Binding molecules
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 soluble, antigen-specific V H binding domain comprising:
(a) immunising a transgenic rodent expressing a heterologous V H heavy chain locus with an antigen wherein:
(i) the V H heavy chain locus comprises a variable region comprising at least one V H gene segment, at least one D gene segment, at least one J gene segment and at least one heavy chain constant region;
(ii) each constant region does not encode a functional C H 1 domain;
(iii) a V H gene segment, a D gene segment and a J gene segment are capable of recombining to form a VDJ coding sequence;
(iv) the recombined V H heavy chain locus, when expressed upon antigen challenge, is capable of forming a soluble, heavy chain-only antibody comprising a soluble, antigen-specific V H binding domain and a constant effector region devoid of a functional C H 1 domain with an antigen;
(b) cloning a V H locus resulting from recombination between single V, D and J gene segments encoding a soluble, antigen-specific V H binding domain from an antibody-producing cell of said immunised transgenic rodent after affinity maturation via somatic mutation; and
(c) producing said soluble, V H binding domain from the clone of step (b).
2. The method of claim 1 , wherein steps (a) to (c) of claim 1 are carried out by:
(a) injecting an antigen into said transgenic rodent;
(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) cloning the heavy chain-only antibody mRNA from said hybridoma; and
(e) producing said soluble, antigen-specific V H binding domain by isolating and expressing mRNA encoding said soluble, antigen-specific heavy chain only antibody from the mRNA of step (d).
3. The method of claim 1 , wherein steps (a) to (c) of claim 1 are carried out by:
(a) injecting an antigen into said transgenic rodent;
(b) isolating a cell or tissue expressing an antigen-specific, soluble heavy chain-only antibody of interest;
(c) cloning the V H locus from mRNA derived from the isolated cell or tissue; displaying the encoded VH domain using phage or similar library;
(d) identifying soluble, antigen-specific V H binding domain(s); and
(e) expressing the soluble, antigen-specific V H domain(s) alone or as a fusion protein.
4. The method of any one of claims 1 - 3 , wherein the heterologous V H heavy chain locus comprises naturally-occurring human V H , human D and human J gene segments.
5. The method of claim 4 , wherein the heterologous V H heavy chain locus comprises more than one human V H gene segment, 5 or more human D gene segments and more than one human J gene segment.
6. The method of claim 5 , wherein the heterologous V H heavy chain locus comprises 5 human V H gene segments, all human D gene segments, and all human J gene segments.
7. The method of claim 6 , wherein the constant region is from the rat.
8. The method of any one of claims 1 - 3 , wherein the endogenous heavy chain locus of the transgenic rodent has been silenced.