Reducing imunogenicities of immunoglobulins by framework-patching

Abstract

Framework (FR)-patching is a novel approach to modify immunoglobulin for reducing potential immunogenicity without significant alterations in specificity and affinity. Unlike previous described methods of humanization, which graft CDRs from a donor onto the frameworks of a single acceptor immunoglobulin, we patch segments of framework (FR1, FR2, FR3 and FR4), or FRs, to replace the corresponding FRs of the parent immunoglobulin. Free assortment of these FRs from different immunoglobulins and from different species can be mixed and matched into forming the final immunoglobulin chain. A set of criteria in the choice of these FRs to minimize or eliminate the need to reintroduce framework amino acids from the parent immunoglobulin for patching is described. The approach gives greater flexibility in the choice of framework sequences, minimizes the need to include parent framework amino acids, and, most importantly, reduces the chances of creating new T- and B-cell epitopes in the resultant immunoglobulin.

1 . A framework (FR)-patched immunoglobulin in which one or more framework sequences, defined as FR1, FR2, FR3 and FR4, of at least one heavy or light chain of a parent immunoglobulin are replaced by corresponding framework sequences from a donor immunoglobulin which is either an immunoglobulin from a different species or a different immunoglobulin from the same species, and such FR-patched immunoglobulin binds specifically to an antigen with affinity within 3-fold of that of the parent immunoglobulin, with the proviso that not all of the replaced FR1, FR2 and FR3 of the FR-patched immunoglobulin heavy chain are from the same framework of a single immunoglobulin heavy chain and not all the replaced FR1, FR2 and FR3 of the FR-patched immunoglobulin light chain are from the same framework of a single immunoglobulin light chain.  
     
     
         2 . A FR-patched immunoglobulin according to  claim 1 , in which the particular FR chosen for patching or replacing each corresponding FR in the parent immunoglobulin: 
 a. exhibits the highest degree of homology, or at least 60%, to the corresponding parent FR;    b. exhibits identical sequence homology to the corresponding parent FR at the three amino acids immediately adjacent to the flanking CDR's; and    c. contains identical amino acid to the corresponding parent FR at positions known to be close to, or have interactions with the CDR's/antigen binding site, as evaluated by computer modeling, crystal structure, or published information.    
     
     
         3 . A FR-patched immunoglobulin according to  claim 1 , in which the particular FR chosen for patching or replacing each corresponding FR in the parent immunoglobulin: 
 a. exhibits the highest degree of homology, or at least 60%, to the corresponding parent FR;    b. exhibits identical sequence homology to the corresponding parent FR at the four amino acids immediately adjacent to the flanking CDR's; and    c. contains identical amino acid to the corresponding parent FR at positions known to be close to, or have interactions with the CDR's/antigen binding site, as evaluated by computer modeling, crystal structure, or published information.    
     
     
         4 . A FR-patched immunoglobulin according to  claim 1 , in which the particular FR chosen for patching each corresponding FR in the parent immunoglobulin: 
 a. exhibits the highest degree of homology, or at least 60%, to the corresponding parent FR;    b. exhibits the highest degree of sequence homology to the corresponding parent FR, preferably identical, or contains conservatively similar amino acids at the four amino acids immediately adjacent to the flanking CDR's; and    c. contains identical, or conservatively similar amino acids to the corresponding parent FR at positions known to be close to, or have interactions with the CDR's/antigen binding site, as evaluated by computer modeling, crystal structure, or published information.    
     
     
         5 . A FR-patched immunoglobulin according to  claim 1 , in which the particular FR chosen for patching or replacing each corresponding FR in the parent immunoglobulin: 
 a. exhibits the highest degree of homology, or at least 60%, to the corresponding parent FR;    b. exhibits the highest degree of sequence homology to the corresponding parent FR, preferably identical, or contains conservatively similar amino acids at the three amino acids immediately adjacent to the flanking CDR's; and    c. contains identical, or conservatively similar amino acids to the corresponding parent FR at positions known to be close to, or have interactions with the CDR's/antigen binding site, as evaluated by computer modeling, crystal structure, or published information.    
     
     
         6 . A FR-patched immunoglobulin according to  claim 1 , wherein one or more amino acids from said parent immunoglobulin replace corresponding amino acids in one or more of said donor immunoglobulin sequences.  
     
     
         7 . A FR-patched immunoglobulin according to  claim 1 , which specifically binds to an antigen with an affinity of between 10 7  M −1  and 10 11 M −1 .  
     
     
         8 . A FR-patched immunoglobulin according to  claim 1 , which specifically binds to an antigen with an affinity of between 10 8  M −1  and 10 10  M −1 .  
     
     
         9 . A FR-patched immunoglobulin according to  claim 1 , which is substantially pure.  
     
     
         10 . A pharmaceutical composition comprising a FR-patched immunoglobulin according to  claim 1 , in a pharmaceutically acceptable carrier.