Compositions and methods for the depletion of cells

Abstract

The invention provides compositions and methods useful for the depletion of cells, such as CD45+, CD135+, CD34+, CD90+, and/or CD110+ cells, and for the treatment of various hematopoietic diseases, metabolic disorders, cancers, and autoimmune diseases, among others. Described herein are antibodies, antigen-binding fragments, ligands, and conjugates thereof that can be applied to effect the treatment of these conditions, for instance, by depleting a population of CD45+, CD135+, CD34+, CD90+, or CD110+ cells in a patient, such as a human. The compositions and methods described herein can be used to treat a disorder directly, for instance, by depleting a population of CD45+, CD135+, CD34+, CD90+, or CD110+ cancer cells or autoimmune cells. The compositions and methods described herein can also be used to prepare a patient for hematopoietic stem cell transplant therapy and to improve the engraftment of hematopoietic stem cell transplants by selectively depleting endogenous hematopoietic stem cells prior to the transplant procedure.

What is claimed is: 
     
         1 . A method of depleting a population of CD45+ cells in a human patient, the method comprising administering to the human patient an effective amount of a conjugate represented by the formula Ab-Am, wherein Ab is an antibody or antigen-binding fragment thereof that specifically binds to human CD45 and Am is an amatoxin, wherein the antibody or antigen-binding fragment thereof comprises an Fc domain and is internalized by a CD45+ cell, wherein the amatoxin is conjugated to the antibody or antigen binding fragment thereof by way a cysteine residue in the Fc domain of the antibody or antigen-binding fragment thereof, and wherein the amatoxin is represented by Formula (I) 
       
         
           
           
               
               
           
         
         wherein R 1  is H, OH, OR A , or OR C ; 
         R 2  is H, OH, OR B , or OR C ; 
         R A  and R B , together with the oxygen atoms to which they are bound, combine to form an optionally substituted 5-membered heterocyclolalkyl group; 
         R 3  is H, R C , or R D ; 
         R 4  is H, O, OR C , OR D , R C , or R D ; 
         R 5  is H, O, OR C , OR D , R C , or R D ; 
         R 6  is H, O, OR C , OR D , R C , or R D ; 
         R 7  is H, O, OR C , OR D , R C , or R D ; 
         R 8  is OH, NH 2 , OR C , OR D , NHR C , or NR C R D ; 
         R 9  is H, OH, OR C , or OR D ; 
         X is —S—, —S(O)—, or —SO 2 —; 
         R C  is -L-Z; 
         R D  is optionally substituted alkyl (e.g., C 1 -C 6  alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6  heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6  alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6  heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6  alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6  heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; 
         L is a linker, such as optionally substituted alkylene (e.g., C 1 -C 6  alkylene), optionally substituted heteroalkylene (C 1 -C 6  heteroalkylene), optionally substituted alkenylene (e.g., C 2 -C 6  alkenylene), optionally substituted heteroalkenylene (e.g., C 2 -C 6  heteroalkenylene), optionally substituted alkynylene (e.g., C 2 -C 6  alkynylene), optionally substituted heteroalkynylene (e.g., C 2 -C 6  heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene; and 
         Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody, antigen-binding fragment thereof. 
       
     
     
         2 . The method of  claim 1 , wherein the cysteine residue is introduced by way of a mutation in the Fc domain of the antibody or antigen-binding fragment thereof. 
     
     
         3 . The method of  claim 2 , wherein the cysteine residue is selected from the group consisting of Cys118, Cys239, and Cys265. 
     
     
         4 . The method of  claim 1 , wherein the cysteine residue is naturally occurring in the Fc domain of the antibody or antigen-binding fragment thereof. 
     
     
         5 . The method of  claim 4 , wherein the Fc domain is an IgG Fc domain and the cysteine residue is selected from the group consisting of Cys261, Csy321, Cys367, and Cys425. 
     
     
         6 . The method of  claim 1 , wherein R 1  is H, OH, or OR A ;
 R 2  is H, OH, or OR B ;   R A  and R B , together with the oxygen atoms to which they are bound, combine to form:   
       
         
           
           
               
               
           
         
         R 3 , R 4 , R 6 , and R 7  are each H; 
         R 5  is OR C ; 
         R 8  is OH or NH 2 ; and 
         R 9  is H or OH. 
       
     
     
         7 . The method of  claim 1 , wherein R 1  and R 2  are each independently H or OH;
 R 3  is R C ;   R 4 , R 6 , and R 7  are each H;   R 5  is H, OH, or OC 1 -C 6  alkyl;   R 8  is OH or NH 2 ; and   R 9  is H or OH.   
     
     
         8 . The method of  claim 1 , wherein R 1  and R 2  are each independently H or OH;
 R 3 , R 6 , and R 7  are each H;   R 4  is OR C , or R C ;   R 5  is H, OH, or OC 1 -C 6  alkyl;   R 8  is OH or NH 2 ; and   R 9  is H or OH.   
     
     
         9 . The method of  claim 1 , wherein R 1  and R 2  are each independently H or OH;
 R 3 , R 6 , and R 7  are each H;   R 4  and R 5  are each independently H or OH;   R 8  is OR C  or NHR C ; and   R 9  is H or OH.   
     
     
         10 . The method of  claim 1 , wherein the antibody or antigen-binding fragment thereof binds CD45 with a K d  of from about 0.1 μM to about 1 μM. 
     
     
         11 . The method of  claim 1 , wherein the antibody or antigen-binding fragment thereof binds CD45 with a k on  of from about 9×10 −2  M −1  s −1  to about 1×10 2  M −1  s −1 . 
     
     
         12 . The method of  claim 1 , wherein the antibody or antigen-binding fragment thereof competitively inhibits the binding of CD45 to a second antibody or antigen binding fragment thereof, wherein the second antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):
 a. a CDR-H1 having the amino acid sequence SYAMS (SEQ ID NO: 16);   b. a CDR-H2 having the amino acid sequence AISGSGGSTFYADSVRG (SEQ ID NO: 17);   c. a CDR-H3 having the amino acid sequence EVMGPIFFDY (SEQ ID NO: 18);   d. a CDR-L1 having the amino acid sequence RASQSIISSALA (SEQ ID NO: 19);   e. a CDR-L2 having the amino acid sequence GASSRAT (SEQ ID NO: 20); and   f. a CDR-L3 having the amino acid sequence QQYGSTPLT (SEQ ID NO: 21).   
     
     
         13 . The method of  claim 1 , wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′) 2  molecule, and a tandem di-scFV. 
     
     
         14 . A conjugate represented by the formula Ab-Am, wherein Ab is an antibody or antigen-binding fragment thereof that specifically binds to human CD45, and Am is an amatoxin, wherein the antibody or antigen-binding fragment thereof comprises an Fc domain and is internalized by a CD45+ cell, wherein the amatoxin is conjugated to the antibody or antigen binding fragment thereof by way of a cysteine residue in the Fc domain of the antibody or antigen-binding fragment thereof, and wherein the amatoxin is represented by Formula (I) 
       
         
           
           
               
               
           
         
         wherein R 1  is H, OH, OR A , or OR C ; 
         R 2  is H, OH, OR B , or OR C ; 
         R A  and R B , together with the oxygen atoms to which they are bound, combine to form an optionally substituted 5-membered heterocyclolalkyl group; 
         R 3  is H, R C , or R D ; 
         R 4  is H, O, OR C , OR D , R C , or R D ; 
         R 5  is H, O, OR C , OR D , R C , or R D ; 
         R 6  is H, O, OR C , OR D , R C , or R D ; 
         R 7  is H, O, OR C , OR D , R C , or R D ; 
         R 8  is OH, NH 2 , OR C , OR D , NHR C , or NR C R D ; 
         R 9  is H, OH, OR C , or OR D ; 
         X is —S—, —S(O)—, or —SO 2 —; 
         R C  is -L-Z; 
         R D  is optionally substituted alkyl (e.g., C 1 -C 6  alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6  heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6  alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6  heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6  alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6  heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; 
         L is a linker, such as optionally substituted alkylene (e.g., C 1 -C 6  alkylene), optionally substituted heteroalkylene (C 1 -C 6  heteroalkylene), optionally substituted alkenylene (e.g., C 2 -C 6  alkenylene), optionally substituted heteroalkenylene (e.g., C 2 -C 6  heteroalkenylene), optionally substituted alkynylene (e.g., C 2 -C 6  alkynylene), optionally substituted heteroalkynylene (e.g., C 2 -C 6  heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene; and 
         Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody, antigen-binding fragment thereof. 
       
     
     
         15 . The conjugate of  claim 14 , wherein the cysteine residue is introduced by way of a mutation in the Fc domain of the antibody or antigen-binding fragment thereof. 
     
     
         16 . The conjugate of  claim 15 , wherein the cysteine residue is selected from the group consisting of Cys118, Cys239, and Cys265. 
     
     
         17 . The conjugate of  claim 14 , wherein the cysteine residue is naturally occurring in the Fc domain of the antibody or antigen-binding fragment thereof. 
     
     
         18 . The conjugate of  claim 17 , wherein the Fc domain is an IgG Fc domain and the cysteine residue is selected from the group consisting of Cys261, Csy321, Cys367, and Cys425. 
     
     
         19 . The conjugate of  claim 14 , wherein R 1  is H, OH, or OR A ;
 R 2  is H, OH, or OR B ;   R A  and R B , together with the oxygen atoms to which they are bound, combine to form:   
       
         
           
           
               
               
           
         
         R 3 , R 4 , R 6 , and R 7  are each H; 
         R 5  is OR C ; 
         R 8  is OH or NH 2 ; and 
         R 9  is H or OH. 
       
     
     
         20 . The conjugate of  claim 14 , wherein R 1  and R 2  are each independently H or OH;
 R 3  is R C ;   R 4 , R 6 , and R 7  are each H;   R 5  is H, OH, or OC 1 -C 6  alkyl;   R 8  is OH or NH 2 ; and   R 9  is H or OH.   
     
     
         21 . The conjugate of  claim 14 , wherein R 1  and R 2  are each independently H or OH;
 R 3 , R 6 , and R 7  are each H;   R 4  is OR C , or R C ;   R 5  is H, OH, or OC 1 -C 6  alkyl;   R 8  is OH or NH 2 ; and   R 9  is H or OH.   
     
     
         22 . The conjugate of  claim 14 , wherein R 1  and R 2  are each independently H or OH;
 R 3 , R 6 , and R 7  are each H;   R 4  and R 5  are each independently H or OH;   R 8  is OR C  or NHR C ; and   R 9  is H or OH.   
     
     
         23 . The conjugate of  claim 14 , wherein the antibody or antigen-binding fragment thereof binds CD45 with a K d  of from about 0.1 μM to about 1 μM. 
     
     
         24 . The conjugate of  claim 14 , wherein the antibody or antigen-binding fragment thereof binds CD45 with a k on  of from about 9×10 −2  M −1  s −1  to about 1×10 2  M −1  s −1 . 
     
     
         25 . The conjugate of  claim 14 , wherein the antibody or antigen-binding fragment thereof competitively inhibits the binding of CD45 to a second antibody or antigen binding fragment thereof, wherein the second antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):
 a. a CDR-H1 having the amino acid sequence SYAMS (SEQ ID NO: 16);   b. a CDR-H2 having the amino acid sequence AISGSGGSTFYADSVRG (SEQ ID NO: 17);   c. a CDR-H3 having the amino acid sequence EVMGPIFFDY (SEQ ID NO: 18);   d. a CDR-L1 having the amino acid sequence RASQSIISSALA (SEQ ID NO: 19);   e. a CDR-L2 having the amino acid sequence GASSRAT (SEQ ID NO: 20); and   f. a CDR-L3 having the amino acid sequence QQYGSTPLT (SEQ ID NO: 21).   
     
     
         26 . The conjugate of  claim 14 , wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′) 2  molecule, and a tandem di-scFV.