Methods for producing antibody libraries using universal or randomized immunoglobulin light chains
AssigneeThe Scripps Research Institute
InventorsCarlos F. Barbas, Dennis R. Burton, Richard A. Lerner
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Abstract
The present invention describes methods for producing antibody libraries, and particularly for increasing antibody library diversity by inducing mutagenesis within the CDR regions of immunoglobulin heavy or light chains that are displayed on the surface of filamentous phage particles comprising the library. The invention also describes oligonucleotides useful for increasing the library diversity, and universal light chains useful in the library production methods.
Claims19
A method for producing an antibody combining site in a polypeptide comprising:
- a) inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene which comprises amplifying a CDR portion of the immunoglobulin gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide, said oligonucleotide having 3′ and 5′ termini and comprising:
- i) a nucleotide sequence at said 3′ terminus capable of hybridizing to a first framework region of the immunoglobulin gene;
- ii) a nucleotide sequence at said 5′ terminus capable of hybridizing to a second framework region of an the immunoglobulin gene;
- and
- iii) a nucleotide sequence between said 3′ and 5′ termini according to the formula:
- [NNK]n (SEQ ID NO:73)
- wherein N is independently any nucleotide, K is G or T, and n is 3 to about 24, said 3′ and 5′ terminal nucleotide sequences having a length of about 6 to 50 nucleotides, or an oligonucleotide having a sequence complementary thereto;
- b) isolating the amplified CDR to form a library of mutagenized immunoglobulin light chain genes;
- c) expressing the isolated library of mutagenized light chain genes in combination with one or more heavy chain genes to form a combinatorial antibody library of expressed heavy and light chain genes;
- and
- d) selecting species of said combinatorial antibody library for the ability to bind a preselected antigen.
A method for producing an antibody combining site in a polypeptide comprising:
- a) inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene which comprises amplifying a CDR portion of the immunoglobulin gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide, said oligonucleotide having 3′ and 5′ termini and comprising:
- i) a nucleotide sequence at said 3′ terminus capable of hybridizing to a first framework region of the immunoglobulin gene;
- ii) a nucleotide sequence at said 5′ terminus capable of hybridizing to a second framework region of the immunoglobulin gene;
- and
- iii) a nucleotide sequence between said 3′ and 5′ termini according to the formula:
- [MNN]n (SEQ ID NO:74),
- wherein N is independently any nucleotide, M is A or C, n is 3 to about 24, said 3′ and 5′ terminal nucleotide sequences having a length of about 6 to 50 nucleotides, or an oligonucleotide having a sequence complementary thereto;
- b) isolating the amplified CDR to form a library of mutagenized immunoglobulin light chain genes;
- c) expressing the isolated library of mutagenized light chain genes in combination with one or more heavy chain genes to form a combinatorial antibody library of expressed heavy and light chain genes;
- and
- d) selecting species of said combinatorial antibody library for the ability to bind a preselected antigen.
Dependencies are inferred from the claim text.