Antibody-based redirection of universal Fabrack-CAR T cells selectively kill antigen bearing tumor cells

Background Chimeric antigen receptor (CAR) T cells engineered to recognize and target tumor associated antigens have made a profound impact on the quality of life for many patients with cancer. However, tumor heterogeneity and intratumoral immune suppression reduce the efficacy of this approach, allowing for tumor cells devoid of the target antigen to seed disease recurrence. Here, we address the complexity of tumor heterogeneity by developing a universal CAR. Method We constructed a universal Fabrack-CAR with an extracellular domain composed of the non-tumor targeted, cyclic, twelve residue meditope peptide that binds specifically to an engineered binding pocket within the Fab arm of monoclonal antibodies (mAbs). As this site is readily grafted onto therapeutic mAbs, the antigen specificity of these universal Fabrack-CAR T cells is simply conferred by administering mAbs with specificity to the heterogeneous tumor. Results Using in vitro and in vivo studies with multiple meditope-engineered mAbs, we show the feasibility, specificity, and robustness of this approach. These studies demonstrate antigen- and antibody-specific T cell activation, proliferation, and IFNγ production, selective killing of target cells in a mixed population, and tumor regression in animal models. Conclusion Collectively, these findings support the feasibility of this universal Fabrack-CAR T cell approach and provide the rationale for future clinical use in cancer immunotherapy.


Fig. S2. The sequence difference between parental and corresponding meditope-enabled antibody. (A)
To enable antibody to bind to meditope, we make mutations shown in red color. (B) Superposition of our meditope enabled trastuzumab (PDB: 4ioi) and parental trastuzumab bound to Her2 (PDB: 1n8z). The CDR loops are circled in red and the meditope binding site is circled in green.
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Fig. S8. Fabrack T cells validated for increased expression of CD25 and CD69 after incubation with target cells and a corresponding memAb.
The percentage of T cells with increased CD25 or CD69 was shown. Increased expression of CD25 (left) and CD69 (right) was observed in Fabrack T cells after αHER2 memAb redirected Fabrack T cells to ovarian (OVCAR3 and SKOV3) or breast (MCF7, SKBR3 and BT474) cancer cells. Cells were incubated with or without 0.5 nM αHER2 memAb for 5 hours at a 1:1 E:T ratio. Activation markers at Fabrack T cell suface were analyzed by flow cytometry after staining cells with fluorescent dye-conjugated antibodies. Mock was gated on all CD3+ cells and Fabrack conditions were gated on CD19+(CAR+) cells. Experiments were done in technical duplicates. (Mean ± SEM, *** = P < 0.001) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Fig. S9. Tumor killing and T cell proliferation after Fabrack T cells directed by HER2 memAb or meFab to target tumor. OVCAR3 cells (A) or SKBR3 cells (B)
were treated with 0.5 nM HER2 memAb or meFab at indicated ET ratio for 24, 48 or 72h. The tumor killing and T cell proliferation were analyzed by flow cytometry. Experiments were done in technical duplicates. Killing was based on tumor counts cococultured with Mock T cells. "#" indicates significance between memAb and meFab treated cells. "*" indicates significance versus Fabrack treated cells. (Mean ± SEM, # and * = P < 0.05, ## and ** = P < 0.01, ### and *** = P < 0.001) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Fig. S12 The effect of high and low dose of Fabrack T cells on OVCAR3 xenograft tumor model. (A)
Mouse images of OVCAR3 tumor burden were shown on day 5, 13, 22 and 28. Mice were intraperitoneally given first dose of 10M or 2M Fabrack T cells on day 6 and second dose of 2M Fabrack T cells on day 23. EGFR/HER3 memAb at a dose of 1.25mg/kg was intraperitoneally given on day 5, 8, 11, 14, 17, 22 and 25. (B) Tumor burden of mice in different treatment groups was plotted based on total flux on day 28. (n=4 or 5, mean ± SEM, * = P < 0.05) (C) The percentage of human CD3+ T cells in mouse blood cells on day 20 was analyzed by flow cytometry. "*" indicates the significance versus tumor only group. (n=5, mean ± SEM, ** = P < 0.01) (D) The percentage of human CD19+ Fabrack T cells in mouse blood cells on day 20 was analyzed by flow cytometry. "*" indicates the significance versus tumor only group. (n=5, mean ± SEM, * = P < 0.05, ** = P < 0.01) (E) The percentage of human CD19+ Fabrack T cells in human CD3+ cells from mouse blood on day 20 was analyzed by flow cytometry. "*" indicates the significance versus Fabrack treated group. (n=5, mean ± SEM, ** = P < 0.01) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer doi: 10.1136/jitc-2021-003752 :e003752.