Patent ID: 6955900
Filing Date: 2005-10-18
Classification: C07H,C07K,C12N,C40B

Abstract:
1. A method for producing a polypeptide having a binding site that binds a preselected antigen, the method comprising the steps of: i) amplifying a complementarity determining region (CDR) encoding portion of a template immunoglobulin variable domain gene selected from the group consisting of a template immunoglobulin heavy chain variable domain gene and a template immunoglobulin light chain variable domain gene, wherein said template immunoglobulin heavy or light chain genes each have a framework encoding region and said CDR encoding portion and encode respective heavy and light chain variable domain polypeptides having a preselected antigen binding specificity to a first antigen, and wherein said amplifying is by a primer extension reaction using a primer extension reaction oligonucleotide or its complement, for mutagenizing a preselected nucleotide in said CDR encoding portion, thereby forming a library of amplified CDR-mutagenized encoding immunoglobulin gene fragments, said primer extension reaction oligonucleotide having a 3′ and 5′ termini and comprising: where the sum of a and b is from 5 to 50, X is a trinucleotide encoding cysteine or a native amino acid residue coded by the immunoglobulin gene, N is independently any nucleotide, M is adenine (A) or cytosine (C), Y is a nucleotide sequence that encodes a preselected polypeptide sequence of from 3 to 50 amino acid residues, Y includes the tripeptide RGD, and said 5′ and 3′ terminal nucleotide sequences have a length of about 6 to 50 nucleotides in length; ii) inserting individual members of the library of amplified CDR encoding portions formed in step (i) into a dicistronic phagemid expression vector comprising immunoglobulin heavy and light chain variable domain genes that lack the immunoglobulin gene portion corresponding to the fragment to be inserted, wherein upon insertion said vector is capable of expressing heavy and light chain variable domain polypeptides encoded by said vector, thereby forming a library of dicistronic expression vectors containing amplified CDR-mutagenized immunoglobulin gene fragments; iii) expressing said immunoglobulin heavy and light chain genes in the library of dicistronic expression vectors formed in step (ii) in a host cell whereby said encoded heavy and light chain variable domain polypeptides assemble on the surface of a phage to form a phage-displayed immunoglobulin heterodimer, thereby producing a library of CDR-mutagenized phage-displayed immunoglobulin heterodimers; and iv) immunoreacting members of the library of CDR-mutagenized phage-displayed immunoglobulin heterodimers formed in step (iii) on a preselected second antigen, said second antigen being different than said first antigen, to allow for selection of a CDR-mutagenized phage-displayed immunoglobulin heterodimer containing a polypeptide having a binding site capable of binding said preselected second antigen; wherein said binding site is an RGD-dependent binding site and wherein said oligonucleotide has the formula: 5′ CTCCTCCTCCTCCTCGACGTCCATATAATAATT[MNN] a ATCGCCACG[MNN] b TGGCCCCAC TCTCGCACAATAATA3′ (SEQ ID NO 49).