Prolactin receptor binding proteins and uses thereof

Abstract

The present invention encompasses PRLR binding proteins. Specifically, the invention relates to antibodies that are chimeric, CDR grafted and humanized antibodies. Preferred antibodies have high affinity for hPRLR and neutralize hPRLR activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. Methods of making and methods of using the antibodies of the invention are also provided. The antibodies, or antibody portions, of the invention are useful for detecting hPRLR and for inhibiting hPRLR activity, e.g., in a human subject suffering from a disorder in which hPRLR activity is detrimental. Also included in the invention are anti-PRLR antibody drug conjugates (ADCs).

We claim: 
     
         1 . A binding protein comprising an antigen binding domain, said binding protein capable of binding prolactin receptor (PRLR), said antigen binding domain comprising at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID Nos:97, 98, 99, 100, 101, 102, 151, 152, 40-42, 46, 47, 49-51, 56-58, 62, 63, 65-67, 71-73, 77, 79-81, 85-87, 92-94, 149 and 150. 
     
     
         2 . The binding protein according to  claim 1 , wherein said binding protein comprises at least 3 CDRs. 
     
     
         3 . The binding protein according to  claim 2 , wherein said at least 3 CDRs are
 (a) a heavy chain variable domain CDR set (CDR1, CDR2, and CDR3) selected from the group consisting of SEQ ID NOs: 40, 41, and 42; SEQ ID NOs: 46, 47, and 42; SEQ ID NOs: 56, 57, and 58; SEQ ID NOs: 62, 63, and 58; SEQ ID NOs: 71, 72, and 73; SEQ ID NOs: 71, 77, and 73; SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 149, 150, and 87; and/or   (b) a light chain variable domain CDR set (CDR1, CDR2, and CDR3) selected from the group consisting of SEQ ID NOs: 49, 50, and 51; SEQ ID NOs: 65, 66, and 67; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 92, 93, and 94.   
     
     
         4 . The binding protein according to  claim 1 , further comprising a human acceptor framework, for example, comprising an amino acid sequence selected from the group consisting of SEQ ID Nos:14-38 or 158. 
     
     
         5 . The binding protein according to  claim 4 , wherein said human acceptor framework comprises at least one Framework Region amino acid substitution, wherein the amino acid sequence of the framework is at least 65% identical to the sequence of said human acceptor framework and comprises at least 70 amino acid residues identical to said human acceptor framework-, for example, wherein said human acceptor framework comprises at least one Framework Region amino acid substitution at a key residue, said key residue selected from the group consisting of:
 a residue adjacent to a CDR;   a glycosylation site residue;   a rare residue;   a residue capable of interacting with human PRLR;   a residue capable of interacting with a CDR;   a canonical residue;   a contact residue between heavy chain variable region and light chain variable region;   a residue within a Vernier zone; and   a residue in a region that overlaps between a Chothia-defined variable heavy chain CDR1 and a Kabat-defined first heavy chain framework.   
     
     
         6 . The binding protein according to  claim 1 , wherein said binding protein comprises at least one variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 39; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 55; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 70; SEQ ID NO: 74; SEQ ID NO: 75; SEQ ID NO: 76; SEQ ID NO: 84; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90; SEQ ID NO: 121; SEQ ID NO: 122 and SEQ ID NO: 123, and/or at least one variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 48; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 64; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 78; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 91; SEQ ID NO: 95; and SEQ ID NO: 96. 
     
     
         7 . The binding protein according to  claim 1 , wherein said binding protein comprises a heavy chain sequence and a light chain sequence selected from the group consisting of:
 (a) a heavy chain having the amino acid sequence of SEQ ID NO: 124; and a light chain having the amino acid sequence of SEQ ID NO: 125;   (b) a heavy chain having the amino acid sequence of SEQ ID NO: 124; and a light chain having the amino acid sequence of SEQ ID NO: 126;   (c) a heavy chain having the amino acid sequence of SEQ ID NO: 124; and a light chain having the amino acid sequence of SEQ ID NO: 127;   (d) a heavy chain having the amino acid sequence of SEQ ID NO: 124; and a light chain having the amino acid sequence of SEQ ID NO: 128;   (e) a heavy chain having the amino acid sequence of SEQ ID NO: 129; and a light chain having the amino acid sequence of SEQ ID NO: 125;   (f) a heavy chain having the amino acid sequence of SEQ ID NO: 129; and a light chain having the amino acid sequence of SEQ ID NO: 126;   (g) a heavy chain having the amino acid sequence of SEQ ID NO: 129; and a light chain having the amino acid sequence of SEQ ID NO: 127;   (h) a heavy chain having the amino acid sequence of SEQ ID NO: 129; and a light chain having the amino acid sequence of SEQ ID NO: 128;   (i) a heavy chain having the amino acid sequence of SEQ ID NO: 130; and a light chain having the amino acid sequence of SEQ ID NO: 125;   (j) a heavy chain having the amino acid sequence of SEQ ID NO: 130; and a light chain having the amino acid sequence of SEQ ID NO: 126;   (k) a heavy chain having the amino acid sequence of SEQ ID NO: 130; and a light chain having the amino acid sequence of SEQ ID NO: 127;   (l) a heavy chain having the amino acid sequence of SEQ ID NO: 130; and a light chain having the amino acid sequence of SEQ ID NO: 128;   (m) a heavy chain having the amino acid sequence of SEQ ID NO: 131; and a light chain having the amino acid sequence of SEQ ID NO: 132;   (n) a heavy chain having the amino acid sequence of SEQ ID NO: 131; and a light chain having the amino acid sequence of SEQ ID NO: 133;   (o) a heavy chain having the amino acid sequence of SEQ ID NO: 131; and a light chain having the amino acid sequence of SEQ ID NO: 134;   (p) a heavy chain having the amino acid sequence of SEQ ID NO: 135; and a light chain having the amino acid sequence of SEQ ID NO: 132;   (q) a heavy chain having the amino acid sequence of SEQ ID NO: 135; and a light chain having the amino acid sequence of SEQ ID NO: 133;   (r) a heavy chain having the amino acid sequence of SEQ ID NO: 135; and a light chain having the amino acid sequence of SEQ ID NO: 134;   (s) a heavy chain having the amino acid sequence of SEQ ID NO: 136; and a light chain having the amino acid sequence of SEQ ID NO: 132;   (t) a heavy chain having the amino acid sequence of SEQ ID NO: 136; and a light chain having the amino acid sequence of SEQ ID NO: 133;   (u) a heavy chain having the amino acid sequence of SEQ ID NO: 136; and a light chain having the amino acid sequence of SEQ ID NO: 134;   (v) a heavy chain having the amino acid sequence of SEQ ID NO: 137; and a light chain having the amino acid sequence of SEQ ID NO: 138;   (w) a heavy chain having the amino acid sequence of SEQ ID NO: 137; and a light chain having the amino acid sequence of SEQ ID NO: 139;   (x) a heavy chain having the amino acid sequence of SEQ ID NO: 137; and a light chain having the amino acid sequence of SEQ ID NO: 140;   (y) a heavy chain having the amino acid sequence of SEQ ID NO: 141; and a light chain having the amino acid sequence of SEQ ID NO: 138;   (z) a heavy chain having the amino acid sequence of SEQ ID NO: 141; and a light chain having the amino acid sequence of SEQ ID NO: 139;   (aa) a heavy chain having the amino acid sequence of SEQ ID NO: 141; and a light chain having the amino acid sequence of SEQ ID NO: 140;   (bb) a heavy chain having the amino acid sequence of SEQ ID NO: 142; and a light chain having the amino acid sequence of SEQ ID NO: 138;   (cc) a heavy chain having the amino acid sequence of SEQ ID NO: 142; and a light chain having the amino acid sequence of SEQ ID NO: 139;   (dd) a heavy chain having the amino acid sequence of SEQ ID NO: 142; and a light chain having the amino acid sequence of SEQ ID NO: 140;   (ee) a heavy chain having the amino acid sequence of SEQ ID NO: 143; and a light chain having the amino acid sequence of SEQ ID NO: 144;   (ff) a heavy chain having the amino acid sequence of SEQ ID NO: 143; and a light chain having the amino acid sequence of SEQ ID NO: 145;   (gg) a heavy chain having the amino acid sequence of SEQ ID NO: 143; and a light chain having the amino acid sequence of SEQ ID NO: 146;   (hh) a heavy chain having the amino acid sequence of SEQ ID NO: 147; and a light chain having the amino acid sequence of SEQ ID NO: 144;   (ii) a heavy chain having the amino acid sequence of SEQ ID NO: 147; and a light chain having the amino acid sequence of SEQ ID NO: 145;   (jj) a heavy chain having the amino acid sequence of SEQ ID NO: 147; and a light chain having the amino acid sequence of SEQ ID NO: 146;   (kk) a heavy chain having the amino acid sequence of SEQ ID NO: 148; and a light chain having the amino acid sequence of SEQ ID NO: 144;   (ll) a heavy chain having the amino acid sequence of SEQ ID NO: 148; and a light chain having the amino acid sequence of SEQ ID NO: 145;   (mm) a heavy chain having the amino acid sequence of SEQ ID NO: 148; and a light chain having the amino acid sequence of SEQ ID NO: 146;   (nn) a heavy chain having the amino acid sequence of SEQ ID NO: 153; and a light chain having the amino acid sequence of SEQ ID NO: 139;   (oo) a heavy chain having the amino acid sequence of SEQ ID NO: 154; and a light chain having the amino acid sequence of SEQ ID NO: 139; and   (pp) a heavy chain having the amino acid sequence of SEQ ID NO: 155; and a light chain having the amino acid sequence of SEQ ID NO: 139.   
     
     
         8 . The binding protein according to  claim 1 , wherein the binding protein
 (a) inhibits binding of prolactin to PRLR;   (b) is capable of modulating a biological function of PRLR; and/or   (c) is capable of neutralizing PRLR.   
     
     
         9 . The binding protein according to  claim 1 , wherein said binding protein has
 (a) an on rate constant (K on ) to PRLR selected from the group consisting of: at least about 10 2 M −1 s −1 ; at least about 10 3 M −1 s −1 ; at least about 10 4 M −1 s −1 ; at least about 10 5 M −1 s −1 ; and at least about 10 6 M −1 s −1 ; as measured by surface plasmon resonance;   (b) an off rate constant (K off ) to PRLR selected from the group consisting of: at most about 10 −3 s −1 ; at most about 10 −4 s −1 ; at most about 10 −5 s −1 ; and at most about 10 −6 s −1 , as measured by surface plasmon resonance; and/or   (c) a dissociation constant (K D ) to PRLR selected from the group consisting of: at most about 10 −7  M; at most about 10 −8  M; at most about 10 −9  M; at most about 10 −10  M; at most about 10 −11  M; at most about 10 −12  M; and at most 10 −13 M.   
     
     
         10 . A binding protein capable of binding PRLR that competes with an antibody comprising a heavy chain variable domain and a light chain variable domain selected from the group consisting of:
 (1) a variable heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 39; SEQ ID NO: 43; SEQ ID NO: 44 and SEQ ID NO: 45; and a variable light chain having an amino acid sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO: 53 and SEQ ID NO:54;   (2) a variable heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 55; SEQ ID NO: 59; SEQ ID NO: 60 and SEQ ID NO: 61; and a variable light chain having an amino acid sequence selected from the group consisting of SEQ ID NO:64, SEQ ID NO:68 and SEQ ID NO: 69;   (3) a variable heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 84; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90; SEQ ID NO: 121; SEQ ID NO: 122; and SEQ ID NO: 123; and a variable light chain having an amino acid sequence selected from the group consisting of SEQ ID NO:91, SEQ ID NO:95 and SEQ ID NO:96;   (4) the variable heavy chain amino acid sequence set forth in SEQ ID NO:112 and the variable light chain amino acid sequence set forth in SEQ ID NO:103;   (5) the variable heavy chain amino acid sequence set forth in SEQ ID NO:113 and the variable light chain amino acid sequence set forth in SEQ ID NO:104;   (6) the variable heavy chain amino acid sequence set forth in SEQ ID NO:114 and the variable light chain amino acid sequence set forth in SEQ ID NO:105;   (7) the variable heavy chain amino acid sequence set forth in SEQ ID NO:116 and the variable light chain amino acid sequence set forth in SEQ ID NO:107;   (8) the variable heavy chain amino acid sequence set forth in SEQ ID NO:117 and the variable light chain amino acid sequence set forth in SEQ ID NO:108;   (9) the variable heavy chain amino acid sequence set forth in SEQ ID NO:118 and the variable light chain amino acid sequence set forth in SEQ ID NO:109;   (10) the variable heavy chain amino acid sequence set forth in SEQ ID NO:119 and the variable light chain amino acid sequence set forth in SEQ ID NO:110; and   (11) the variable heavy chain amino acid sequence set forth in SEQ ID NO:120 and the variable light chain amino acid sequence set forth in SEQ ID NO:111.   
     
     
         11 . The binding protein of  claim 1 , wherein the binding protein is an antibody. 
     
     
         12 . An antibody construct comprising the binding protein of  claim 1 , said antibody construct further comprising a linker polypeptide or an immunoglobulin constant domain. 
     
     
         13 . The antibody construct according to  claim 12 , wherein said binding protein is selected from the group consisting of:
 an immunoglobulin molecule, a disulfide linked Fv,   a monoclonal antibody, a scFv,   a chimeric antibody, a single domain antibody,   a CDR-grafted antibody, a diabody,   a humanized antibody, a multispecific antibody,   a Fab,   a Fab′,   a F(ab′)2,   a Fv,   a dual specific antibody,   a bispecific antibody.   
     
     
         14 . An antibody conjugate comprising the antibody construct of  claim 12 , said antibody conjugate further comprising
 (a) an agent selected from the group consisting of: an immunoadhension molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent;   (b) an imaging agent selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin;   (c) a radiolabel selected from the group consisting of:  3 H,  14 C,  35 S,  90 Y,  99 Tc,  111 In,  125 I,  131 I,  177 Lu,  166 Ho, or  153 Sm;   (d) a therapeutic or cytotoxic agent selected from the group consisting of: an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent; or   (e) an anti-mitotic agent is selected from the group consisting of a dolastatin, an auristatin, a maytansinoid, a plant alkaloid, a taxane, and a  vinca  alkaloid.   
     
     
         15 . The binding protein according to  claim 1 , wherein said binding protein is a crystallized binding protein. 
     
     
         16 . The antibody construct according to  claim 12 , wherein said antibody construct is a crystallized antibody construct. 
     
     
         17 . The antibody construct according to  claim 16 , wherein said crystallized antibody construct
 (a) is a carrier-free pharmaceutical controlled release crystallized antibody construct;   (b) has a greater half life in vivo than the soluble counterpart of said antibody construct; and/or   (c) retains biological activity.   
     
     
         18 . An isolated nucleic acid encoding a binding protein amino acid sequence of  claim 12 . 
     
     
         19 . An isolated nucleic acid encoding an antibody construct amino acid sequence of  claim 1 . 
     
     
         20 . A vector comprising an isolated nucleic acid according to  claim 1 , optionally selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ. 
     
     
         21 . A host cell comprising a vector according to  claim 20 . 
     
     
         22 . The host cell according to  claim 21 , wherein said host cell is a prokaryotic cell or a eukaryotic cell. 
     
     
         23 . The host cell according to  claim 22 , wherein
 (a) said prokaryotic host cell is  E. Coli.;      (b) said eukaryotic cell is selected from the group consisting of a protist cell, an animal cell, a plant cell and a fungal cell;   (c) said eukaryotic cell is an animal cell selected from the group consisting of a mammalian cell, an avian cell, and an insect cell; or   (d) said host cell is selected from the group consisting of a CHO cell, a COS cell, a yeast cell, an insect Sf9 cell, and  Saccharomyces cerevisiae.      
     
     
         24 . A method of producing a protein capable of binding PRLR, comprising culturing a host cell of  claim 21  in culture medium under conditions sufficient to produce a binding protein capable of binding PRLR. 
     
     
         25 . A protein produced according to the method of  claim 26 . 
     
     
         26 . A composition for the release of a binding protein said composition comprising:
 (a) a formulation, wherein said formulation comprises a crystallized binding protein, according to  claim 15 , and an ingredient, optionally selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol; and   (b) at least one polymeric carrier, optionally selected from one or more of the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo) phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.   
     
     
         27 . A method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition according to  claim 26 . 
     
     
         28 . A pharmaceutical composition comprising the binding protein of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         29 . The pharmaceutical composition of  claim 28 , further comprising at least one additional therapeutic agent for treating a disorder in which PRLR activity is detrimental; optionally selected from the group consisting of: therapeutic agent, imaging agent, cytotoxic agent, angiogenesis inhibitors; kinase inhibitors; co-stimulation molecule blockers; adhesion molecule blockers; anti-cytokine antibody or functional fragment thereof; methotrexate; cyclosporin; rapamycin; FK506; detectable label or reporter; a TNF antagonist; an anti-rheumatic; a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an oral steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist. 
     
     
         30 . A method for reducing human PRLR activity comprising contacting human PRLR with the binding protein of  claim 1 , such that human PRLR activity is reduced. 
     
     
         31 . An anti-PRLR antibody, or antigen binding fragment thereof, that specifically competes with an anti-PRLR binding protein of  claim 1 , wherein said competition can be detected in a competitive binding assay using said antibody, the human PRLR polypeptide, and the anti-PRLR binding protein. 
     
     
         32 . An anti-PRLR Antibody Drug Conjugate (ADC) comprising an anti-PRLR antibody, or antigen-binding fragment thereof, and at least one drug, wherein the antibody, or antigen-binding portion thereof, comprises at least 3 CDRs. 
     
     
         33 . The anti-PRLR Antibody Drug Conjugate (ADC) of  claim 32 , wherein the antibody, or antigen-binding portion thereof, comprises at least 3 CDRs selected from a heavy chain variable domain CDR set (CDR1, CDR2, and CDR3) consisting of SEQ ID NOs: 40, 41, and 42; SEQ ID NOs: 46, 47, and 42; SEQ ID NOs: 56, 57, and 58; SEQ ID NOs: 62, 63, and 58; SEQ ID NOs: 71, 72, and 73; SEQ ID NOs: 71, 77, and 73; SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 149, 150, and 87; and/or at least 3 CDRs selected from a light chain variable domain CDR set (CDR1, CDR2, and CDR3) consisting of SEQ ID NOs: 49, 50, and 51; SEQ ID NOs: 65, 66, and 67; SEQ ID NOs: 79, 80, and 81; and SEQ ID NOs: 92, 93, and 94. 
     
     
         34 . The ADC of  claim 32 , wherein the drug is selected from the group consisting of a mitotic inhibitor, an antitumor antibiotic, an immunomodulating agent, a vector for gene therapy, an alkylating agent, an antiangiogenic agent, an antimetabolite, a boron-containing agent, a chemoprotective agent, a hormone, an antihormone agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a topoisomerase inhibitor, a tyrosine kinase inhibitor, and a radiosensitizer. 
     
     
         35 . A pharmaceutical composition comprising the ADC of  claim 32 . 
     
     
         36 . A method of treating cancer in a subject in need thereof, said method comprising administering the ADC of  claim 32 , such that the subject is treated; optionally wherein the cancer is selected from the group consisting of melanoma, endometrial cancer, lymphoma, breast cancer, ovarian cancer, renal carcinoma, gastrointestinal cancer, colon cancer, lung cancer, pancreatic cancer, and prostate cancer.