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Abstract
Background Chimeric antigen receptor (CAR) T cell therapy has transformed therapy for hematological malignancies but has not yet been established as standard of care for any solid tumors. One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant, immunocompetent animal models. In this study, we sought to establish CAR T cells for naturally occurring canine sarcomas in client owned animals as a model for human CAR T cell therapy.
Methods Archived FFPE, freshly isolated canine solid tumor samples as well as tumor lines were tested for B7H3 expression by immunohistochemistry (IHC) and flow cytometry analysis. We designed CARs using the scFv from the human B7H3-specific antibody MGA271 and confirmed the cross-reactivity to canine B7H3 (construct information see figure 1A). A truncated EGFR (tEGFR) was included in the construct to allow for IHC and flow cytometry testing for the presence of CAR T cells. Killing efficiency was evaluated using 3D tumor spheroid killing assays to monitor dynamics. Safety of the CAR products following lymphodepletion was confirmed in two healthy dogs (figure 1B).
Results Canine solid tumors were confirmed to be B7H3 positive in almost all cases. Using the GALV-pseudotyped retrovirus system, transduction was efficient with up to 70% CAR+ cells. Post-transduction expansion was over 100 folds. B7H3 CAR transduced canine T cells were able to eliminate B7H3+ canine tumor spheroids effectively (figure 2). Safety of the CAR T cells (dose: 1 × 109/m2) were confirmed in both healthy animals following cyclophosphamide lymphodepletion. After week 6, cetuximab was given to the subjects to deplete EGFR+ cells. Subject 2 experienced fever after CAR T cell administration. Both dogs showed elevated serum ALP and ALT levels and returned to normal (figure 3). No other treatment-related adverse events were observed. Information of the CAR T cell products can be found in table 1.
Construct information and safety trial design(A) Four 2nd generation CAR constructs were generated. Two B7H3 CARs were candidates for the treatment, and two HER2 CARs served as controls, as they have been shown to kill canine cancer cells. The CARs are consisted of a single chain variable fragment (scFv, either B7H3-specific MGA271 or HER2-specific FRP5), a short hinge, a transmembrane domain (tm), a canine costimulatory signaling domain (either canine CD28 or 4-1BB) and canine CD3? signaling domain. Truncated EGFR is added in the construct for CAR+ T cell detection and facilitate the depletion of CAR T cells in vivo as a safety measure. (B) Blood from the subjects were drawn 3 weeks prior to the treatment for CAR T cell production. Cyclophosphamide (Cy, 400 mg/m2) and Fludarabine (Flu, 10 mg/m2) were given to the subjects for 2 days for lymphodepletion. CAR T cells (1 × 109/m2) and cetuximab (200 mg/m2) were given to the subjects as indicated. Blood, lymph node (LN) and bone marrow (BM) aspirates were collected for CAR T cell homing and persistence analysis
Killing of canine OSA spheroids by canine CAR T ce(A) Scheme of tumor cell spheroid forming and killing. The loss of GFP can be measured for cytotoxicity readout (B) FRP5 and MGA271 CAR T cells can effectively kill canine cancer spheroids. Experiments were done in triplicates and error bars indicate SD
Dynamics of peripheral lymphocytes, serum ALP and Current treatment regimen effectively decreased peripheral lymphocytes number after cyclophosphamide and fludarabine administration (D-4 and D-3) and increased serum ALP and ALT level after CAR T cell infusion (D0). Dashed line in both graphs show the upper limit of ALP and ALT levels, which are both 68U/L
Infused CAR T cell product information
Conclusions We demonstrated that, similar to human cancers, B7H3 is a target in canine solid tumors. We successfully generated canine B7H3 specific CAR T cell products that are highly efficient at killing canine 3D tumor spheroids using a production protocol that closely models human CAR T cell production procedure and confirmed the safety in vivo. We plan to test and optimize various approaches to enhance CAR T cell efficacy for solid tumor treatment both in vitro and in canine sarcoma patients.
Ethics Approval The study was approved by Fred Hutchinson Cancer Research Center‘s Institutional Animal Care and Use Committee (IACUC), approval number PROTO201900860
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