Method for generating and selecting antibodies against target protein

Abstract

Compositions are provided that comprise antibody against membrane proteins such as chemokine receptors. In particular, monoclonal human antibodies against human CXCR4 are provided that are capable of inhibiting HIV infection and chemotaxis in human breast cancer cells. The antibodies can be used as prophylactics or therapeutics to prevent and treat HIV infection and cancer, for screening drugs, and for diagnosing diseases or conditions associated with interactions with chemokine receptors.

1 - 31 . (canceled) 
     
     
         32 . A method for selecting an antibody that binds to a target membrane protein by yeast display, comprising the step of:
 contacting yeast cells that display a library of antibodies with a target membrane protein or a peptide fragment of the target membrane protein;   selecting the yeast cell that binds to the target membrane or the peptide fragment of the target membrane protein; and   identifying the antibody displayed by the selected yeast cell.   
     
     
         33 . The method of  claim 32 , wherein the antibodies are single-chain antibodies each of which is fused via its C-terminus to a binding subunit of a yeast adhesion receptor. 
     
     
         34 . The method of  claim 33 , wherein the yeast adhesion receptor is a-agglutinin or α-agglutinin. 
     
     
         35 . The method of  claim 34 , wherein the yeast adhesion receptor is an Aga2p mating adhesion receptor of  Saccharomyces cerevisiae.    
     
     
         36 . The method of  claim 33 , wherein the single-chain antibodies each comprises an antibody heavy chain variable region, an antibody light chain variable region, and a linker peptide that links the antibody heavy chain variable region and the antibody light chain variable region. 
     
     
         37 . The method of  claim 32 , wherein the diversity of the library of antibodies is at least 1×10 6 . 
     
     
         38 . The method of  claim 32 , wherein the diversity of the library of antibodies is at least 1×10 7 . 
     
     
         39 . The method of  claim 32 , wherein the diversity of the library of antibodies is at least 1×10 7 -1×10 10 . 
     
     
         40 . The method of  claim 32 , wherein the diversity of the library of antibodies is at least 1×10 8 -1×10 10 . 
     
     
         41 . The method of  claim 32 , wherein the sequence of the antibody heavy or light chain variable region is fully human. 
     
     
         42 . The method of  claim 32 , wherein the V H  and V L  are encoded by variable regions of immunoglobulin genes of a human, non-human primate, or rodent. 
     
     
         43 . The method of  claim 32 , wherein the V H  and V L  are encoded respectively by a heavy-chain variable region and a light-chain variable region of a human immunoglobulin gene. 
     
     
         44 . The method of  claim 32 , wherein the V H  is encoded by a heavy-chain variable region of a first human immunoglobulin gene, and the V L  is encoded by a light chain variable region of a second human immunoglobulin gene different from the first human immunoglobulin gene. 
     
     
         45 . The method of  claim 32 , wherein the antibody heavy or light chain variable region is encoded by a human IgG gene. 
     
     
         46 . The method of  claim 32 , wherein the target membrane protein is selected from the group consisting of receptors for growth factors, insulin receptor, MHC proteins, CD3 receptor, T cell receptors, cytokine receptors, tyrosine-kinase-associated receptors and G-protein coupled receptors. 
     
     
         47 . The method of  claim 46 , wherein receptors for growth factors are selected from the group consisting of receptors for vascular endothelial growth factor, epidermal growth factor, transforming growth factor, fibroblast growth factor, platelet derived growth factor, and insulin-like growth factor. 
     
     
         48 . The method of  claim 46 , wherein the MHC protein is class I or class II MHC protein. 
     
     
         49 . The method of  claim 46 , wherein the cytokine receptor is selected from interleukin-1 receptor, interleukin-2 receptor, interleukin-8 receptor, and interleukin-12 receptor, 
     
     
         50 . The method of  claim 46 , wherein the tyrosine-kinase-associated receptors is selected from the group consisting of Src, Yes, Fgr, Flt, Lck, Lyn, Hck, and Blk. 
     
     
         51 . The method of  claim 32 , wherein the peptide fragment of the target membrane protein has a length of 5-100 aa. 
     
     
         52 . The method of  claim 32 , wherein the peptide fragment of the target membrane protein has a length of 10-80 aa. 
     
     
         53 . The method of  claim 32 , wherein the peptide fragment of the target membrane protein has a length of 20-60 aa. 
     
     
         54 . The method of  claim 32 , wherein the peptide fragment of the membrane protein is an extracellular domain of the target membrane protein. 
     
     
         55 . The method of  claim 32 , wherein the yeast cell that binds to the target membrane or the peptide fragment of the target membrane protein is selected by flow cytometric cell sorting. 
     
     
         56 . A library of yeast cells displaying a library of antibodies on the surface of the yeast cells, comprising: yeast cells transformed with a library of yeast expression vectors encoding a library of antibodies each comprising an antibody heavy chain variable region (V H ) and an antibody light chain variable region (V L ), wherein at least one yeast expression vector comprises one or more homologous recombination sites and has undergone homologous recombination such that a polynucleotide sequence from a first polynucleotide sequence encoding the V H  or a second polynucleotide sequence encoding the V L  is incorporated into the yeast expression vector, and also a sequence encoding a binding subunit of a yeast adhesion receptor. 
     
     
         57 . The library of  claim 56 , wherein the yeast cells are  Saccharomyces cerevisiae  cells. 
     
     
         58 . The library of  claim 56 , wherein the yeast adhesion receptor is a-agglutinin or α-agglutinin. 
     
     
         59 . The library of  claim 56 , wherein a region of the V H  or V L  is mutagenized. 
     
     
         60 . The library of  claim 59 , wherein the region of the VH or VL is mutagenized through mutagenesis selected from the group consisting of error-prone PCR mutagenesis, site-directed mutagenesis, cassette mutagenesis, DNA shuffling and combinations thereof. 
     
     
         61 . The library of  claim 56 , wherein the diversity of the library of antibodies is at least 1×10 6 . 
     
     
         62 . The library of  claim 56 , wherein the diversity of the library of antibodies is at least 1×10 7 . 
     
     
         63 . The library of  claim 56 , wherein the diversity of the library of antibodies is at least 1×107-1×10 10 . 
     
     
         64 . The library of  claim 56 , wherein the diversity of the library of antibodies is at least 1×10 8 -1×10 10 . 
     
     
         65 . The library of  claim 56 , wherein the sequence of the antibody heavy or light chain variable region is fully human. 
     
     
         66 . The library of  claim 56 , wherein the V H  and V L  are encoded by variable regions of immunoglobulin genes of a human, non-human primate, or rodent. 
     
     
         67 . The library of  claim 56 , wherein the V H  and V L  are encoded respectively by a heavy-chain variable region and a light-chain variable region of a human immunoglobulin gene. 
     
     
         68 . The library of  claim 56 , wherein the V H  is encoded by a heavy-chain variable region of a first human immunoglobulin gene, and the V L  is encoded by a light chain variable region of a second human immunoglobulin gene different from the first human immunoglobulin gene. 
     
     
         69 . The library of  claim 56 , wherein the antibody heavy or light chain variable region is encoded by a human IgG gene.