Antibody diversity generation

An Ig expression cassette comprising:

• one or more cloning sites for heavy chain (VH) and light chain (VL) sequences;
• an encoded N-terminal fusion of VH and VL with an stII signal sequence;
• an encoded C-terminal fusion of VH and VL to human CH1 and a human CL regions;
• an encoded C-terminal fusion of VH/CL to a phage gIII protein;
• an amber stop codon at a CL/gIII border;
• and, a promoter selected from the group consisting of:
• a lacZ promoter, an alkaline phosphatase promoter, and an arabinose promoter.

A method of evolving HIV envelope proteins with improved antigenicity, the method comprising:

• (a) providing a population of DNA fragments, which DNA fragments comprise at least one polynucleotide derived from at least one HIV envelope protein;
• (b) recombining the population of DNA fragments to produce a library of recombinant DNA segments;
• (c) optionally repeating the recombining of steps (a) and (b) one or more times;
• (d) screening the library of recombinant DNA fragments to identify at least one recombinant DNA segment encoding an evolved HIV envelope protein which has acquired or evolved a desired property;
• (e) repeating the recombining of steps (a) through (d) until the evolved HIV envelope protein has acquired the desired property.

A method for modifying the effector function of an antibody, the method comprising:

• (a) providing at least one nucleic acid derived from at least one immunoglobulin heavy chain constant region;
• (b) recombining the at least one nucleic acid to produce a library of recombinant immunoglobulin constant region nucleic acids;
• (c) optionally repeating the recombination process of steps (a) and (b) one or more times;
• (d) selecting at least one recombinant immunoglobulin constant region nucleic acid encoding a Fc region with a desired property;
• (e) optionally repeating steps (a) through (d) one or more time until the Fc region has acquired a desired property.

A method of humanizing an antibody, the method comprising:

• (i) selecting at least one non-human antibody with a desired antigen binding specificity;
• (ii) determining or inferring the amino acid sequence of the variable domain of the selected antibody;
• (iii) aligning a plurality of human antibody amino acid sequences with the amino acid sequence of the variable domain of the selected antibody;
• (iv) providing oligonucleotides corresponding to at least one CDR domain of the non-human antibody and at least two corresponding variable domain framework regions of the human antibody sequences;
• (v) producing at least one library of synthetically shuffled humanized antibody sequences by randomly assembling the at least one CDR sequence with the at least two framework region sequences maintaining amino acid positions relative to the plurality of human antibody amino acid sequences.