PSMA formulations and uses thereof

Abstract

The invention includes stable multimeric, particularly dimeric, forms of PSMA protein, compositions and kits containing dimeric PSMA protein as well as methods of producing, purifying and using these compositions. Such methods include methods for eliciting or enhancing an immune response to cells expressing PSMA, including methods of producing antibodies to dimeric PSMA, as well as methods of treating cancer, such as prostate cancer.

1 . A composition comprising isolated PSMA protein, wherein at least 5% of the isolated PSMA protein is an isolated PSMA protein multimer.  
     
     
         2 . The composition of  claim 1 , wherein the isolated PSMA protein multimer is an isolated PSMA protein dimer.  
     
     
         3 - 12 . (canceled)  
     
     
         13 . The composition of  claim 1 , wherein the composition further comprises at least 0.25 molar equivalents of metal ion to PSMA protein.  
     
     
         14 - 16 . (canceled)  
     
     
         17 . The composition of  claim 1 , wherein the composition is in a liquid or lyophilized form.  
     
     
         18 . The composition of  claim 1 , wherein the composition further comprises an adjuvant.  
     
     
         19 . The composition of  claim 18 , wherein the adjuvant is alum; monophosphoryl lipid A; a saponin; QS-7; QS-17; QS-18; QS-21; a saponin fraction; a saponin-based adjuvant; SaponImmune™; PolysaccImmune™; SynthImmune™; an immunostimulatory oligonucleotide; incomplete Freund's adjuvant; complete Freund's adjuvant; montanide; MONTANIDE ISA51; MONTANIDE ISA720; vitamin E, a water-in-oil emulsions prepared from a biodegradable oil; Quil A; a micellular mixture of Quil A and cholesterol known as immunostimulating complexes (ISCOMS); a MPL and mycobacterial cell wall skeleton combination; ENHANZYN™; RC-529; RC-552; CRL-1005, L-121, alpha-galactosylceramide; a composition of biodegradable particles composed of poly-lactide-co-glycolide (PLG); a composition of aluminum or iron oxide beads or a combination thereof.  
     
     
         20 - 21 . (canceled)  
     
     
         22 . The composition of  claim 1 , wherein the composition further comprises a cancer therapeutic agent  
     
     
         23 - 24 . (canceled)  
     
     
         25 . The composition of  claim 1 , wherein the composition further comprises a cytokine.  
     
     
         26 . (canceled)  
     
     
         27 . The composition of  claim 1 , wherein the composition is free of chelating agents.  
     
     
         28 . The composition of  claim 1 , wherein the composition further comprises at least one buffer.  
     
     
         29 . (canceled)  
     
     
         30 . The composition of  claim 1 , wherein the composition further comprises a free amino acid, wherein the free amino acid is naturally occurring or non-naturally occurring.  
     
     
         31 - 32 . (canceled)  
     
     
         33 . The composition of  claim 1 , wherein the composition further comprises a surfactant.  
     
     
         34 . (canceled)  
     
     
         35 . The composition of  claim 1 , wherein the composition further comprises a cryoprotectant, an antioxidant, a preservative or a combination thereof.  
     
     
         36 - 46 . (canceled)  
     
     
         47 . A composition comprising isolated multimeric PSMA protein, wherein the composition comprises less than 35% of a monomeric PSMA protein.  
     
     
         48 . The composition of  claim 47 , wherein the isolated multimeric PSMA protein is an isolated dimeric PSMA protein.  
     
     
         49 - 51 . (canceled)  
     
     
         52 . A composition comprising PSMA protein in a solution that promotes or preserves multimeric association of PSMA protein.  
     
     
         53 . The composition of  claim 52 , wherein the solution that promotes or preserves multimeric association of PSMA protein is a solution that promotes or preserves dimeric association of PSMA protein.  
     
     
         54 . The composition of  claim 52 , wherein the solution that promotes or preserves dimeric association of PSMA protein has a pH that ranges from 4 to 8.  
     
     
         55 - 57 . (canceled)  
     
     
         58 . The composition of  claim 52 , wherein the solution that promotes or preserves dimeric association of PSMA protein comprises a salt.  
     
     
         59 - 63 . (canceled)  
     
     
         64 . The composition of  claim 58 , wherein the composition further comprises an adjuvant.  
     
     
         65 . (canceled)  
     
     
         66 . The composition of  claim 52 , wherein the solution that promotes or preserves dimeric association of PSMA protein comprises metal ions.  
     
     
         67 . The composition of  claim 66 , wherein the metal ions are zinc ions, calcium ions, magnesium ions, cobalt ions, manganese ions or a combination thereof.  
     
     
         68 . The composition of  claim 67 , wherein the metal ions are zinc ions and calcium ions.  
     
     
         69 . (canceled)  
     
     
         70 . The composition of  claim 68 , wherein the zinc ions are present at a concentration that is lower than the concentration of the calcium ions.  
     
     
         71 - 74 . (canceled)  
     
     
         75 . The composition of  claim 67 , wherein the metal ions are magnesium ions and calcium ions.  
     
     
         76 . The composition of  claim 52 , wherein the solution that promotes or preserves dimeric association of PSMA protein is free of chelating agents.  
     
     
         77 - 99 . (canceled)  
     
     
         100 . A composition comprising isolated PSMA protein in a solution that promotes or preserves dimeric association of PSMA protein wherein the solution comprises: 
 (a) 5 to 20 mM of sodium phosphate, sodium acetate or a combination thereof,    (b) 100 to 300 mM sodium chloride or sodium sulfate, and    (c) 0.1 to 2 mM of at least one metal ion.    
     
     
         101 . The composition of  claim 100 , wherein the solution has a pH in the range of 4 to 8.  
     
     
         102 - 103 . (canceled)  
     
     
         104 . The composition of  claim 101 , wherein the composition further comprises an adjuvant.  
     
     
         105 . The composition of  claim 104 , wherein the adjuvant is alum or a saponin-based adjuvant.  
     
     
         106 . (canceled)  
     
     
         107 . The composition of  claim 100 , wherein the composition further comprises a cancer therapeutic agent.  
     
     
         108 - 110 . (canceled)  
     
     
         111 . A composition comprising isolated PSMA protein in a solution that promotes or preserves dimeric association of PSMA protein wherein the solution comprises: 
 (a) 1.47 mM potassium phosphate, monobasic,    (b) 8.1 mM sodium phosphate, dibasic,    (c) 2.68 mM potassium chloride,    (d) 0.14 M sodium chloride,    (e) 0.9 mM calcium chloride, and    (f) 0.49 mM magnesium chloride;    and wherein the solution has a pH of 7.0.    
     
     
         112 . The composition of  claim 111 , wherein the isolated PSMA protein is at a concentration of between 0.2 mg/mL and 10 mg/mL.  
     
     
         113 - 115 . (canceled)  
     
     
         116 . The composition of  claim 111 , wherein the composition further comprises an adjuvant.  
     
     
         117 . The composition of  claim 116 , wherein the adjuvant is a saponin-based adjuvant.  
     
     
         118 - 121 . (canceled)  
     
     
         122 . A method of promoting or preserving dimeric association of PSMA protein in a solution comprising: 
 obtaining a solution of PSMA protein, and    adjusting the pH to be in the range of 4 to 8.    
     
     
         123 . The method of  claim 122 , wherein the pH is adjusted to be in the range of 5 to 7.  
     
     
         124 - 125 . (canceled)  
     
     
         126 . A method of processing a PSMA protein comprising: 
 contacting the PSMA protein in a solution with a first agent that promotes or preserves dimeric association of PSMA protein in an amount effective to promote or preserve PSMA protein dimer formation.    
     
     
         127 . The method of  claim 126 , wherein the amount effective to promote or preserve PSMA protein dimer formation is enough to promote or maintain at least 5%, 25%, 50%, 75% or 95% of the PSMA protein in PSMA dimer form.  
     
     
         128 . The method of  claim 126 , wherein the first agent that promotes or preserves dimeric association of PSMA protein is a salt, metal ion or a pH adjusting agent.  
     
     
         129 - 132 . (canceled)  
     
     
         133 . The method of  claim 126 , further comprising combining the PSMA protein solution with an adjuvant or diluent.  
     
     
         134 . The method of  claim 133 , wherein the adjuvant or diluent is combined with the PSMA protein in an amount to dilute the salt concentration to 100 mM to 300 mM.  
     
     
         135 - 143 . (canceled)  
     
     
         144 . The method of  claim 128 , wherein the pH of the solution is adjusted to be in the range of 4 to 8.  
     
     
         145 - 147 . (canceled)  
     
     
         148 . The method of  claim 128 , wherein the method further comprises contacting the PSMA protein with a second agent that promotes or preserves dimeric association of PSMA protein, and wherein the second agent is different than the first agent.  
     
     
         149 . The method of  claim 148 , wherein the second agent that promotes or preserves dimeric association of PSMA protein is a metal ion, salt or pH adjusting agent.  
     
     
         150 - 152 . (canceled)  
     
     
         153 . The method of  claim 148 , wherein the pH of the solution is adjusted to be in the range of 4 to 8.  
     
     
         154 - 156 . (canceled)  
     
     
         157 . A method of purifying a sample containing PSMA protein comprising: 
 subjecting the sample containing PSMA to chromatography in the presence of an agent that preserves or promotes the dimeric association of PSMA.    
     
     
         158 . The method of  claim 157 , wherein the agent that promotes or preserves the dimeric association of PSMA is a metal ion, a salt or a solution with a pH in the range of 4 to 8 or a combination thereof.  
     
     
         159 . The method of  claim 158 , wherein the metal ion is a zinc ion, calcium ion, magnesium ion, cobalt ion, manganese ion or a combination thereof.  
     
     
         160 . The method of  claim 159 , wherein the metal ion is a combination of calcium ion and magnesium ion.  
     
     
         161 - 168 . (canceled)  
     
     
         169 . A method of purifying a sample containing PSMA protein comprising: 
 applying the sample to a first column,    washing the first column with a first wash solution containing salt and metal ions,    and collecting the PSMA protein that elutes from the first column.    
     
     
         170 . The method of  claim 169 , wherein the metal ions are zinc ions, calcium ions, magnesium ions, cobalt ions, manganese ions or a combination thereof.  
     
     
         171 . The method of  claim 170 , wherein the metal ions are calcium and magnesium ions.  
     
     
         172 - 173 . (canceled)  
     
     
         174 . The method of  claim 169 , wherein the cationic component of the salt is sodium, potassium, ammonium, magnesium, calcium, zinc or a combination thereof, and wherein the anionic component of the salt is chloride, sulfate acetate or a combination thereof.  
     
     
         175 . The method of  claim 174 , wherein the salt is ammonium sulfate at a saturation of no more than 35% in the wash solution.  
     
     
         176 . The method of  claim 169 , further comprising dialyzing or diafiltering the eluted PSMA protein with a first salt solution at a pH in the range of 6 to 7.5 to yield a dialyzed or diafiltrated solution containing PSMA protein.  
     
     
         177 . The method of  claim 176 , wherein the first salt solution has a salt concentration of at least 5 mM.  
     
     
         178 . (canceled)  
     
     
         179 . The method of  claim 169 , further comprising: 
 loading the eluted PSMA protein, dialyzed or diafiltrated solution containing PSMA protein onto a second column,    washing the second column with a second salt solution,    and collecting the PSMA eluted by the second salt solution.    
     
     
         180 . The method of  claim 179 , wherein the second salt solution has a salt concentration of 100 mM to 2 M.  
     
     
         181 . (canceled)  
     
     
         182 . The method of  claim 179 , wherein the second salt solution has a pH in the range of 6 to 7.5.  
     
     
         183 . The method of  claim 179 , further comprising 
 dialyzing or diafiltrating the PSMA eluted by the second salt solution with a metal ion solution,    applying the dialyzed or diafiltrated PSMA eluted by the second salt solution onto a third column,    washing the third column with a second wash solution containing salt and metal ions and collecting the PSMA eluted.    
     
     
         184 . The method of  claim 183 , wherein the pH is maintained in the range of 6 to 7.5 through all of the purification steps.  
     
     
         185 . The method of  claim 183 , further comprising separating the different forms of PSMA protein, wherein the different forms of PSMA protein are monomeric, dimeric or other multimeric forms of PSMA.  
     
     
         186 . (canceled)  
     
     
         187 . A method of identifying an agent which promotes or preserves dimeric association of PSMA protein comprising: 
 determining the amount of a form of PSMA protein in a sample prior to exposure to a candidate agent,    exposing the sample to the candidate agent,    determining the amount of the form of PSMA protein in the sample after the exposure, and    comparing the amount of the form of PSMA protein in the sample prior to and after the exposure.    
     
     
         188 . (canceled)  
     
     
         189 . A method of treating a subject to elicit or enhance an immune response to cells in the subject expressing PSMA, comprising administering to the subject an effective amount of the composition of any one of claims  1 ,  47 ,  52 ,  100  and  111 .  
     
     
         190 . The method of  claim 189 , wherein the expressed PSMA is expressed on the cell surface.  
     
     
         191 . The method of  claim 189 , wherein the method further comprises administering one or more booster doses of a composition comprising PSMA protein.  
     
     
         192 . The method of  claim 191 , wherein the composition comprising PSMA protein is a composition of PSMA protein dimer.  
     
     
         193 . The method of  claim 191 , wherein the booster dose composition further comprises an adjuvant.  
     
     
         194 - 196 . (canceled)  
     
     
         197 . The method of  claim 189 , wherein the subject has cancer or has been treated for cancer.  
     
     
         198 . (canceled)  
     
     
         199 . The method of  claim 197 , wherein the subject has prostate cancer.  
     
     
         200 . A method of eliciting an immune response, comprising administering to a subject an effective amount of the composition of any one of claims  1 ,  47 ,  52 ,  100  and  111 .  
     
     
         201 . The method of  claim 200 , wherein the method further comprises administering one or more booster doses of a composition comprising PSMA protein.  
     
     
         202 . The method of  claim 201 , wherein the composition comprising PSMA protein is a composition PSMA protein dimer.  
     
     
         203 . The method of  claim 201 , wherein the booster dose composition further comprises an adjuvant.  
     
     
         204 - 206 . (canceled)  
     
     
         207 . A kit which comprises the composition of any one of claims  1 ,  47 ,  52 ,  100  and  111  and instructions for use.  
     
     
         208 . A kit which comprises the composition of any one of claims  1 ,  47 ,  52 ,  100  and  111 , an adjuvant and instructions for mixing.  
     
     
         209 - 210 . (canceled)  
     
     
         211 . A kit which comprises the composition of any one of claims  1 ,  47 ,  52 ,  100  and  111 , a diluent and instructions for mixing.  
     
     
         212 - 213 . (canceled)  
     
     
         214 . A pharmaceutical composition comprising the composition of any one of the compositions of claims  1 ,  47 ,  52 ,  100  and  111 , and a pharmaceutically acceptable carrier.  
     
     
         215 . A method of treating prostate cancer in a subject, comprising: 
 administering to the subject a therapeutically effective amount of a composition comprising isolated PSMA protein in a solution that promotes or preserves dimeric association of the PSMA protein, wherein the composition is effective in treating prostate cancer.    
     
     
         216 . The method of  claim 215 , wherein the composition that is administered further comprises an adjuvant.  
     
     
         217 . The method of  claim 216 , wherein the adjuvant is a saponin-based adjuvant.  
     
     
         218 . The method of  claim 215 , wherein the method further comprises administering to the subject a conventional prostate cancer therapy.  
     
     
         219 . (canceled)  
     
     
         220 . A method of inhibiting metastasis in a subject with prostate cancer, comprising: 
 administering to the subject a therapeutically effective amount of a composition comprising isolated PSMA protein in a solution that promotes or preserves dimeric association of the PSMA protein, wherein the composition is effective in inhibiting metastasis.    
     
     
         221 . The method of  claim 220 , wherein the composition that is administered further comprises an adjuvant.  
     
     
         222 . The method of  claim 221 , wherein the adjuvant is a saponin-based adjuvant.  
     
     
         223 . The method of  claim 220 , wherein the method further comprises administering to the subject a conventional prostate cancer therapy.  
     
     
         224 . (canceled)