Article Text

Download PDFPDF

310 Targeted delivery of a VEGF-blocking scFv to enhance CAR-T cell potency
  1. Valentina Supper1,
  2. Hannah Donner1,
  3. Michael Kann1,
  4. Filippo Birocchi1,
  5. Felix Korell1,
  6. Diego Salas-Benito1,
  7. Stefanie Bailey1,
  8. Tamina Kienka1,
  9. Merle Philips1,
  10. Nicholas Haradhvala2,
  11. Amanda Bouffard1,
  12. Bryan Choi1,
  13. Marcela Maus1 and
  14. Mark Leick1
  1. 1Massachusetts General Hospital, Boston, MA, USA
  2. 2The Broad Institute, Cambridge, MA, 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 Blockade of the vascular endothelial growth factor (VEGF) pathway represents a long-standing strategy for anti-tumor activity, with drugs employing this approach for a variety of solid tumor types. It is also known that VEGF directly drives T cell exhaustion and dysfunction.1 However, systemic VEGF blockade causes significant cardiovascular and renal toxicity and limits the dose of these medications, restricting their maximal theoretical benefit.

In an updated analysis of our previously published single cell RNA sequencing data from infusion products of Tisagenlecleucel,2 we discovered expression of VEGF and VEGFR1 on CAR-T cell infusion products. Building on this observation, we found that our previously reported CD703 and Mesothelin CAR-T cells produce VEGF, as detected by ELISA, and upregulate VEGFR1 upon activation (p<.0001), suggesting active signaling along this pathway may impact CAR-T cell biology.

Methods To improve the therapeutic window of VEGF blockade and mitigate putative VEGF mediated T cell dysfunction, we generated CD70 and Mesothelin targeted CAR-T cells that secrete a VEGF-blocking single chain variable fragment (scFv, 70VEGF and MesoVEGF respectively).

Results 70VEGF and MesoVEGF completely abrogated VEGF signaling in a HEK293 reporter assay (p<0.0001) and blocked all detectable VEGF by ELISA during CAR-T production (p<.0001) compared to CD19 scFv expressing control constructs (7019 and Meso19). 70VEGF and MesoVEGF demonstrated superior activation (by CD69 expression) and expansion in both short term plate bound antigen assays and long term repetitive stimulation assays with CD70 or Mesothelin expressing K562 targets. 70VEGF and MesoVEGF also mediated superior cytotoxicity and CAR-T expansion against RCC, AML, NSCLC, and ovarian cancer cell lines in vitro compared to 7019 and Meso19. Preliminary data suggest that neither CRISPR-Cas9 VEGFA knockout of 7019 (7019,VEGFKO) nor exogenous administration of anti-VEGF scFv alone were sufficient to phenocopy the proliferative advantage of 70VEGF when co-cultured with tumor cells; however, when exogenous anti-VEGF scFv was added to 7019,VEGFKO CAR-T cells the performance was similar to 70VEGF, arguing that a combination of intracrine and exogenous VEGF modulate CAR-T cell function. Nanostring gene expression profiling revealed distinct expression programs between MesoVEGF and Meso19 including lower apoptosis of the former. In vivo experiments with AML PDX, orthotopic RCC, and ovarian/lung cancer metastatic models are ongoing and will be reported at the conference.

Conclusions Targeted delivery of a VEGF blocking scFv is a promising therapeutic strategy that augments CAR-T cell function in a tumor and target agnostic fashion and warrants further development.

Acknowledgements *These first authors contributed equally


  1. Kim C, Jang M, Kim Y. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers. Sci Immunol. 2019;4(41)

  2. Haradhvala N, Leick M, Maurer K. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nature Medicine. 2022;(9):1848–1859

  3. Leick M, Silva H, Scarfò S. Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia, Cancer Cell. 2022;40(5):494–508.

Ethics Approval All care and conducted experiments on mice were carried out with protocols approved by the MGH Institutional Animal Care and Use Committee.

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

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.