University of Pennsylvania Perelman School of Medicine Philadelphia, Pennsylvania, United States
Background/Case Studies: Chimeric antigen receptor T cell (CART) therapy has resulted in unprecedented durable clinical responses in patients with blood cancers, but target antigen loss and reduced persistence and effectiveness within the immunosuppressive tumor microenvironment represent challenges for improving efficacy particularly against solid tumors. A plethora of novel approaches has been proposed for identifying tumor targets, enhancing CART function, and improving cell manufacturing processes, but conventional mouse models fall short in their ability to recapitulate human biology and predict safety and efficacy before entering the clinic. Pet dogs are closely related phylogenetically to humans, spontaneously developing cancers that share remarkably similar biological, behavioral and genetic features with human counterparts, and performing clinical trials in dogs is considerably less expensive and complex than in humans. Thus, dog patients can serve as a relevant parallel patient population to help unravel the complexities of immunotherapies as embraced by the Beau Biden Cancer Moonshot Initiative that launched a canine immunotherapy clinical trials network designed to accelerate human clinical translation. Progress, though, has been hampered by a lack of fully canine antibody fragments for in vivo use, as murine or human antibodies in dogs elicits anti-species immune responses. To solve this problem, an extraordinarily large naïve/non-immune canine antibody phage display library was created to enable the isolation of fully canine antibodies to canine targets of therapeutic interest.
Study
Design/Methods: By analyzing the canine immunoglobulin locus, 36 heavy and 109 light chain PCR primer pairs were designed to amplify variable region gene segments from a billion B cells derived from 7 dogs. Heavy and light chains from over 1000 individual PCR reactions were randomly combined, cloned into the pComb3X phagemid, and electroporated into E coli to create a 40-billion-member IgG/IgM kappa/lambda phage display library. Nucleotide sequencing of randomly selected clones with mapping to their germline Ig genes validated the natural diversity and comprehensive nature of the library.
Results/Findings: To date, dozens of antibody library selection campaigns have been completed using conventional phage display methods to provide canine antibodies to targets of high therapeutic value including those to canine checkpoint inhibitors CTLA4 and PD1 which have entered canine clinical trials. Of note, anti-canine CD19 antibodies have been isolated for incorporation into fully canine CD28z CART constructs for treating canine patients with relapsed, refractory B-cell NHL. Conclusions: This platform technology for comparative oncology will serve a dual purpose of enabling pet dogs with spontaneous malignancies to accelerate next generation antibody-directed human therapies while providing greatly needed treatments for canine patients.
Importance of research: CAR-Ts have led to unpresented clinical responses and 6 recent FDA approvals for blood cancers. Application to solid tumors lags behind due to lack of suitable animal models whose cancers arise and behave like those of humans. Dog patients fit the bill, but lack of immune compatible antibodies prevents this area of investigation. Work reported here describes the first and only solution to this barrier. It will not only help to advance human health but help our beloved four-legged family members.
