A phage display approach for rapid antibody humanization: Designed combinatorial V gene libraries

Abstract

The development of a new strategy for antibody humanization is described. This strategy incorporates key recognition sequences from the parental rodent antibody into a phage display-based selection strategy. The original sequences of the third complementarity-determining regions (CDRs) of heavy and light chains, HCDR3 and LCDR3, were maintained and all other sequences were replaced by human sequences selected from phage-displayed antibody libraries. This approach was applied to the humanization of mouse mAb LM609 that is directed to human integrin ␣ v ␤ 3 and has potential applicability in cancer therapy as an antiangiogenic agent. We demonstrate this approach (i) provides a rapid route for antibody humanization constraining the content of original mouse sequences in the final antibodies to the most hypervariable of the CDRs; (ii) generates several humanized versions with different sequences at the same time; (iii) results in affinities as high as or higher than the affinity of the original antibody; and (iv) retains the antigen and epitope specificity of the original antibody. The production of multiple humanized variants may present advantages in the selection of antibodies that are more readily expressed on a large scale and could be important in therapeutic regimens that call for long-term treatment with antibodies in which antiidiotypic responses might be avoided by administration of alternative antibodies.Since the development of the hybridoma approach (1), a large number of rodent mAbs with specificity for antigens of therapeutic interest have been generated and characterized. The fact that rodent antibodies are highly immunogenic in humans, however, severely limits their clinical applications, especially when repeated administration is required for therapy. As a means of circumventing this limitation, several strategies have been developed to convert rodent antibody sequences into human antibody sequences, a process termed antibody humanization. Ideally, antibody humanization must not diminish specificity and affinity toward the antigen whereas immunogenicity must be completely eliminated. It has become apparent that the accomplishment of both aims is usually a timeconsuming and costly undertaking with even the most current humanization strategies. Here, we report the development of a new humanization strategy that combines rational design with combinatorial selections using phage display. We demonstrate that this approach provides a rapid route to antibody humanization and demonstrate its application to the humanization of mouse mAb LM609 which is directed against the human integrin ␣ V ␤ 3 . We chose LM609 as a model antibody for our humanization strategy because of its clinical potential. Recent findings by in a chorioallantoic membrane model and a severe combined immunodeficient mouse/human skin chimeric model have shown that LM609, when administered i.v., is able to reduce growth and metastasis of human tumors due to the inhibition of angiogenesis induced by the tumors. These findings sugge...

Neutralization ofBurkholderia pseudomalleiProtease by Fabs Generated through Phage Display

“…16,17) Therapeutically, chimeric mouse/human Fab can readily be channeled into previously reported strategies for complete humanization. 14,18) The phage library displaying chimeric mouse/human Fab was panned against immobilized B. pseudomallei protease. Two different mouse Fab libraries were used in the panning, and the size for both mouse libraries was estimated at approximately 3:9 Â 10 8 cfu.…”

Potential nuclease-based strategies for HIV gene therapy

“…Libraries of phage displaying peptides can also be screened for secreted cytotoxic nucleases having better binding affinities to a receptor. As already discussed, combinatorial phage libraries can be used to select for high affinity human scFvs (155,156). In one study, an scFv was obtained with a five to six fold higher affinity compared to antibodies produced from mouse hybridoma cell lines generated with the same antigen (155).…”

“…Combinatorial phage libraries may be used to select for high affinity human scFvs. A human scFv gene library may be constructed by heavy and light chain shuffling followed by selection of phage that bind to antigens immobilized on a solid support (155,156). Protein design can also be used to build better packageable nucleases.…”

Mining human antibody repertoires

“…108 A similar method, also based on phage display, preserves only the complementarity determining region (CDR) 3 sequences of mouse heavy and light chains, i.e., HCDR3 and LCDR3, while converting everything else to human sequences. 109 A general concern about the de novo generation of human mAbs from naïve and synthetic repertoires in vitro is their lack of exposure to negative selection processes in vivo which are integral to immune tolerance mechanisms and efficiently remove undesirable off-target reactivities. Addressing this concern, phage display has also been used to mine non-combinatorial antibody libraries, i.e., antibody libraries that retain original heavy and light chain pairings of human antibody repertoires through in-cell RT-PCR.…”