionally, the dose is in the range of about 200 mg to about 250 mg, such as about 210 mg.

[0289] Conversely 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. Optionally, 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. Optionally the intervals are about once per week.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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; and 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; Avastin® against VEGF; or antibodies to the Epidermal Growth Factor (EGF) receptor such as Erbitux® (cetuximab) and Vectibix® (panitumumab).

[0294] 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

VII. FORMULATIONS

[0295] Pharmaceutical 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).

Pharmaceutical 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. For injection, 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). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0296] Formulations are provided 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. 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). The 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

concentrations in the reconstituted formulation.

[0297] Optionally, 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. Such a formulation should preferably stabilize the antibody throughout freezing, lyophilization, storage, and reconstitution as well as being suitable for parenteral administration. In an exemplary work flow, 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.

[0298] Some formulations include a bulking agent, which may or may not be the same as the sugar/polyol component. Typically, the formulations are sterile, for example, as accomplished by sterile filtration using a 0.2 μηι or a 0.22 μηι 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.

[0299] In some formulations, 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.

[0300] The formulation can comprise any of the antibodies described herein. In some formulations, 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. In such formulations, position 1 (Kabat numbering) of the mature heavy chain variable region can be occupied by E. In some formulations, 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. For example, in some formulations, 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.

[0301] In other formulations, the formulated antibody is an isolated anti-MCAM antibody described herein. In such a formulation, the isolated anti-MCAM antibody binds to human MCAM (SEQ ID NO: 11) at an epitope including amino acid residue 141.

[0302] 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. In some formulations, 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.

[0303] 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. For example, 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.

[0304] 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%. For example, 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%. Alternatively, in some formulations, 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.

[0305] 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. In one example, 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. In another example, 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.

[0306] 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. Other 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.

[0307] 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).

[0308] 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). [0309] 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).

[0310] 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.

[0311] Also provided are 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. More particularly, 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. 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.

VIII. MONITORING REGIMES

[0312] 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.

Although understanding of mechanism is not required for practice of the invention, it is believed that anti-MCAM antibodies reduce expression of MCAM on CD3+ T cells by inducing isotype switching such that more MCAM is expressed as a soluble extracellular domain. 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. In some methods, 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.

[0313] The level of change of MCAM if any responsive to treatment can be used as an indicator of whether to continue or change treatment. In general, patients experiencing a positive treatment response (i.e., decreased expression of MCAM on CD3+ cells or increased expression in blood) are likely to continue receiving treatment with anti-MCAM antibody. 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.

[0314] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

EXAMPLES

Materials and Methods

Antibody generation / characterization

[0315] For the generation of antibodies capable of binding to murine MCAM, 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 μg 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.

[0316] Supernatants from hybridomas that screened positive for MCAM specific antibodies were pre-incubated with fluorescently labeled mouse MCAM-Fc protein (5 μg/mL) for 30 minutes before addition to the laminin a4 expressing cell line WM2664 and neutralization of binding of the MCAM-Fc protein to the cell line was determined by flow cytometry.

[0317] For the generation of rat antibodies capable of binding to human MCAM, 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 μg 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.

[0318] For the generation of mouse antibodies capable of binding to human MCAM, 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 μg 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).

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.

[0319] Supernatants from hybridomas that screened positive for human MCAM specific antibodies were pre-incubated with fluorescently labeled hMCAM-Fc protein (5 μg/mL) for 30 minutes before addition to the laminin a4 expressing cell line WM2664 and neutralization of binding of the hMCAM-Fc protein to the cell line was determined by flow cytometry.

Nucleic Acid and protein manipulation

[0320] For determination of CDRs, total 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.

[0321] For determination of IL-17 concentrations in the supernatant, ELISA was performed using a commercial kit (R&D Systems).

Example 1. Generation of anti-MCAM monoclonal antibodies

[0322] 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.

[0323] Using these techniques, 823 independent mouse fusions clones were isolated and shown to express an antibody capable of binding to human MCAM. Additionally, 152 independent rat fusions clones were isolated and shown to express an antibody capable of binding to human MCAM.

[0324] Next, 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 μg/mL) was pre-incubated with isotype control antibody, or 10 μg/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.

Furthermore, parental CHO cells (CHOKl) or CHO cells transfected with a human MCAM gene were preincubated with CHO culture media (DMEM), recombinant laminin 411 (10 μg/ml), or recombinant laminin 511 (i.e., laminin 10 (α5β1γ1)) (10 μg/ml) at 37°C for 45 minutes. Cells were washed, and specific binding of laminin 411, but not laminin 511, to MCAM was detected with a pan-laminin antibody by flow cytometry. Pre-incubation of human MCAM transfected CHO cells with the anti-MCAM antibody (at 20 μg/ml), prior to laminin incubation, abolished the binding of human MCAM to laminin 411.

[0325] Using this technique, it was shown that 87 of the 823 independent mouse fusion clones and 26 of the 152 independent rat fusion clones described above expressed an antibody that was capable of blocking the interaction between human MCAM protein and its ligand, a-4 chain of laminin.

Example 2. Further characterization of anti-MCAM monoclonal antibodies

[0326] 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. First, 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.

[0327] Using the above described assay, six independent anti-human MCAM monoclonal antibody clones were identified as binding to human MCAM and having the greatest ability to block the interaction between human MCAM expressed on the surface of cells and its binding ligand, human laminin 411. These six anti-MCAM monoclonal antibody clones are herein referred to as (i) the mouse anti-human MCAM monoclonal clones 1174.1.3, 1414.1.2, 1415.1.1, and 1749.1.3, and (ii) the rat anti-human MCAM monoclonal antibody clones 2120.4.19 and 2107.4.10. 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. Moreover, experiments performed to determine the specific binding affinity of each monoclonal antibody demonstrated that each was capable of binding to human MCAM protein with high affinity (data not shown). As such, each of these specific monoclonal antibodies was very capable of binding to human MCAM and inhibiting the interaction of cell-expressed human MCAM with its laminin a4 binding ligand. In contrast, two control antibodies, a non-specific human IgGl antibody and a previously described, fully human anti-MCAM antibody referred to as ABX-MAl (e.g., see Mills et al, Cancer Res. 62:5106 (2002), and US Patent Nos. 6,924,360, 7,067,131, and

7,090,844) were both incapable of blocking the binding interaction between human MCAM and its laminin 411 counterpart. As such, the six specific monoclonal antibodies identified above possess the novel ability to both (i) bind with high affinity to human MCAM on the surface of living cells, and (ii) block the interaction of cell expressed human MCAM with a laminin protein comprising a laminin a4 polypeptide chain. Example 3. Generation of Humanized anti-MCAM 2120 Antibodies

[0328] Various humanized anti-MCAM antibodies were generated according to the following protocol. First, a three-dimensional molecular model of the variable regions was constructed using JN Biosciences' proprietary algorithm. Second, the framework amino acid residues important for the formation of the CDR structure or necessary for the binding to antigen were identified using the molecular model. In parallel, cDNA-derived human VH and VL amino acid sequences with high homology to the VH and VL amino acid sequences, respectively, were selected. Lastly, CDR sequences together with framework amino acid residues important for CDR structure or antigen binding were grafted from VH and VL into the corresponding selected human framework sequences.

[0329] 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.

[0330] 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. 3B (boxed residues between CDR-L1 and CDR-L2 (L46V and Y49F) and between CDR-L2 and CDR-L3 (V58I) affect CDR contact; boxed residues between CDR-L1 and CDR-L2 (L46V and Y49F) affect CDR contact; and L46V, Y49F, and V58I mutations combined with an additional mutation before CDR-L1 (T22N) affect antibody/antigen interaction).

[0331] Several versions of each chain were designed (standard vs. aggressive or

conservative). For those antibodies that contained N-deamidation motifs (NG), 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). [0332] The heavy and light chain plasmids were co-transfected into 293F cells with the FreeStyle™ 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.

[0333] 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.

[0334] 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.

[0335] Table 5 summarizes the design of humanized sequences.

Table 5

[0336] The heavy and light chain plasmids were co-transfected into 293F cells with the FreeStyle™ 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. [0337] 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:

[0338] 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.

[0339] The 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.

[0340] 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.

[0341] Table 6 provides the constructs used for transfection.

Table 6

Table 6

[0342] Table 7 describes the specific transfection experiments.

Table 7

[0343] 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 8

[0344] 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.

Table 9

[0345] Overall, the data demonstrates that the various 2120 humanized antibodies have a >5x reduction in affinity as measured by ForteBio, and most have a >2-3x reduction in apparent affinity and potency as measured by the competitive ELISA and laminin blocking assay, with the exception of VH5VL3 (G-A N-deamidation mutant VH/conservative VL), which had a <2x reduction in affinity and potency. [0346] Certain candidate antibodies were re-expressed and tested for their affinity by ForteBio and their IC50. The results are provided in Table 10 below.

Table 10

Example 4. Modification of humanized 2120 antibodies

[0347] Utilizing the DNA manipulation methods described above and according to Liu et al. JBC. 286: 11211-7, 2011, 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 and 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

+h2120VL3; h2120VH2.QlE +h2120VLl; h2120VH2.QlE +h2120VL2; h2120VH2.QlE +h2120VL3; h2120VH3.QlE +h2120VLl; h2120VH3.QlE +h2120VL2; h2120VH3.QlE +h2120VL3; h2120VH4.QlE +h2120VLl; h2120VH4.QlE +h2120VL2; h2120VH4.QlE +h2120VL3; h2120VH5.QlE +h2120VLl; h2120VH5.QlE +h2120VL2; and h2120VH5.QlE +h2120VL3.

Example 5. MCAM expression is dose- and time-dependently downregulated by PRX003 treatment in hMCAM transgenic mice

• Whole blood samples were analyzed using FACS with gating for NK1.1 and NK cells after single and the final of 4- weekly doses of PRX003 • In samples taken from one homozygous hMCAM transgenic mouse before or after (at 6 and 96 hours) injection of PRX003 (250 mg/kg), MCAM expression levels and the percentage of MCAM ⁺ cells were time-dependently decreased (FIGS. 5A-C)

• Expression and subsequent reduction of MCAM were measured on NK cells by flow cytometry and were monitored over 7 days. After a single PRX003 dose, a reduction in MCAM expression on NK cells was apparent within the first 6 hours, and maximum MCAM reduction (<30% of baseline) was achieved by 48 hours in all dose groups. MCAM expression returned as the concentration of PRX003 fell to subsaturation levels in serum and approached full recovery at the 10-mg/kg dose (n=32 mice/dose) (FIGS 6A & B)

• After the last of 4 weekly doses of PRX003, time to maximum reduction of MCAM was more rapid at the middle and high PRX003 doses, possibly because of sustained PRX003 exposure and continued partial downregulation through the 168-hour dosing interval with repeated administration. MCAM expression on NK cells varied normally over time in hMCAM transgenic mice treated with vehicle alone (n=32 mice/dose) (FIGS 6A & B)

Example 6. PRX003 selectively downregulates MCAM and does not affect the viability of

MCAM ⁺ cells

• Heterozygous hMCAM transgenic mice, with both mMCAM and hMCAM on the surfaces of NK cells, were used to assess whether the loss of MCAM expression resulted from deletion of the cells

• PRX003 resulted in hMCAM downregulation (FIG. 7 A) but did not affect the level of mMCAM (FIG. 7 B), indicating that these cells remain viable in circulation. Conversely, clone 15 downregulates mMCAM without affecting levels of hMCAM

Example 7. Dynamic changes in soluble MCAM induced by PRX003 are tightly coupled with cellular downregulation of MCAM

• After a single dose of PRX003, serum soluble MCAM increased in a dose-dependent fashion in magnitude and duration • Although baseline values were not determined, levels of soluble MCAM appeared to rise within 6 hours of dose administration and continued to increase approximately threefold to fourfold over mean vehicle control values at peak levels. This increase appeared to be reversible in nearly all dose groups and generally corresponded to PRX003 concentration and clearance from serum (FIG. 8)

Example 8. Anti-MCAM lowers inflammation and clinical score in a mouse model of

EAE, known to involve TH17 cells.

• Two days after the onset of EAE (12-14 days after immunization) and each day thereafter, the animals received either clone 15 neutralizing antibody or isotype control antibody. Mice treated with anti-MCAM had a less severe form of EAE (FIG 9A)

• Anti-MCAM treatment also reduced the numbers of infiltrating MCAM ⁺ T cells in the central nervous system (CNS) detected histologically (FIG. 9B) and MCAM ⁺ /CD3+ T cells isolated from the CNS and analyzed by flow cytometry (FIG 9C)

Example 9. Nonclinical safety in hMCAM transgenic mice

• 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). However, the off -target tissue cross-reactivity profile (e.g., PRX003 binding to vascular, adipocyte, and smooth muscle cells) in hMCAM transgenic mice appears similar to that seen in humans and allows for supplemental safety assessments of PRX003 for potential effects of binding off-target tissues with repeated administration o Weekly intravenous injection of PRX003 in hMCAM ^{+ +} transgenic mice once weekly for 4 weeks was well tolerated at all dose levels (10, 50, and 250 mg/kg/week) o Neither PRX003 -related change in any of the safety end points evaluated (clinical observation, clinical pathology, gross and microscopic pathology) nor target organ toxicity was observed o Although antidrug antibodies were detected in several repeat-dose animals, systemic exposure occurred throughout the dosing interval o Consequently, the no-observed-adverse-effect-level (NOAEL) for PRX003 in hMCAM ^+/+ transgenic mice was the highest dose level tested (250 mg/kg/week)

Example 10. Nonclinical safety in hMCAM cynomolgus monkeys

• A 3 -month GLP repeat-dose toxicology study in cynomolgus monkeys was conducted o Intravenous injection of PRX003 once every 2 weeks was well tolerated at all dose levels tested (10, 50 and 300 mg/kg/dose) o Dose-related increases in serum PRX003 exposure were maintained to the end of the treatment period o No PRX003 treatment-related adverse changes occurred in any of the study parameters evaluated o Consequently, the NOAEL for PRX003 in cynomolgus monkeys was the highest dose level tested (300 mg/kg/dose)

Example 11. Pharmacokinetic and Pharmacodynamic Study of PRX003 in Chimpanzee

[0348] The objective of this study was to model the pharmacokinetics and

pharmacodynamics relationship of 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) exposure with MCAM saturation on circulating T cells following single IV infusions of PRX003 (0, 1.5, 5, 15 and 50 mg/kg) in chimpanzees.

[0349] Method: A total of 9 chimpanzees (4 males and 5 females) were dosed on Day 1 via slow IV infusion of vehicle or PRX003.

Table 11

[0350] 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.

[0351] 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. [0352] 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. In selected earlier time points (predose, 504, 1008 hours) in this animal, also with no quantifiable PRX003 to interfere, no ADA titers were detected and there was no apparent meaningful effect on exposures or PK profile.

Table 12

ADA = anti-drug antibody; F = female; M = male; OD = optical density (1 : 50 screening dilution; cutpoint = 0.055)

* OD > 0.055

[0353] PRX003 Pharmacokinetics. Individual free serum PRX003 concentrations in chimpanzees were plotted over time in. Dose-dependent, non-linear kinetics was observed over the dose range tested in chimpanzees. Loss of exposure (BLQ = 0.08 μg/mL) occurred within 1-2 weeks following Γν administration of the low doses (1.5 and 5 mg/kg). Loss of exposure at 15 mg/kg occurred after 3 weeks while exposures at the 50 mg/kg high dose were maintained for 6-7 weeks. Consistent with the route of administration, peak concentrations of PRX003 were generally observed at the first sampling time point (5 minutes or 0.0833 hours) post end-of- infusion, except in one 15 mg/kg male (Tmax = 8 hours). Consistent with the observed nonlinear kinetics, there were generally greater than proportional increases in exposure with increasing dose, mainly with respect to AUC, where a nearly 200-fold increase was observed with a 33 -fold increase from the low (1.5 mg/kg) to high (50 mg/kg) dose. No apparent gender differences in systemic exposure were observed.

[0354] 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 μg/mL) that were well above experimental concentrations (1-3 μg/mL) required to saturate MCAM in chimpanzee whole blood, in vitro. Through the first 24 hours post EOI, a concurrent time-dependent loss of signal from a labeled non-competing anti-MCAM commercial antibody (P1H12) was observed, indicating a reduction of MCAM expression on the cell surface. For this reason, the expression and subsequent reduction of MCAM were measured independently on CD3+ memory T cells with a more sensitive FACS assay and monitored over time to establish the relationship of PRX003 exposure with reduction/recovery of MCAM expression as a potential PD biomarker of PRX003 activity.

[0355] Both time to maximum reduction in MCAM expression (<10% of baseline) on CD3+ memory T cells and duration of reduction were dose-dependent. Maximum MCAM reduction was achieved by 48 hours at the low dose compared to nearly 2 weeks at the high dose, with duration of effect lasting several days up to 4 weeks, respectively, before recovery back to baseline MCAM expression levels. The vehicle-treated control animal shows normal variability of MCAM expression on CD3+ memory T cells over time. As seen with occupancy and consistent with MCAM saturation levels in vitro, serum PRX003 levels above ~1 μg/mL were generally required for maximum reduction of MCAM on CD3+ memory T cells. Recovery of MCAM was observed as soon as serum PRX003 levels dropped below the predicted saturation concentration and took approximately 2 weeks to return to baseline MCAM expression levels in all dose groups. [0356] 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.

[0357] 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.

Example 12. Selection of Doses and Dosing Schedule

[0358] 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.

[0359] 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.

[0360] The no-observed-adverse-effect-level (NOAEL) in cynomolgus monkeys provides a dose multiple of 1000-fold over the starting dose of 0.3 mg/kg and 10-fold over the highest dose planned of 30 mg/kg PRX003 in the current study." Half-log doses were added in between to arrive at 1 mg/kg, 3 mg/kg and 10 mg/kg in addition to 0.3 mg/kg and 30 mg/kg. Example 13. Single Ascending Dose Study of PRX003 in Healthy Subjects

[0361] This single ascending dose study is to determine safety, tolerability,

pharmacokinetics, and immunogenicity of PRX003.

[0362] Method: Randomized, double-blind, placebo-controlled, single ascending dose- escalation study of PRX003 administered by intravenous infusion over approximately 60 minutes in healthy subjects. Patients enrolled in the trial met all of the following criteria:

1. Healthy subjects

2. Body mass index (B-MI) between 18-32 kg/m2 with a minimum weight of 45 kg

3. Female subjects must be surgically sterile or post-menopausal or if of child- bearing potential must use contraception

4. Male subjects and their partners of childbearing potential must use contraception [0363] Anyone of the following criteria excluded a patient from being enrolled in the trial:

1. Positive test for drug of abuse

2. Past or current history of alcohol abuse

3. Positive for TB, hepatitis B, hepatitis C or HIV infection

[0364] Based on preclinical models of markers of target engagement, doses were selected to evaluate a range of PD responses spanning from submaximal responses of short duration to maximal responses with durations that meet or exceed the target dose interval of 4 weeks. Five escalating dose cohorts received 0.3, 1.0, 3.0, 10 and 30 mg/kg of PRX003 or placebo and were monitored in an inpatient unit for 24 hours and by periodic follow-up for 12 weeks. This study provides data on the safety, tolerability, pharmacokinetics, pharmacodynamics and

immunogenicity of PRX003 in healthy human subjects. Example 14. Multiple Ascending Dose Study of PRX003 in Subjects with Psoriasis

[0365] This study determines safety, tolerability, pharmacokinetics, and immunogenicity of PRX003.

[0366] Methods: Randomized, Double-blind, Placebo-controlled, Multiple Ascending Dose Study of PRX003 Administered by Intravenous Infusion in Subjects with Psoriasis. Patients enrolled in the trial met all of the following criteria:

1. Male or female, 18 to 80 years of age (inclusive), body weight range of > 45 kg (99 lb) to < 120 kg (264 lb) and a body mass index (BMI) of 18 - 35 kg/m2

2. Provide written informed consent

3. PASI score of >12

4. Plaque psoriasis covering >10% of BSA

5. s-PGA score of 3 or 4

6. Able to perform all protocol-specified assessments and comply with the study visit schedule

7. Female subjects who are not postmenopausal or surgically sterile must use

physician approved contraception for at least 60 days prior to Baseline (Day

1 /Visit 2) to 12 weeks following the last study drug administration. Unless they are at least 2 years postmenopausal or surgically sterile, women must have a pregnancy test with follicle stimulating hormone (FSH) >40 R7/L and estradiol <20 pg/mL (unless on hormone-replacement therapy). Women of childbearing potential must be non lactating and have a negative serum pregnancy test (beta human chorionic gonadotropin [β HCG]) at Screening (Visit 1).

8. If male, must be surgically sterile or must agree to use physician-approved

contraception from Baseline (Day 1/Visit 2) to 12 weeks following the last study drug administration one of the following criteria excluded a patient from being enrolled in the trial: Presents with psoriasis that is predominantly guttate, erythrodermic, inverse, pustular or palmo-plantar, or an unstable form of psoriasis Receipt of any of the following within the specified time frame prior to Baseline (Day 1 /Visit 2): o Topical psoriasis treatments (other than low-potency topical

corticosteroids or emollients, which are permitted during the study) within 2 weeks o Systemic (nonbiologic) psoriasis treatments within 4 weeks or 5 half-lives (whichever is longer) o Biologic psoriasis treatments within 12 weeks or 5 half-lives (whichever is longer) o Drugs that appear to have a strong causal relationship to psoriasis (e.g., beta-blockers and lithium) within 4 weeks or 5 half-lives (whichever is longer) o Phototherapy within 4 weeks Participation in recreational sunbathing or use of a sun-bed (e.g., tanning salon) within 7 days prior to Baseline (Day 1) Any major medical illness or unstable medical condition that, in the opinion of the Investigator or Sponsor, may interfere with the subject's ability to comply with study procedures or abide by study restrictions, or with the ability to interpret safety data, including, but not limited to: o Within 5 years of Screening (Visit 1) o History of cancer with the exception of fully excised non-melanoma skin cancer o History of stroke o History of epilepsy or seizure disorder other than febrile seizures as a child, or any seizure or loss of consciousness o History of or active autoimmune disease (other than psoriasis or PsA) o Within 2 years of Screening (Visit 1) o Myocardial infarction o Clinically significant cardiovascular disease including any of the

following: unstable angina, decompensated congestive heart failure, clinically significant arrhythmias

0 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) Uncontrolled hypertension as indicated by a resting systolic BP >150 mmHg or diastolic BP >95 mmHg at Screening (Visit 1) or Baseline (Day 1/Visit 2) predose or a known history or documentation of uncontrolled hypertension on more than one occasion within 3 months prior to Baseline (Day 1/Visit 2) Clinically significant systemic infection (e.g., chronic or acute infection, urinary tract infection, upper respiratory infection) within 30 days of Baseline (Day 1/ Visit 2), or a history or presence of recurrent or chronic infection (e.g., viral infections [including hepatitis B or C, human immunodeficiency virus (HIV)], bacterial infections, systemic fungal infections, or syphilis)

History of any inflammatory bowel disease Any current psychiatric diagnosis according to Diagnostic and Statistical Manual of Mental Disorders IV Text Revision (DSM-IV-TR) that may interfere with the subject's ability to perform the study and all assessments (e.g., alcohol or drug- related abuse or alcohol dependence, or alcohol or drug- related dementia, major depression, developmental disability, schizophrenia, bipolar disorder). Note: 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. A positive tuberculosis skin test (TST) during Screening (Visit 1) or a positive Interferon Release Assay Test during Screening for subjects with a history of Bacillus Calmette Guerin (BCG) vaccination. Note: In the event a subject has had a TST within 3 months before Baseline (Day 1 /Visit 2), this does not need to be repeated during screening and the previous result can be carried forward and used in this study. Prior standard treatment for latent tuberculosis and prior exposure to tuberculosis with subsequent standard prophylactic treatment is allowed if recent (within < 30 days) negative chest X-ray. 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 ^χ ULN o Serum creatinine >133 μηιοι/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

Use of an investigational product or device or participation in a drug research study within a period of 30 days (or 5 half-lives of the drug, whichever is longer) prior to Screening (Visit 1); for investigational products or drug research studies relating to psoriasis or arthritis, the duration will be extended to 12 weeks (or 5 half-lives of the drug, whichever is longer) prior to Screening (Visit 1)

13. Allergy to any of the components of PRX003 such as histidine, sucrose and polysorbate 20

14. Receipt of any vaccine (with the exception of seasonal influenza) within 30 days prior to Screening (Visit 1)

15. Donation of >500 mL of blood within 3 months prior to Screening (Visit Treatment at 1, 3, 10, or 30 mg/kg was administered three times at intervals of 28 days.

Example 15. Formulation of the PRX003 Drug Product

[0368] Description: 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. 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.

[0369] Composition: The qualitative and quantitative composition of a unit vial of the PRX003 drug product (200 mg/vial) is described in Table 13.

Table 13

Table 13

[0370] 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

[0371] A single dose of PRX003 was reconstituted and administered to healthy adults at the following doses: 1 mg/kg, 3 mg/kg, 10 mg/kg or 30 mg/kg along with a placebo (0 mg/kg antibody). The study was a double-blind trial of 40 individuals of the characteristics shown in Table 14. PRX003 was administered by intravenous infusion.

Table 14

0.3 3.0 30 All

Characteristic Placebo 1.0 mg/kg 10 mg/kg

mg/kg mg/kg mg/kg Subjects

American Indian

0 (0) 0 (0) 2 (33.3) 0 (0) 0 (0) 0 (0) 2 (5) or Alaska Native

Asian+Black or

0 (0) 1 (16.7) 0 (0) 0 (0) 0 (0) 0 (0) 1 (2.5) African American

Asian+ White 0 (0) 0 (0) 0 (0) 1 (16.7) 0 (0) 0 (0) 1 (2.5)

70.5 75.3 73.9

Median body weight 66.8 (56- 71.4 (65.9- 74.8 (50- 77.3 (62.7- (48.9- (68.1- (48.9- (range), kg 98.1) 95.1) 88.1) 87.5)

102) 85.4) 102)

[0372] Serum was extracted from the patients 0-90 day(s) post infusion. Next, CD3+ lymphocytes were isolated from the extracted serum and cell surface expression levels of MCAM was determined by FACS analysis. 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.

[0373] 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.

[0374] FIG. 12 shows the mean concentration of PRX003 in μg/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. [0375] 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.

Table 15

[0376] FIG. 14A depicts the dose-dependent increase in peak MCAM expression with increasing concentrations of PRX003. In FIG. 14B, this effect is depicted by the area under the curve (AUC), where MCAM expression increases with increasing concentrations of PRX003.

[0377] Taken together, these results provide evidence that elevated levels PRX003 have a mobilizing effect of TH17 lymphocytes from the vascular wall and tissue resulting in initially elevated levels of MCAM on CD3+ lymphocytes following treatment. These results provide evidence to support a two part treatment regimen in which an initially high dose of PRX003 induces demargination of TH17 lymphocytes from the vascular cell wall and tissue, and subsequent and repeated treatments of low doses PRX003 maintain the demargination and prevent extravasation of TH17 lymphocytes from blood to tissue.

Example 16: PRX003 safety and tolerability in healthy adults.

[0378] After treatment as described in Example 14, patients were assessed by a qualified medical profession and questioned to determine if they experienced any adverse effects after their treatment with PRX003. In addition, other medical complications such as infusion-related or systemic hypersensitivity reactions, and toxicity were also investigated.

[0379] 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. There was no systemic hypersensitivity reactions or dose-limiting toxicity observed with PRX003 treatment.

[0380] Taken together, these results suggest that there were no serious or severe adverse effects experienced with PRX003 treatment.

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