Binding molecules

Abstract

The present invention relates to the manufacture of a diverse repertoire of functional heavy chain-only antibodies that undergo affinity maturation, and uses thereof. The invention also relates to the manufacture and use of a diverse repertoire of class-specific heavy chain-only antibodies and to the manufacture and use of multivalent polypeptide complexes with antibody heavy chain functionality, preferably antibody heavy chain binding functionality, constant region effector activity and, optionally, additional effector functions. The present invention also relates to a method of generation of fully functional heavy chain-only antibodies in transgenic mice in response to antigen challenge. In particular, the present invention relates to a method for the generation of human antigen-specific, high affinity, heavy chain-only antibodies of any class, or mixture of classes and the isolation and expression of fully functional VH antigen-binding domains.

1 . A method for the production of a V H  heavy chain-only antibody in a non-human transgenic mammal comprising the step of expressing a heterologous V H  heavy chain locus in that mammal, wherein the V H  heavy chain locus comprises one or more V gene segments selected or engineered to show improved solubility characteristics and a heavy chain constant region which does not encode a C H 1 domain and which locus, when expressed, is capable of forming heavy chain-only antibodies of defined class or classes. 
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of  claim 1  where each V gene segment is derived from a human. 
     
     
         6 - 10 . (canceled) 
     
     
         11 . The method of  claim 1  wherein the heavy chain constant region comprises one or more of the following heavy chain constant regions: Cα, Cε, Cδ, Cγ Cμ, and isotypes thereof. 
     
     
         12 - 17 . (canceled) 
     
     
         18 . The method of claim  12  wherein the D and J gene segments are derived from a human. 
     
     
         19 - 23 . (canceled) 
     
     
         24 . The method of  claim 1  wherein the heavy chain constant region gene of the heterologous heavy chain locus is of human origin. 
     
     
         25 - 26 . (canceled) 
     
     
         27 . A heavy chain-only antibody, or a fragment thereof produced according to the method of  claim 1 . 
     
     
         28 - 31 . (canceled) 
     
     
         32 . A vector comprising a heterologous heavy chain locus as in  claim 1 . 
     
     
         33 . A host cell transformed with a vector according to  claim 32 . 
     
     
         34 . A non-human transgenic mammal expressing a heterologous heavy chain locus as in  claim 1 . 
     
     
         35 - 37 . (canceled) 
     
     
         38 . A pharmaceutical composition comprising the heavy chain-only antibody or fragment thereof according to  claim 27  and a pharmacologically appropriate carrier. 
     
     
         39 . The method of  claim 1 , further comprising the steps of:
 (a) injecting an antigen into the transgenic mammal;
 b) isolating a cell or tissue expressing an antigen-specific, heavy chain-only antibody of interest; 
   c) producing a hybridoma from the cell or tissue of step (b);   d) optionally cloning the heavy chain-only antibody mRNA from said hybridoma for subsequent production in a heterologous expression system such as a mammalian, plant, insect, microbial, fungal or alternative system.   
     
     
         40 . A method of production and selection of a V H  binding domain comprising the steps of:
 (a) injecting an antigen into the transgenic mammal of  claim 34 ;
 b) isolating a cell or tissue expressing an antigen-specific heavy chain-only antibody of interest; 
   c) cloning the V H  locus from mRNA derived from the isolated cell or tissue;   d) displaying the encoded protein using a phage or similar library;   e) identifying antigen-specific V H  domain(s); and   f) expressing the V H  domain(s) alone or as a fusion protein in bacterial, yeast or alternative expression systems.   
     
     
         41 . A binding polypeptide complex comprising a dimer of a first heavy chain and a second heavy chain wherein:
 each heavy chain comprises a binding domain, an effector moiety and a dimerisation domain;   the binding domain in the first heavy chain may be of the same specificity as, or of a different specificity from, the specificity of the binding domain in the second heavy chain;   the effector moiety in the first heavy chain may have the same or different function from the effector moiety on the second heavy chain.   
     
     
         42 . The binding polypeptide complex of  claim 41 , wherein the effector moiety on each heavy chain is a second binding domain, which may be of the same specificity as, or of a different specificity from, the binding domain of the heavy chain of which it is a part, and may be of the same specificity as, or a different specificity from, either one or both of the binding domains on the other heavy chain. 
     
     
         43 . The binding polypeptide complex of  claim 41  wherein the or each dimerisation domain comprises at least C H 2, C H 3 and, optionally, C H 4 antibody constant domains derived from the any class of immunoglobulin heavy chain gene. 
     
     
         44 . The polypeptide complex of  claim 41 , further comprising a pair of effector (light) chains, wherein:
 one of the effector (light) chains is associated with one of the heavy chains and the other of the effector (light) chains is associated with the other of the heavy chains;   the effector (light) chain comprises a complementary assembly domain having attached to it an effector moiety; and   the effector domain of the heavy chain and the complementary assembly domain of the effector (light) chain associate with one another through non-covalent interactions.   
     
     
         45 . The binding polypeptide complex of  claim 44  wherein the dimerisation domain comprises at least C H 2, C H 3 and, optionally, C H 4 antibody constant regions derived from any class of immunoglobulin heavy chain gene. 
     
     
         46 . The polypeptide complex of  claim 44 , wherein at least one effector moiety of the effector (light) chain is an enzyme, toxin, binding domain, serum stabilising agent, cell or an imaging agent. 
     
     
         47 . The polypeptide complex of  claim 41  or  44 , wherein at least one effector moiety of the heavy chain is an enzyme, toxin, binding domain, a serum stabilising agent or an imaging agent. 
     
     
         48 - 51 . (canceled) 
     
     
         52 . A binding polypeptide complex comprising a plurality of heavy chain dimers and a J chain, wherein:
 the plurality of heavy chain dimers are assembled by the J chain;   each heavy chain comprises a soluble binding domain and identical μ C H 2, C H 3 and C H 4 domains; and   there are at least two soluble binding domains having different specificities in the binding polypeptide complex.   
     
     
         53 . The binding polypeptide complex of  claim 52 , wherein there are only two binding domains of different specificity. 
     
     
         54 . The binding polypeptide complex of  claim 41 ,  44 , or  52 , wherein each heavy chain further includes a hinge domain, or a flexible hinge-like domain, at the carboxyl terminus of the C H 3 domain or, if present, the C H 4 domain. 
     
     
         55 . The polypeptide complex of  claim 41 ,  44 , or  52 , wherein one or more of the binding domains is a V HH  domain. 
     
     
         56 . The polypeptide complex of  claim 41 ,  44 , or  52 , wherein one or more of the binding domains is a V H  domain from any organism selected or engineered to show improved solubility characteristics. 
     
     
         57 . The polypeptide complex of  claim 41 ,  44 , or  52  wherein one or more of the binding domains is a mammalian cell adhesion polypeptide complex, a prokaryotic cell adhesion polypeptide complex or a viral cell adhesion polypeptide complex. 
     
     
         58 . The polypeptide complex of,  claim 41 ,  44 , or  52  wherein one or more of the binding domains is a cytokine, a growth factor, a receptor antagonist or agonist or a ligand. 
     
     
         59 . An isolated polynucleotide encoding one of the chains of a polypeptide complex according to  claim 41 ,  44 , or  52 . 
     
     
         60 . A cloning or expression vector comprising an isolated polynucleotide of  claim 59 . 
     
     
         61 - 62 . (canceled) 
     
     
         63 . A host cell transformed with at least one expression vector of  claim 60 . 
     
     
         64 . A method for the production of a polypeptide complex, comprising culturing the host cell of  claim 63  and isolating the polypeptide complex. 
     
     
         65 . A method of producing a polypeptide complex of  claim 41 ,  44 , or  52 , which comprises:
 transforming a host cell with a first polynucleotide encoding a first heavy chain and a second polynucleotide encoding a second heavy chain;   growing the host cell under conditions which allow for the expression of the coding sequences of the two polynucleotides; and   harvesting the polypeptide complex from the host cell.   
     
     
         66 . The method of  claim 65  further comprising transforming the host cell with a polynucleotide encoding a light chain. 
     
     
         67 - 68 . (canceled) 
     
     
         69 . A pharmaceutical composition comprising a polypeptide binding complex according to  claim 41 ,  44 , or  52 . 
     
     
         70 - 71 . (canceled) 
     
     
         72 . A method of treating a patient, comprising administering a composition comprising the polypeptide binding complex of  claim 41 ,  44 , or  52 . 
     
     
         73 . (canceled) 
     
     
         74 . The polypeptide complex of  claim 41 ,  44 , or  52 , comprising a flexible linker at the carboxyl terminus of the C H 3 domain or, if present, the C H 4 domain. 
     
     
         75 . The polypeptide complex of  claim 54 , wherein the cysteines in the hinge are replaced with prolines.