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299 SNAP CAR T cells for programmable antigen targeting
  1. Elisa Ruffo,
  2. Victor So,
  3. Michael Kvorjak,
  4. Olivera J Finn,
  5. Alexander Deiters and
  6. Jason Lohmueller
  1. University of Pittsburgh, Pittsburgh, PA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Universal chimeric antigen receptors (CARs) are synthetic receptors that instead of directly binding to an antigen, recognize one or more adaptor molecules that bind to target antigens. Universal CARs are of high clinical interest due to their abilities to be tuned by adaptor dose, potentially avoiding toxicities, and to be targeted toward multiple antigens through combinatorial use of different adaptors, potentially avoiding relapse due to antigen loss.

Methods We developed a universal CAR, ‘SNAP-CAR,’ that carries out a self-labeling reaction to covalently attach to adaptor antibodies conjugated to a benzylguanine (BG) tag. SNAP-CAR-T2A-LNGFR was cloned and packaged into a gamma-retroviral expression system, for efficient transduction of primary human T cells. CAR T cells were co-incubated with different antibody adaptors at varying concentrations and target cells and then assayed by flow cytometry for T cell activation and target cell lysis and ELISA for cytokine production. To assess the in vivo activity of SNAP-CAR, NSG mice were challenged with HER2+ or CD20+ human leukemia or ovarian tumor xenografts. Mice were then treated with SNAP-CAR T cells and adaptor injections, and tumor size was measured by IVIS imaging.

Results In vitro experiments showed potent and specific SNAP-CAR function with co-administered adaptors targeting HER2, EGFR, and CD20 on cancer cell lines including activation of CD69 and CD107a markers, specific target cell lysis, and IFN-gamma production. Testing SNAP-CAR T cells in vivo in a human leukemia tumor xenograft NSG mouse model targeting HER2, we observed that SNAP-CAR T cells were able to significantly reduce tumor burden, leading to a lack of detectable tumors in the majority of mice. In another leukemia model targeting the CD20 antigen, SNAP-CAR T cells showed significant inhibition of tumor growth. While tumors in these mice relapsed, investigation demonstrated that relapsed cancer cells were CD20 negative, suggesting the importance of future multi-antigen targeting. Finally, evaluating two anti-HER2 adaptors with distinct binding epitopes in a human ovarian cancer xenograft model, we observed a significant tumor reduction with both adaptors compared to adaptor only and SNAP-CAR T cell only controls.

Conclusions Overall, these data demonstrate the potent and versatile antigen targeting abilities of SNAP-CAR T cells both in vitro and in vivo in human tumor xenograft models, suggesting future potential for treating liquid and solid tumor malignancies. Development of SNAP-CAR T cells for human use is underway.

Acknowledgements This work was supported by NIH grant R01 GM142007 (J.L., A.D.); NIH grant R35 CA210039 (O.J.F.); NIH grant R21 AI130815 (A.D.); AIRC postdoctoral fellowship 22321 (E.R.); the Michael G. Wells Prize (J.L.); and by Coeptis Therapeutics SRA00003007 (J.L., A.D.). This work benefitted from using the SPECIAL BD LSRFORTESSA funded by NIH 1S10OD011925–01. This project also used the Hillman Animal Facility, In Vivo Imaging Facility, and Cytometry Facility that are supported in part by award P30CA047904.

Ethics Approval All work in this study was approved by the University of Pittsburgh Institutional Biosafety Committee (IBC201900130). Animal work in this study was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (23053145), and procedures were performed under their guidelines.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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