Genetastix Corporation

Adimab, LLC

Shaobing Benjamin Hua

Li Zhu

Michelle Haynes Pauling

Shuanghong Wei

James T Sheridan

Yu-Huei Lin

Compositions, methods, and kits are provided for efficiently generating and screening humanized antibody with high affinity against a specific antigen. The library of humanized antibody is generated by mutagenizing a chimeric antibody template that combines human antibody framework and antigen binding sites of a non-human antibody. Alternatively, the library of humanized antibody is generated by grafting essential antigen-recognition segment(s) such as CDRs of the non-human antibody into the corresponding position(s) of each member of a human antibody library. This library of humanized antibody is then screened for high affinity binding toward a specific antigen in vivo in organism such as yeast or in vitro using techniques such as ribosome display or mRNA display. The overall process can be efficiently performed in a high throughput and automated manner, thus mimicking the natural process of antibody affinity maturation.

High throughput generation and affinity maturation of humanized antibody

Methods and compositions are provided for efficient, high throughput screening of antibody libraries against proteins targets, especially membrane proteins in yeast. In particular, methods are provided for screening a diverse antibody library for antibodies that bind to a membrane protein such as a chemokine receptor in yeast.

Method for generating and selecting antibodies against target protein

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.

Human monoclonal antibodies against human CXCR4

Compositions are provided that comprise antibody against coreceptors for human immunodeficiency virus such as CCR5 and CXCR4. In particular, monoclonal human antibodies against human CCR5 are provided that bind to CCR5 with high affinity and are capable of inhibiting HIV infection at low concentrations. The antibodies can be used as prophylactics or therapeutics to prevent and treat HIV infection, for screening drugs, and for diagnosing diseases or conditions associated with interactions with HIV coreceptors.

Human monoclonal antibody against coreceptors for human immunodeficiency virus

Compositions, methods, and kits are provided for efficiently generating and screening a library of highly diverse protein complexes for their ability to bind to other proteins or oligonucleotide sequences. In one aspect of the invention, a library of expression vectors is provided for expressing the library of protein complexes. The library comprises a first nucleotide sequence encoding a first polypeptide subunit; and a second nucleotide sequence encoding a second polypeptide subunit. The first and second nucleotide sequences each independently varies within the library of expression vectors. In addition, the first and second polypeptide subunit are expressed as separate proteins which self-assemble to form a protein complex, such as a double-chain antibody fragment (dcFv or Fab) and a fully assembled antibody, in cells into which the library of expression vectors are introduced. The library of expression vectors can be efficiently generated in yeast cells through homologous recombination; and the encoded proteins complexes with high binding affinity to their target molecule can be selected by high throughput screening in vivo or in vitro.

Assembly and screening of highly complex and fully human antibody repertoire in yeast

Methods are provided for generating highly diverse libraries of expression vectors encoding fusion proteins such as single-chain antibodies via homologous recombination in yeast. The method comprises: transforming into yeast cells a linearized yeast expression vector having a 5&prime;- and 3&prime;-terminus sequence at the site of linearization and a library of insert nucleotide sequences that are linear and double-stranded; and having homologous recombination occur between the vector and the insert sequence such that the insert sequence is included in the vector in the transformed yeast cells. The insert sequence comprises a first nucleotide sequence encoding a first polypeptide subunit, a second nucleotide sequence encoding a second polypeptide subunit, a linker sequence encoding a linker peptide that links the first and second polypeptide subunits, and a 5&prime;- and 3&prime;-flanking sequence at the ends of the insert sequence which are sufficiently homologous to the 5&prime;- and 3&prime;-terminus sequences of the linearized yeast expression vector, respectively, to enable homologous recombination to occur. The first polypeptide subunit, the second polypeptide subunit, and the linker polypeptide are expressed as a single fusion protein; and the first and second nucleotide sequences each independently varies within the library of expression vectors.

Generation of highly diverse library of expression vectors via homologous recombination in yeast

A highly diverse library of yeast expression vectors encoding a library of fusion proteins such as antibodies is provided. The yeast expression vector formed in the library comprises: a first nucleotide sequence encoding a first polypeptide subunit; a second nucleotide sequence encoding a second polypeptide subunit; and a linker sequence encoding a linker peptide that links the first nucleotide sequence and the second nucleotide sequence. The first polypeptide subunit, the second polypeptide subunit, and the linker polypeptide are expressed as a single fusion protein within the library of fusion proteins. The first and second nucleotide sequences each independently varies within the library of expression vectors. The library of fusion proteins expressed by the library expression vectors can be used for screening against target molecules such as proteins, peptides, DNAs and small molecules in vitro and in vivo.

Highly diverse library of yeast expression vectors

Compostions, kits and methods are provided for generating highly diverse libraries of proteins such as antibodies via homologous recombination in vivo, and screening these libraries against protein, peptide and nucleic acid targets using a two-hybrid method in yeast. The method for screening a library of tester proteins against a target protein or peptide comprises: expressing a library of tester proteins in yeast cells, each tester protein being a fusion protein comprised of a first polypeptide subunit whose sequence varies within the library, a second polypeptide subunit whose sequence varies within the library independently of the first polypeptide, and a linker peptide which links the first and second polypeptide subunits; expressing one or more target fusion proteins in the yeast cells expressing the tester proteins, each of the target fusion proteins comprising a target peptide or protein; and selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the tester fusion protein to the target fusion protein.

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Cupertino, CA, United States of America

Shaobing Benjamin Hua