High throughput generation and affinity maturation of humanized antibody
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.
1. A method for screening a library of human or humanized antibodies in yeast, comprising:
(i) assembling a library of antibodies in yeast cells, wherein the step of assembling the library comprises:
(a) mutagenizing at least one of a V H and a V L to produce a plurality of V H and/or V L ; and
(b) introducing the plurality of V H and/or V L into an expression vector by homologous recombination;
(ii) expressing the library of antibodies in the yeast cells, wherein each of the antibodies comprises a V H and a V L that form a V H /V L heterodimer in the yeast cells;
(iii) contacting a specific target protein with the library of antibodies expressed in the yeast cells; and
(iv) selecting those antibodies based on their binding to the target protein.
2. The method of claim 1 , wherein members of the antibody library comprise:
a V H having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V H grafted into a human antibody framework, and
a V L having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V L grafted into a human antibody framework; wherein
the one or more heavy chain CDR or CDRs and the one or more light chain CDR or CDRs are from the same non-human antibody, and the selected antibodies bind to the same target protein as the antibody from which the non-human CDRs are obtained.
3. The method of claim 1 , wherein the antibody library is generated by mutagenizing a chimeric antibody that comprises:
a V H having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V H grafted into a human antibody framework, and
a V L having 3 CDRs, wherein at least one of the CDRs is a CDR3 of a non-human V L grafted into a human antibody framework, wherein
the one or more heavy chain CDR or CDRs and the one or more light chain CDR or CDRs are from the same non-human antibody, and the selected antibodies bind to the same target protein as the antibody from which the non-human CDRs are obtained.
4. The method of claim 1 , wherein the V H and the V L each is linked with a zipper domain by which the V H and the V L are associated with each other to form a heterodimer.
5. The method of claim 4 , wherein the zipper domains of the V H and the V L facilitate the heterodimerization of the V H and the V L through coiled-coil interactions.
6. The method of claim 4 , wherein the zipper domains of the V H and the V L are leucine zippers.
7. The method of claim 4 , wherein the zipper domains of the V H and the V L are leucine zippers formed by the leucine zippers from Fos and Jun.
8. The method of claim 4 , wherein the zipper domains of the V H and the V L are leucine zippers formed by the leucine zippers from Myc and Max.
9. The method of claim 4 , wherein the zipper domains of the V H and the V L each is linked to the C-terminus of the V H and the V L , respectively.
10. The method of claim 1 , wherein the plurality of V H and/or V L is introduced into an expression vector by gap repair homologous recombination.
11. The method of claim 1 , wherein the at least one of a V H and V L are mutagenized by one or more of site-directed mutagenesis, error-prone PCR mutagenesis, cassette mutagenesis, random PCR mutagenesis, DNA shuffling, and chain shuffling.
12. The method of claim 1 , wherein the expression vector is a yeast-bacteria shuttle vector.
13. The method of claim 12 , wherein the yeast-bacteria shuttle vector comprises at least one of a replicating vector and an integrating vector.
14. The method of claim 13 , wherein the replicating vector comprises at least one of an autonomously replicating vector and a centromeric plasmid.
15. The method of claim 1 , wherein the V H is mutagenized to produce a plurality of V H .