PSMA Targeting Trispecific Proteins and Methods of Use

Abstract

Provided herein are prostate specific membrane antigen (PSMA) targeting trispecific proteins comprising a domain binding to CD3, a half-life extension domain, and a domain binding to PSMA. Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such PSMA targeting trispecific proteins. Also disclosed are methods of using the disclosed PSMA targeting trispecific proteins in the prevention, and/or treatment diseases, conditions and disorders.

1 . A trispecific protein targeting an epitope within a human prostate specific membrane antigen (PSMA), wherein said protein comprises
 (a) a first domain (A) which is a single chain variable fragment (scFv) that specifically binds to a human CD3, comprising a heavy chain complementarity determining region (HC CDR) 1, a HC CDR2, a HC CDR3, a light chain complementarity determining region (LC CDR) 1, a LC CDR 2, and a LC CDR 3, wherein the HC CDR1 comprises the amino acid sequence of SEQ ID NO: 32; the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 42; the HC CDR3 comprises the amino acid sequence of SEQ ID NO: 44, the LC CDR1 comprises the amino acid sequence of SEQ ID NO: 54; the LC CDR2 comprises the amino acid sequence of SEQ ID NO: 79; and the LC CDR3 comprises the amino acid sequence of SEQ ID NO: 80;   (b) a second domain (B) which is a single domain antibody (sdAb) comprising a CDR1, a CDR2 and a CDR3, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 100; the CDR2 comprises the amino acid sequence of SEQ ID NO: 104; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 109; and   (c) a third domain (C) which is a sdAb that specifically binds to the human PSMA, comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 114; the CDR2 comprises the amino acid sequence of SEQ ID NO: 126; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 124; and   wherein the human PSMA is a 100 kD Type II membrane glycoprotein expressed in prostate tissue.   
     
     
         2 . (canceled) 
     
     
         3 . The trispecific protein of  claim 1 , wherein the first domain comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         4 . The PSMA targeting trispecific protein of  claim 1 , wherein the first domain is humanized or human. 
     
     
         5 . The PSMA targeting trispecific protein of  claim 1 , wherein the first domain has a K D  binding of 150 nM or less to CD3 on CD3 expressing cells. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The PSMA targeting trispecific protein of  claim 1 , wherein the second domain comprises the amino acid sequence of SEQ ID NO: 94. 
     
     
         9 . The PSMA targeting trispecific protein of  claim 1 , wherein the third domain comprises the amino acid sequence of SEQ ID No: 130 or 140. 
     
     
         10 . The PSMA targeting trispecific protein of  claim 1 , wherein the domains are linked by linkers L1 and L2, and wherein linkers L1 and L2 are each independently selected from (GS)n (SEQ ID NO: 153), (GGS)n (SEQ ID NO: 154), (GGGS)n (SEQ ID NO: 155), (GGSG)n (SEQ ID NO: 156), (GGSGG)n (SEQ ID NO: 157), or (GGGGS)n (SEQ ID NO: 158), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 
     
     
         11 . The PSMA targeting trispecific protein of  claim 10 , wherein linkers L1 and L2 are each independently (GGGGS) 4  (SEQ ID NO: 159) or (GGGGS) 3  (SEQ ID NO: 160). 
     
     
         12 . The PSMA targeting trispecific protein of  claim 10 , wherein the domains are linked in the order H 2 N-(C)-L1-(B)-L2-(A)-COOH. 
     
     
         13 . The PSMA targeting trispecific protein of  claim 1 , wherein the protein is less than about 80 kDa. 
     
     
         14 . The PSMA targeting trispecific protein of  claim 1 , wherein the protein is about 50 to about 75 kDa. 
     
     
         15 . The PSMA targeting trispecific protein of  claim 1 , wherein the protein is less than about 60 kDa. 
     
     
         16 . The PSMA targeting trispecific protein of  claim 1 , wherein the protein has an elimination half-time of at least about 50 hours. 
     
     
         17 . The PSMA targeting trispecific protein of  claim 1 , wherein the protein has an elimination half-time of at least about 100 hours. 
     
     
         18 . The PSMA targeting trispecific protein of  claim 1 , wherein the protein has increased tissue penetration as compared to an IgG to the same PSMA. 
     
     
         19 . The PSMA targeting trispecific protein of  claim 1 , wherein the protein comprises an amino acid sequence of SEQ ID NO: 147 or 152. 
     
     
         20 . (canceled) 
     
     
         21 . A pharmaceutical composition comprising (i) the PSMA targeting trispecific protein according to  claim 1 , and (ii) a pharmaceutically acceptable carrier. 
     
     
         22 - 28 . (canceled) 
     
     
         29 . A method of treating prostate cancer expressing a PSMA, the method comprising administration of an effective amount of the pharmaceutical composition of  claim 21  to a subject having said prostate cancer, wherein said human PSMA is a 100 kD type II membrane glycoprotein expressed in prostate tissue. 
     
     
         30 . (canceled)